U.S. patent application number 13/330099 was filed with the patent office on 2012-12-06 for materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs.
Invention is credited to Elazer R. Edelman, Heiko Methe, Helen Marie Nugent.
Application Number | 20120308610 13/330099 |
Document ID | / |
Family ID | 36695000 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120308610 |
Kind Code |
A1 |
Edelman; Elazer R. ; et
al. |
December 6, 2012 |
Materials and Methods for Altering an Immune Response to Exogenous
and Endogenous Immunogens, Including Syngeneic and Non-Syngeneic
Cells, Tissues or Organs
Abstract
Disclosed herein are materials and methods for modulating an
immunologically adverse response to an exogenous or endogenous
immunogen, including a cell, tissue, or organ associated immunogen.
An implantable material comprising cells, such as but not limited
to endothelial cells, anchored or embedded in a biocompatible
matrix can modulate an adverse immune or inflammatory reaction to
exogenous or endogenous immunogens, including response to
non-syngeneic or syngeneic cells, tissues or organs, exogenous
immunogens or stimuli, as well as ameliorate an autoimmune
condition. The implantable material can be provided prior to,
coincident with, or subsequent to occurrence of the immune response
or inflammatory reaction. The implantable material can induce
immunological acceptance in a transplant patient, reduce graft
rejection and reduce donor antigen immunogenicity.
Inventors: |
Edelman; Elazer R.;
(Brookline, MA) ; Nugent; Helen Marie; (Needham,
MA) ; Methe; Heiko; (Boston, MA) |
Family ID: |
36695000 |
Appl. No.: |
13/330099 |
Filed: |
December 19, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11918908 |
Oct 19, 2007 |
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PCT/US2006/015555 |
Apr 21, 2006 |
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13330099 |
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60673417 |
Apr 21, 2005 |
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60682217 |
May 18, 2005 |
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Current U.S.
Class: |
424/400 ;
424/278.1; 435/325 |
Current CPC
Class: |
A61K 9/00 20130101; C12N
2533/54 20130101; C12N 5/069 20130101; A61P 29/00 20180101; A61K
35/44 20130101; A61K 45/06 20130101; A61P 37/06 20180101; A61K
39/001 20130101; A61K 2035/122 20130101; A61P 43/00 20180101; A61P
37/02 20180101 |
Class at
Publication: |
424/400 ;
424/278.1; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 9/00 20060101 A61K009/00; A61P 37/02 20060101
A61P037/02; A61P 29/00 20060101 A61P029/00; A61P 37/06 20060101
A61P037/06; C12N 5/071 20100101 C12N005/071; A61K 35/44 20060101
A61K035/44 |
Claims
1. A method of reducing an immune response or an inflammatory
reaction, comprising the step of: providing to a recipient an
implantable material comprising a biocompatible matrix; and,
anchored or embedded endothelial cells, endothelial-like cells, or
analogs thereof, wherein said implantable material is provided to
said recipient in an amount sufficient to reduce the immune
response or inflammatory reaction in said recipient.
2. The method of claim 1 wherein the providing step is prior to,
coincident with, or subsequent to administration to said recipient
of one or more doses of a cell, tissue or organ from a syngeneic or
non-syngeneic donor.
3. The method of claim 1 wherein the providing step is prior to,
coincident with, or subsequent to occurrence of the immune response
or inflammatory reaction.
4. A method of inducing acceptance in a patient, comprising the
step of: providing an implantable material comprising a
biocompatible matrix and anchored or embedded endothelial cells,
endothelial-like cells, or analogs thereof, prior to, coincident
with, or subsequent to transplantation of autograft, xenograft or
allograft cells, tissue or organ in said patient in an amount
effective to induce acceptance in said patient.
5. A method of reducing donor antigen immunogenicity, comprising
the step of: providing an implantable material comprising a
biocompatible matrix and anchored or embedded endothelial cells,
endothelial-like cells, or analogs thereof prior to, coincident
with, or subsequent to introduction of said donor antigen to a
recipient in an amount effective to reduce donor antigen
immunogenicity.
6. The method of claim 1, 4 or 5 wherein said providing step occurs
prior to, coincident with, or subsequent to administration to said
recipient of an immunosuppressive agent.
7. The method of claim 6 wherein said immunosuppressive agent
resides in said implantable material during coincident
administration.
8. The method of claim 5 wherein said donor and recipient are the
same.
9. The method of claim 1, 4, 5 or 8 wherein said recipient has an
autoimmune disease.
10. An implantable material suitable for use with any one of claims
1-9.
11. The implantable material of claim 10 wherein the
endothelial-like cells or analogs are non-endothelial cells.
12. The implantable material of claim 10 wherein the cells are
autogenic, allogenic, xenogeneic or genetically-modified variants
of any one of the foregoing.
13. The implantable material of claim 10 wherein the cells are
vascular endothelial cells.
14. A method of transplanting to a recipient a syngeneic or
non-syngeneic cell, tissue or organ transplant, comprising the step
of: providing to said recipient an implantable material comprising
a biocompatible matrix and anchored or embedded endothelial cells,
endothelial-like cells, or analogs thereof, prior to, coincident
with, or subsequent to transplantation such that said transplanted
syngeneic or non-syngeneic cell, tissue or organ is not rejected by
said recipient.
15. The method of claim 4 or 14 wherein said transplanted cell,
tissue or organ comprises non-endothelial cells.
16. The method of claim 5 wherein said donor antigen comprises a
non-endothelial cell antigen.
17. The method of claim 14 further comprising the step of
administering an immunosuppressive agent prior to, coincident with,
or subsequent to transplantation.
18. A cell suitable for use with the implantable material of any
one of claims 1-17.
19. The cell of claim 18 wherein said endothelial-like cell or its
analog is a non-endothelial cell.
20. The cell of claim 18 wherein said analog is non-natural.
21. The cell of claim 18 wherein said cell, when anchored to or
embedded within a biocompatible matrix, reduces a recipient's
humoral or cellular immune response to a non-syngeneic donor cell,
tissue or organ.
22. The cell of claim 18 wherein said cell, when anchored to or
embedded within a biocompatible matrix, exhibits diminished
immunogenicity.
23. The cell of claim 22 wherein said diminished immunogenicity is
reduced expression of MHC or reduced capacity to bind, activate or
promote maturation of innate immune cells, when anchored to or
embedded within a biocompatible matrix, wherein said innate immune
cells are selected from the group consisting of NK cells,
dendritic, cells, monocytes, and macrophages.
24. The cell of claim 18 wherein said cell, when anchored to or
embedded within a biocompatible matrix, exhibits reduced expression
of MHC, costimulatory molecules or adhesion molecules.
25. The cell of claim 18 wherein said cell, when anchored to or
embedded within a biocompatible matrix and co-cultured with a
dendritic cell, inhibits expression by said dendritic cell of
HLA-DR, IL12, Toll-like receptor or CD83; promotes uptake of
dextran by said dendritic cell; or blocks dendritic cell-induced
lymphocyte proliferation; or when co-cultured with adaptive immune
cells inhibits proliferation, activation or differentiation of said
cells, wherein adaptive immune cells are selected from the group
consisting of B-lymphocytes and T-lymphocytes.
26. An implantable material comprising a biocompatible matrix and
the anchored or embedded endothelial cell, endothelial-like cell,
or analog thereof of claim 22.
27. An implantable material comprising a biocompatible matrix and
the anchored or embedded endothelial cell, endothelial-like cell,
or analog thereof of claim 24.
28. An implantable material comprising a biocompatible matrix and
the anchored or embedded endothelial cell, endothelial-like cell,
or analog thereof of claim 25.
29. A cell bank comprising the cell of claim 18 or any one of
claims 23-25.
30. A bank of implantable material comprising the implantable
material of claim 10.
31. A bank of implantable material, wherein said implantable
material comprises a biocompatible matrix and the anchored or
embedded endothelial cell, endothelial-like cell, or analog thereof
of claim 22.
32. A bank of implantable material, wherein said implantable
material comprises a biocompatible matrix and the anchored or
embedded endothelial cell, endothelial-like cell, or analog thereof
of claim 24.
33. A bank of implantable material, wherein said implantable
material comprises a biocompatible matrix and the anchored or
embedded endothelial cell, endothelial-like cell, or analog thereof
of claim 25.
34. The implantable material of claim 10 wherein said implantable
material is a solid or non-solid.
35. The implantable material of claim 10 wherein said implantable
material is provided to the recipient by implantation, injection or
infusion.
36. An implantable material for reducing an immune response to a
non-syngeneic cell, tissue or organ, wherein said implantable
material comprises: a biocompatible matrix; and, anchored thereto
or embedded therein, endothelial cells, endothelial-like cells, or
analogs thereof; or tissue, or organ, or a segment thereof; wherein
an effective amount of said implantable material reduces the
recipient's immune response to said non-syngeneic cell, tissue or
organ.
37. The implantable material of claim 36 wherein said non-syngeneic
cell, tissue or organ is that of the recipient suffering from an
autoimmune disease.
38. A method of reducing an immune response or an inflammatory
reaction resulting from exposure to an exogenous immunogen,
comprising the step of: providing to a recipient an implantable
material comprising a biocompatible matrix; and, anchored or
embedded endothelial cells, endothelial-like cells, or analogs
thereof, wherein said implantable material is provided to said
recipient in an amount sufficient to reduce the immune response or
inflammatory reaction in said recipient resulting from exposure to
said exogenous immunogen.
39. The method of claim 38 wherein the providing step is prior to,
coincident with, or subsequent to occurrence of the immune response
or inflammatory reaction.
40. The method of claim 38 wherein said exogenous immunogen is
naturally occurring.
41. The method of claim 38 wherein said exogenous immunogen is
selected from the group consisting of pharmaceutical agents,
toxins, surgical implants, infectious agents and chemicals.
42. The method of claim 38 wherein said exogenous immunogen is an
exogenous stimulus selected from the group consisting of
environmental stress, injury and exposure.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/918,908, filed Dec. 19, 2007, which is a
U.S. national phase application of International Patent Application
No. PCT/US2006/015555, filed Apr. 21, 2006, which claims priority
to and the benefit of U.S. Provisional Patent Application No.
60/673,417, filed Apr. 21, 2005, and U.S. Provisional Patent
Application No. 60/682,217, filed May 18, 2005, the contents of
each being incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention is directed to materials and methods for
modulating an immunologically adverse response to an exogenous or
endogenous immunogen, including a cell-, tissue-, or
organ-associated immunogen. For example, the present invention can
modulate an adverse immune response or inflammatory reaction to
exogenous or endogenous immunogens, including non-syngeneic or
syngeneic cells, tissues or organs, as well as ameliorate an
autoimmune condition.
BACKGROUND OF THE INVENTION
[0003] Research on xenotransplantation has been intensified over
the past years to alleviate organ shortage. However, host immune
responses present a formidable barrier to transplantation across
species. Whereas natural antibodies cause immediate rejection of
such discordant transplants, endothelial cell (EC) injury and
activation of graft vessel lining EC play a pivotal role in
initiating chronic graft rejection. Disruption of the integrity of
the endothelial layer is of undoubted importance in numerous
conditions, including syngeneic and non-syngeneic tissue
transplants as well as infectious, neoplastic, inflammatory and
cardiovascular diseases.
[0004] Heretofore immunomodulation and transplant acceptance have
required reliance on systemically-administered immunosuppressive
agents. While such agents permit some degree of transplant
acceptance, success is limited and perhaps of more significance, a
patient's immune system is thoroughly compromised as a result of
such agents. Thus a need still remains for therapeutic materials
and treatment paradigms which can achieve immunomodulation absent
the toxicity and adverse affects on a patient's immune system.
[0005] Similarly, exogenous immunogens or stimuli have posed a
clinical challenge. These, too, can result in adverse immunological
events or inflammatory reactions which necessitate treatment.
Heretofore, clinical management of such adverse events has relied
almost exclusively on treatments with pharmaceutical agents which
suppress the immune system non-specifically.
[0006] Autoimmune diseases and other similar diseases are yet
another clinical manifestation of heightened inflammatory reactions
or adverse immune responses. Successful management of such diseases
has eluded clinicians to date.
[0007] An object of the present invention is to provide a tissue
engineering solution for achieving immunomodulation without
reliance on chemicals or pharmaceuticals which compromise a
patient's immune system. This tissue engineering solution can be
employed to alter, in a clinically practical manner, an immune
response to exogenous and endogenous immunogens, including
non-syngeneic as well as syngeneic cell-, tissue- or
organ-associated immunogens. Another object of the present
invention is to facilitate a patient's acceptance of non-syngeneic
as well as syngeneic cells, tissues or organs. Another object of
the present invention is to employ this tissue engineering solution
to modulate an inflammatory reaction such as that associated with
injury and various diseases. Another object is to utilize the
materials and methods of the present invention to manage
autoimmunity and related diseases.
SUMMARY OF THE INVENTION
[0008] The present invention exploits the discovery that cells
anchored to and/or embedded within a biocompatible matrix,
preferably one having a three-dimensional configuration, can
modulate an immunologically adverse response or inflammatory
reaction to any exogenous or endogenous immunogen. Immunogen
includes any syngeneic or non-syngeneic immunogen, including a
cell-, tissue-, or organ-associated immunogen, as well as injury,
disease and environmental stimuli.
[0009] In one aspect, the present invention is a method of reducing
an immune response or an inflammatory reaction. According to this
method, a recipient is provided an implantable material comprising
a biocompatible matrix and anchored and/or embedded endothelial
cells, endothelial-like cells, or analogs thereof. The implantable
material is provided to the recipient in an amount sufficient to
reduce the immune response or inflammatory reaction in the
recipient.
[0010] According to the invention, the providing step can occur
prior to, coincident with, or subsequent to administration to the
recipient of one or more doses of a cell, tissue or organ from a
syngeneic or non-syngeneic donor. According to another embodiment,
the providing step is prior to, coincident with, or subsequent to
occurrence of an immune response or inflammatory reaction.
According to another embodiment, the method reduces an immune
response or an inflammatory response by modulating immunological
memory.
[0011] In a related aspect, the present invention is a method of
inducing immunological acceptance in a patient. According to this
method, the patient is provided an implantable material comprising
a biocompatible matrix and anchored and/or embedded endothelial
cells, endothelial-like cells, or analogs thereof, prior to,
coincident with, or subsequent to transplantation of autograft,
xenograft or allograft cells, tissue or organ in an amount
effective to induce acceptance in the patient.
[0012] Additionally, the present invention is directed to a method
of reducing donor antigen immunogenicity. According to this method,
an implantable material comprising a biocompatible matrix and
anchored and/or embedded endothelial cells, endothelial-like cells,
or analogs thereof are presented prior to, coincident with, or
subsequent to introduction of the donor antigen to a recipient in
an amount effective to reduce donor antigen immunogenicity.
According to another embodiment, the donor and recipient are the
same. According to a further embodiment, the recipient has an
autoimmune disease. According to yet another embodiment, the donor
antigen comprises a non-endothelial cell antigen.
[0013] According to various other embodiments, the providing step
occurs prior to, coincident with, or subsequent to administration
to the recipient of an immunosuppressive agent. The
immunosuppressive agent can reside in the implantable material.
[0014] Moreover, the present invention is also directed to a method
of transplanting to a recipient a syngeneic or non-syngeneic cell,
tissue or organ transplant. According to this method, a recipient
is provided an implantable material comprising a biocompatible
matrix and anchored and/or embedded endothelial cells,
endothelial-like cells, or analogs thereof, prior to, coincident
with, or subsequent to transplantation such that the transplanted
syngeneic or non-syngeneic cell, tissue or organ is not rejected by
the recipient. According to one embodiment of the method, the
transplanted cell, tissue or organ comprises non-endothelial
cells.
[0015] In another aspect, the present invention is an implantable
material comprising a biocompatible matrix and cells anchored
thereto and/or embedded therein. According to one currently
preferred embodiment, the cells are endothelial cells,
endothelial-like cells and/or analogs of either. In certain other
embodiments, endothelial-like cells or analogs of the implantable
material are non-endothelial cells. According to another
embodiment, the cells of the implantable material are autogenic,
allogenic, xenogeneic or genetically-modified variants of any one
of the foregoing cell types. According to a further preferred
embodiment, the cells of the implantable material are vascular
endothelial cells. According to one embodiment, the implantable
material is a solid or non-solid. According to yet another, the
implantable material is provided to the recipient by implantation,
injection or infusion.
[0016] The present invention is also directed to an implantable
material for reducing an immune response to a syngeneic or
non-syngeneic cell, tissue or organ. According to this aspect of
the invention, the implantable material comprises a biocompatible
matrix and, anchored thereto and/or embedded therein, endothelial
cells, endothelial-like cells, or analogs thereof. According to
this aspect of the invention, an effective amount of the
implantable material reduces the recipient's immune response to the
syngeneic or non-syngeneic cell, tissue or organ. According to one
embodiment of this aspect of the present invention, the cell,
tissue or organ is that of the recipient suffering from an
autoimmune disease.
[0017] The invention is also directed to a variation of the
above-described implantable material which is useful for reducing
an immune response to a non-syngeneic cell, tissue or organ,
wherein said implantable material comprises cells, tissue, or organ
or a segment thereof anchored to and/or embedded within the
biocompatible matrix. An effective amount of this implantable
material reduces the recipient's immune response to a non-syngeneic
cell, tissue or organ. The non-syngeneic cell, tissue or organ is
that of the recipient suffering from an autoimmune disease.
[0018] In a further aspect, the present invention is a cell
suitable for use with the implantable material of any one of
inventions described herein. According to one embodiment, the
endothelial-like cell or its analog is a non-endothelial cell.
According to another embodiment, the analog is non-natural.
According to a further embodiment, the cell, when anchored to
and/or embedded within a biocompatible matrix, reduces a
recipient's humoral or cellular immune response to a syngeneic or
non-syngeneic donor cell, tissue or organ.
[0019] According to another embodiment, the cell, when anchored to
and/or embedded within a biocompatible matrix, exhibits diminished
immunogenicity. According to one embodiment, the cell exhibits
diminished immunogenicity by exhibiting reduced expression of MHC
or reduced capacity to bind, activate or promote maturation of
innate immune cells, when anchored to and/or embedded within a
biocompatible matrix, wherein said innate immune cells are selected
from the group consisting of NK cells, dendritic, cells, monocytes,
and macrophages.
[0020] According to another embodiment, the cell, when anchored to
and/or embedded within a biocompatible matrix, exhibits reduced
expression of costimulatory molecules or adhesion molecules.
According to a further embodiment, the cell, when anchored to
and/or embedded within a biocompatible matrix and co-cultured with
a dendritic cell, inhibits expression by said dendritic cell of
HLA-DR, IL12, Toll-like receptor or CD83; promotes uptake of
dextran by said dendritic cell; or blocks dendritic cell-induced
lymphocyte proliferation; or when co-cultured with adaptive immune
cells inhibits proliferation, activation or differentiation of said
cells, wherein adaptive immune cells are selected from the group
consisting of B-lymphocytes and T-lymphocytes.
[0021] In another aspect, the present invention is a cell bank
comprising any one of the cells described herein. In a further
aspect, the present invention is a bank comprising any one of the
implantable materials described herein.
[0022] In a further aspect, the present invention is a method of
reducing an immune response or an inflammatory reaction resulting
from exposure to an exogenous immunogen. According to this method,
a recipient is provided with an implantable material comprising a
biocompatible matrix and anchored or embedded endothelial cells,
endothelial-like cells, or analogs thereof. The implantable
material is provided to the recipient in an amount sufficient to
reduce the immune response or inflammatory reaction in the
recipient resulting from exposure to the exogenous immunogen.
[0023] According to one embodiment of this method, the providing
step is prior to, coincident with, or subsequent to occurrence of
the immune response or inflammatory reaction. According to another
embodiment, the exogenous immunogen is naturally occurring.
According to a further embodiment, the exogenous immunogen is
selected from the group consisting of pharmaceutical agents,
toxins, surgical implants, infectious agents and chemicals.
According to another embodiment, the exogenous immunogen is an
exogenous stimulus selected from the group consisting of
environmental stress, injury and exposure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A, 1B and 1C graphically depict levels of circulating
PAE-specific antibodies according to an illustrative embodiment of
the invention.
[0025] FIG. 2 graphically depicts lytic activity of splenocytes
according to an illustrative embodiment of the invention.
[0026] FIG. 3A graphically depicts the frequencies of
cytokine-producing cells according to an illustrative embodiment of
the invention.
[0027] FIG. 3B depicts representative ELISPOT wells according to an
illustrative embodiment of the invention.
[0028] FIG. 3C graphically depicts the frequencies of T cells
according to an illustrative embodiment of the invention.
[0029] FIGS. 4A and 4B graphically plot levels of effector cells
according to an illustrative embodiment of the invention.
[0030] FIGS. 5A, 5B and 5C graphically depict antibody levels
according to an illustrative embodiment of the invention.
[0031] FIG. 6 graphically depicts levels of splenocytes according
to an illustrative embodiment of the invention.
[0032] FIGS. 7A and 7B graphically depict antibody levels according
to an illustrative embodiment of the invention.
[0033] FIGS. 8A and 8B graphically depict the frequency of
cytokine-producing cells according to an illustrative embodiment of
the invention.
[0034] FIGS. 9A and 9B graphically depict effector cell levels
according to an illustrative embodiment of the invention.
[0035] FIGS. 10A and 10B depict correlations between the frequency
of Th2-cytokine producing splenocytes and the extent of T cell
differentiation into effector cells according to an illustrative
embodiment of the invention.
[0036] FIG. 11 graphically depicts the degree of damage to
endothelial cells according to an illustrative embodiment of the
invention.
[0037] Figures which refer to "embedded" or "matrix-embedded" PAE,
HAE or EC mean matrix-anchored and/or matrix-embedded PAE, HAE,
EC.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Tissue engineering is a promising approach to exploit
endothelial cells, endothelial-like cells, or analogs of either as
a cellular therapy for diseases accompanied by or typified by
adverse immunological components. For example, certain diseases
such as but not limited to vascular diseases provoke adverse
immunological responses and/or inflammatory reactions. The present
invention is based on the discovery that cells such as endothelial
cells which are anchored to or embedded in three-dimensional
matrices, secrete essential regulatory factors which can ameliorate
or otherwise modulate an adverse immunological response.
[0039] The implantable material of the present invention was
developed on the principals of tissue engineering and represents a
novel approach to addressing the herein-described clinical needs.
The implantable material of the present invention is unique in that
the viable cells anchored to and/or embedded within the
biocompatible matrix are able to supply to the site of
administration multiple cell-based products in physiological
proportions under physiological feed-back control. As described
elsewhere herein, the cells suitable for use with the implantable
material are endothelial, endothelial-like cells, or analogs of
each of the foregoing. Local delivery of multiple compounds by
these cells and physiologically-dynamic dosing provide more
effective regulation of the processes responsible for modulating an
immune response. The implantable material of the present invention
can provide an environment which mimics supportive physiology and
is conducive to modulation of an immune response.
[0040] This is an unexpected discovery since endothelial cells can
play a pivotal role in initiation of adverse allo- and xeno-immune
responses. Moreover, endothelial cells can activate T-cells through
antigen-mediated processes and T-cell activation can modify crucial
endothelial cell function, including antigen presentation via
activation by cytokines, thereby contributing to an adverse immune
response. And, endothelial cells constitutively express class I
Major Histocompatibility Complex (MHC) molecules, and IFN-.gamma.
can induce endothelial cells to express class II MHC molecules
which allows them to provide antigen-dependent signals to CD8.sup.+
and CD4.sup.+ T-cells through the direct pathway. Endothelial cells
also can primarily provide costimulation to T-cells. In addition,
the capacity to capture T-cells via endothelial expression of
adhesion molecules allows formation of contact regions which
furthers the adverse immune response in the form of inflammation.
Furthermore, autoimmunity can exacerbate vascular disease, in
particular in the form of anti-endothelial cell antibodies. The
heightened morbidity of cardiovascular diseases in concert with
diabetes mellitus, hypertension and other disease states reflects
the increased presence and potency of these antibodies.
[0041] In contrast, as disclosed herein, matrix-anchored and/or
-embedded endothelial cells, when implanted in a host, act as
powerful regulators of the immune system as indicated by
significant reduction in the expected systemic immune response
and/or local inflammation. As exemplified herein, the ability of
such cells to ameliorate or modulate immune responsiveness has been
demonstrated by comparing the immune response against free versus
matrix-anchored and/or -embedded endothelial cells in naive mice as
well as mice with heightened endothelial cell immune reactivity.
Matrix-associated endothelial cells as described herein provide
immune protection at multiple levels; human and porcine endothelial
cells demonstrate a marked reduction in elaborated MHC class
molecules; costimulatory molecules; and adhesion molecules when
matrix-anchored and/or -embedded as disclosed herein.
[0042] Matrix anchoring and/or embedding of endothelial cells can
also influence formation of immunological memory as exemplified
herein. Whereas reimplantation of free, saline-suspended
endothelial cell pellets alone or as pellets situated adjacent to
an empty matrix evoked a significant increased humoral and cellular
xenoresponse, rechallenging mice with matrix-anchored and/or
-embedded endothelial cells led to a reduced lytic ability of
splenocytes without enhancing the humoral immune responses.
Moreover, a modest shift in the Th1/Th2 balance towards the former
was obvious in mice receiving matrix-anchored and/or -embedded
xenogeneic endothelial cells.
[0043] Thus, introduction of free endothelial cells adjacent to an
empty matrix failed to reduce the host immune response indicating
the importance of matrix-anchoring and/or -embedding. Failure of
anchored and/or embedded endothelial cells to express MHC II,
costimulatory, and adhesion molecules upon stimulation could
account for the attenuated differentiation of T-cells in effector
cells in response to implanted matrix-anchored and/or -embedded
xenogeneic endothelial cells. As explained herein, activation of
mice splenocytes is muted when exposed to matrix-anchored and/or
-embedded xenogeneic endothelial cells in a MHC class II dependent
manner.
[0044] Overall the isotropic nature of endothelial cells
contributes to this unique form of immunomodulation wherein cell
anchoring and/or embedding in a suitable matrix provides
immunoprotection through isolation or masking of critical antigens.
It is well recognized that in vivo endothelial cell function is
anchorage- and density-dependent. Previous studies have shown that
the endothelial basement membrane (EBM) controls aspects of cell
adhesion, spreading, migration, contractility, differentiation,
proliferation, protein synthesis and secretion. Furthermore, EBM is
altered in many in vivo disease states, from diabetes to
glomerulopathy to atherosclerosis. Dysfunction of endothelial cells
correlates with changes in basement membrane composition cumulating
in the degree of attachment of endothelial cells, and the quality
of basement membrane anchoring plays a role for endothelial cells
immunobiology.
[0045] The present invention is based on the unexpected discovery
that anchoring and/or embedding endothelial cells in a suitable
biocompatible matrix, such as but not limited to a 3-dimensional
collagen-based matrix, can transform xenogeneic endothelial cells
into an immunologically non-offending cell phenotype. Such a
discovery can now be exploited by the skilled practitioner,
following the guidance provided herein, as a tolerance-inducing
approach to syngeneic or non-syngeneic therapies such as but not
limited to allotransplantation or xenotransplantation as
exemplified herein. For example, in a preferred embodiment of the
present invention, a clinician can diminish and/or delay rejection
by implanting matrix-anchored and/or -embedded endothelial cells
prior to transplantation of an allo- or xenograft tissue or organ.
For purposes of the present invention, blood is a type of
tissue.
[0046] Pre-treatment acclimates the recipient's immune system and
can result in a reduced, attenuated and/or delayed immune response
to a graft. The present invention does not require that the
implantable material comprise anchored- and/or embedded cells which
are the same as or similar to those ultimately transplanted in the
recipient. All that is required is that the implantable material
comprising anchored- and/or embedded cells has an immunomodulatory
effect when provided to a recipient. In certain circumstances, a
single administration prior to or coincident with a transplant can
be sufficient. In other circumstances, multiple or serial
administrations are preferred. The skilled clinician will recognize
such circumstances.
[0047] As is well recognized, even transplantation of allogeneic
cells is often accompanied by an immune response. A question of
much interest is whether this is a constitutive and immutable
property of foreign cells or one that can be regulated. The
experiments set forth herein demonstrate that the immunogenicity of
cells that are normally anchored to basement membranes can be
markedly reduced if implanted in a matrix-anchored and/or -embedded
state an effect not seen when these same cells were injected in a
free state. Other experiments set forth herein investigate the
influence of heightened anti-endothelial cell immunity which is a
common clinical feature in a variety of autoimmune and
endocrinological diseases.
[0048] Additionally, certain of the experiments summarized herein
demonstrate that serial injections of free porcine aortic
endothelial cells (PAE) induced circulating anti-PAE antibodies,
elevating immunosensitivity. The response to subsequent PAE
injections was even greater than that observed upon first exposure.
In contrast, when PAE were implanted in a matrix-anchored and/or
-embedded state, the immune response to subsequent exposures was
muted and dropped significantly over time. Also, as illustrated
below, the initial response to endothelial cells is IgM-mediated,
lower than the subsequent IgG response and muted when preceded by
serial injections. The IgM response is more evident in naive than
pre-sensitized animals and takes longer to abate after free PAE
exposure to than after exposure to matrix-anchored and/or -embedded
endothelial cells.
[0049] Pre-sensitization of mice with suspensions of free PAE
resembles the IgG.sub.1-driven anti-endothelial immunity seen in
diabetes mellitus, hypertension and autoimmune diseases. The
cellular immune response to free and matrix-anchored
and/or--embedded cells followed the pattern of humoral immunity.
Repeated exposure to antigens resulted in increased formation of
memory and subsequently in a more vigorous immune reaction by
effector T cells. Hence, the induction of xenoreactive IL-4- and
IL-10-producing splenocytes and effector T cells was elevated over
time and visible after implantation of free endothelial cells in
naive and pre-sensitized mice. In all mice, cytokine levels
correlated linearly and precisely with effector T cell induction
further supporting the notion of a Th2-driven cellular response in
xenoreactivity and accentuating the immunosilencing aspects of
matrix-embedded endothelial cells to activate adaptive immune
mechanisms. Damage to implanted endothelial cells correlated with
the extent of the immune response elicited. Implanted cells were
most profoundly affected after pre-sensitization and with free PAE.
The decreased induction of humoral and cellular immune responses in
naive mice receiving matrix-embedded endothelial cells resulted in
a lesser degree of damage by host immune cells.
[0050] These experiments provide insights into the activation of
and damage to endothelial cells, suggesting a pivotal role for
cell-matrix contact. The honeycomb-like structure of a currently
preferred matrix, Gelfoam, allows endothelial cells to associate
with, or anchor to, or embed within its three-dimensional
configuration and in certain embodiments, line the internal
surfaces of this matrix in a fashion which simulates the appearance
of confluent endothelium in quiescent vessels. Thus in certain
embodiments, anchoring to and/or embedding endothelial cells within
a matrix with the properties of Gelfoam resembles the physiologic
three-dimensional state of intact endothelium. The experiments set
forth below demonstrate that matrix-anchoring and/or embedding not
only protects endothelial cells from host immune reactions but
changes the host's perception of endothelial cell
immunogenicity.
[0051] Thus, during disease for example, phenotypic transformation
of endothelial cells dislocated from an intact, matrix-adherent
endogenous state to a free state is likely critical to initiation
and perpetuation of vascular disease, for example. The teachings
herein indicate that endothelial cell detachment precedes
expression of adhesion, costimulatory and MHC molecules which is
then followed by attraction of immune cells, perpetuating
endothelial activation and cell damage. In this regard, the
immunobiological and immunoreactive qualities of endothelial cells
correlate with morphology and function. Endothelial cells from
different vascular beds and divergent basement membrane
connectivity demonstrate marked differences in constitutive and
inducible expression of adhesion, costimulatory and MHC-molecules.
Further, there is growing appreciation that deposition of
transitional extracellular matrix proteins such as fibronectin and
fibrinogen into the subendothelial matrix as well as detachment of
endothelial cells from the basement membrane affects
intra-endothelial cell signaling.
[0052] As contemplated by the present invention, manipulation of
cell phenotype, immunogenicity, and function can be used to tailor
the properties of tissue engineered constructs developed in vitro
for regenerative purposes; in particular, such a use of the present
invention is clinically beneficial since current cell-based
therapies are limited by profound host immune reactions. For
example, the present invention is particularly useful for treatment
of atherosclerotic disease since the presence of activated immune
cells and inflammation are key pathophysiologic components.
Similarly, heightened anti-endothelial immunity has been identified
as a pivotal rate-limiting effect for endothelial cell-based
therapies, such as but not limited therapies involving seeding of
the interior of a vascular structure with cells or tissue. In
contrast, the present invention can be exploited to manage
endothelial cell phenotypic shifts which occur in vascular
pathology, e.g., via dearrangement of cell-matrix contact, and
appropriately targeted therapeutic options can then be implemented
in the clinic using the materials and methods of the present
invention.
[0053] Taken together, the teachings presented herein also
demonstrate that features of a matrix such as but not limited to
biocompatibility, porosity, three-dimensionality, can support the
growth of a population of endothelial cells and can modulate the
immunogenicity of such cells. Endothelial cells anchored to and/or
embedded within a three-dimensional matrix elicited far less
activation of host immune mechanisms and were subject to far lower
attack and damage from host immune cells. Findings in naive mice
were amplified in hosts with heightened anti-endothelial immunity.
In vivo studies presented herein show a marked decrease in the
Th2-driven immune response in animals implanted with a matrix such
as Gelfoam comprising anchored and/or embedded endothelial cells
versus animals injected with free endothelial cells. In order for
endothelial cells to activate naive host T-cells, two signals are
required: 1) antigen-presentation in the context of MHC molecules
expressed on the donor endothelial cells and 2) a second signal
provided by a costimulatory molecule also expressed on the donor
endothelial cell surface. Therefore, while not wishing to be bound
by theory, one possible explanation for the observed results is
that the interaction between a biocompatible matrix and embedded
endothelial cells results in a decrease in surface expression of
crucial costimulatory, MHC and/or adhesion molecules on the donor
endothelial cells. Indeed, in vitro analysis of critical adhesion,
MHC-II and co-stimulatory molecule expression on both PAE and HAE
(human aortic endothelial cells) show a matrix-anchored and/or
embedded dependent profile. The expression profiles of adhesion
(E-selectin, P-selectin, ICAM-1, VCAM-1, and CD58), costimulatory
(CD40, CD80, CD86) and MHC-II molecules were all reduced in
endothelial cells anchored to and/or embedded with a Gelfoam matrix
as compared to the same endothelial cells grown on standard tissue
culture plates. P-selectin, E-selectin and VCAM-1 are closely
associated with T-cell recruitment at sites of immune inflammation.
Because antigen presentation to CD4+ T-cells via MHC class II
molecules is essential for host immune recognition in the setting
of non-vascularized xenogeneic implants, the observed reduced
MHC-II expression on matrix-anchored and/or -embedded endothelial
cells translated into a reduced proliferative response of host
splenocytes. Furthermore, repeated in vitro exposure of the same
splenocytes to endothelial cells grown on tissue culture plates
elicited a more vigorous secondary response, whereas there was no
increased secondary response and therefore no memory of prior
exposure to matrix-anchored and/or -embedded endothelial cells.
These in vitro findings correlate with the significantly muted
immune reaction observed in rats and mice after implantation and
re-challenge with matrix-anchored and or embedded endothelial cells
as exemplified herein.
[0054] Similarly, a mechanism by which culturing endothelial cells
in a biocompatible matrix such as but not limited to Gelfoam
affects expression of MHC class II molecules, and subsequent
endothelial immunogenicity in vitro, was further elucidated by
investigating intracellular signaling pathways. Endothelial
expression of MHC class II molecules is induced by proinflammatory
cytokines (e.g. interferon (IFN)-.gamma.) that are secreted by
activated immune cells (e.g. T-cells). Binding of proinflammatory
cytokines to their receptors on endothelial cells initiates an
intracellular signaling cascade resulting in phosphorylation of
Janus protein tyrosine kinase (e.g. JAK-1 and 2) and signal
transducer and activators of transcription (e.g. STAT-1).
Activation of JAK and STAT are usually tightly regulated within a
target cell. As set forth below, detailed in vitro analyses
demonstrated differences in IFN-.gamma. induced intracellular
signaling pathways between endothelial cells grown to confluence on
tissue culture plates as compared to those anchored to and/or
embedded or within Gelfoam matrices. Gelfoam-embedded HAE exhibited
lower rates of STAT-phosphorylation and activation of the crucial
interferon-regulatory factor-1 (IRF-1) with no change in surface
IFN-.gamma. receptor expression. Lower rates of JAK activation were
also seen upon stimulation of HAE in Gelfoam with IFN-.gamma..
[0055] Upon further investigation, it was observed that
non-IFN-.gamma. stimulated HAE grown on a Gelfoam matrix expressed
significantly higher levels of the counteracting inhibitory
molecule, Suppressor of Cytokine Signaling (SOCS)-1 and 3, than HAE
grown on tissue culture plates. One explanation therefore for the
muted IFN-.gamma. induced intracellular signaling in
Gelfoam-embedded HAE is that the increased levels of SOCS-1 and 3
resulted in an increase in the threshold for cytokine-induced
activation of endothelial cells.
[0056] In a currently preferred embodiment, the implantable
material of the present invention comprising anchored and/or
embedded endothelial cells is implanted at any non-luminal site.
Thus, immediate exposure of the donor cells to the host circulation
is not required. Recent evidence has demonstrated the importance of
the soluble endothelial factor CX.sub.3CL1 (fractalkine) for
attraction of immune cells (i.e., natural killer cells) and surface
expressed forms of fractalkine for adherence of those immune cells.
Given that only modest cellular infiltration in and around
implantation sites of xeno- and allogeneic matrix-anchored and/or
embedded endothelial cells was observed, release of soluble and
surface expression of fractalkine on HAE was quantified. As
illustrated in experiments set forth below, matrix-anchored and/or
embedded endothelial cells showed reduced secretion and
down-regulation of fractalkine surface expression upon cytokine
stimulation as compared to HAE grown on tissue culture plates. This
resulted in significantly less adherence of human natural killer
cells to matrix-anchored and/or embedded HAE in vitro.
[0057] Taken together, the changes in intracellular signaling,
increased levels of SOCS-1 and 3 (resulting in attenuated
expression of MHC-II molecules and subsequent T-cell activation) as
well as reduced secretion and surface expression of fractalkine in
matrix-anchored and/or embedded endothelial cells as compared to
cells grown on tissue culture plates indicated an altered
endothelial cell immunogenicity attributable to
matrix-embedding.
[0058] Cell Source.
[0059] As described herein, the implantable material of the present
invention comprises cells which can be syngeneic, allogeneic,
xenogeneic or autologous. In certain embodiments, a source of
living cells can be derived from a suitable donor. In certain other
embodiments, a source of cells can be derived from a cadaver or
from a cell bank.
[0060] In one currently preferred embodiment, cells are endothelial
cells. In a particularly preferred embodiment, such endothelial
cells are obtained from vascular tissue, preferably but not limited
to arterial tissue. As exemplified below, one type of vascular
endothelial cell suitable for use is an aortic endothelial cell.
Another type of vascular endothelial cell suitable for use is
umbilical cord vein endothelial cells. And, another type of
vascular endothelial cell suitable for use is coronary artery
endothelial cells. Yet other types of vascular endothelial cells
suitable for use with the present invention include pulmonary
artery endothelial cells and iliac artery endothelial cells.
[0061] In another currently preferred embodiment, suitable
endothelial cells can be obtained from non-vascular tissue.
Non-vascular tissue can be derived from any tubular anatomical
structure as described elsewhere herein or can be derived from any
non-vascular tissue or organ.
[0062] In yet another embodiment, endothelial cells can be derived
from endothelial progenitor cells or stem cells; in still another
embodiment, endothelial cells can be derived from progenitor cells
or stem cells generally. In a preferred embodiment, the cells can
be progenitor cells or stem cells. In other preferred embodiments,
cells can be non-endothelial cells that are syngeneic, allogeneic,
xenogeneic or autologous derived from vascular or non-vascular
tissue or organ. The present invention also contemplates any of the
foregoing which are genetically altered, modified or
engineered.
[0063] In a further embodiment, two or more types of cells are
co-cultured to prepare the present implantable material. For
example, a first cell can be introduced into the biocompatible
matrix and cultured until confluent. The first cell type can
include, for example, smooth muscle cells, fibroblasts, stem cells,
endothelial progenitor cells, a combination of smooth muscle cells
and fibroblasts, any other desired cell type or a combination of
desired cell types suitable to create an environment conducive to
endothelial cell growth. Once the first cell type has reached
confluence, a second cell type is seeded on top of the first
confluent cell type in, on or within the biocompatible matrix and
cultured until both the first cell type and second cell type have
reached confluence. The second cell type may include, for example,
endothelial cells or any other desired cell type or combination of
cell types. It is contemplated that the first and second cell types
can be introduced step wise, or as a single mixture. It is also
contemplated that cell density can be modified to alter the ratio
of smooth muscle cells to endothelial cells. Similarly, matrices
can be seeded initially with a mixture of different cells suitable
for the intended indication or clinical regimen.
[0064] All that is required of the anchored and/or embedded cells
of the present invention is that they exhibit one or more preferred
phenotypes or functional properties. The present invention is based
on the discovery that a cell having a readily identifiable
phenotype (described elsewhere herein) when associated with a
preferred matrix can reduce, ameliorate, and/or otherwise modulate
an immune response or inflammatory reaction via systemic and/or
local effects.
[0065] For purposes of the present invention, one such preferred,
readily identifiable phenotype typical of cells of the present
invention is an altered immunogenic phenotype as measured by the in
vitro assays described elsewhere herein. Another readily
identifiable phenotype typical of cells of the present invention is
an ability to block or interfere with dendritic cell maturation as
measured by the in vitro assays described elsewhere herein. Each
phenotype is referred to herein as an immunomodulatory
phenotype.
[0066] Evaluation of Immunomodulatory Functionality:
[0067] For purposes of the invention described herein, the
implantable material can be tested for indicia of immunomodulatory
functionality prior to implantation. For example, samples of the
implantable material are evaluated to ascertain their ability to
reduce expression of MHC class II molecules, to reduce expression
of co-stimulatory molecules, to inhibit the maturation of
co-cultured dendritic cells, and to reduce the proliferation of T
cells. In certain preferred embodiments, the implantable material
can be used for the purposes described herein when the material is
able to reduce expression of MHC class II molecules by at least
about 25-80%, preferably 50-80%, most preferably at least about
80%; to reduce expression of co-stimulatory molecules by at least
about 25-80%, preferably 50-80%, most preferably at least about
80%; inhibit maturation of co-cultured dendritic cells by at least
about 25-95%, preferably 50-95%, most preferably at least about
95%; and/or reduce proliferation of lymphocytes by at least about
25-90%, preferably 50-90%, most preferably at least about 90%.
[0068] Levels of expression of MHC class II molecules and
co-stimulatory molecules can be quantitated using routine flow
cytometry analysis, described in detail below. Proliferation of
lymphocytes can be quantitated by in-vitro coculturing
.sup.3[H]-thymidine-labeled CD3+-lymphocytes with the implantable
composition via scintillation-counting as described below in
detail. Inhibition of dendritic cell maturation can be quantitated
by either co-culturing the implantable material with dendritic
cells and evaluating surface expression of various markers on the
dendritic cells by flow cytometry and FACS analysis, or by
measuring dendritic cell uptake of FITC-conjugated dextran by flow
cytometry. Each of these methods is described in detail below.
[0069] In a typical operative embodiment of the present invention,
cells need not exhibit more than one of the foregoing phenotypes.
In certain embodiments, cells can exhibit more than one of the
foregoing phenotypes.
[0070] While the foregoing phenotypes each typify a functional
endothelial cell, such as but not limited to a vascular endothelial
cell, a non-endothelial cell exhibiting such a phenotype(s) is
considered endothelial-like for purposes of the present invention
and thus suitable for use with the present invention. Cells that
are endothelial-like are also referred to herein as functional
analogs of endothelial cells; or functional mimics of endothelial
cells. Thus, by way of example only, cells suitable for use with
the materials and methods disclosed herein also include stem cells
or progenitor cells that give rise to endothelial-like cells; cells
that are non-endothelial cells in origin yet perform functionally
like an endothelial cell using the parameters set forth herein;
cells of any origin which are engineered or otherwise modified to
have endothelial-like functionality using the parameters set forth
herein.
[0071] Typically, cells of the present invention exhibit one or
more of the aforementioned phenotypes when present in confluent,
near-confluent or post-confluent populations and associated with a
preferred biocompatible matrix such as those described elsewhere
herein. As will be appreciated by one of ordinary skill in the art,
confluent, near-confluent or post-confluent populations of cells
are identifiable readily by a variety of techniques, the most
common and widely-accepted of which is direct microscopic
examination. Others include evaluation of cell number per surface
area using standard cell counting techniques such as but not
limited to a hemocytometer or coulter counter.
[0072] Additionally, for purposes of the present invention,
endothelial-like cells include but are not limited to cells which
emulate or mimic functionally and phenotypically confluent,
near-confluent or post-confluent endothelial cells as measured by
the parameters set forth herein.
[0073] Thus, using the detailed description and guidance set forth
below, the practitioner of ordinary skill in the art will
appreciate how to make, use, test and identify operative
embodiments of the implantable material disclosed herein. That is,
the teachings provided herein disclose all that is necessary to
make and use the present invention's implantable materials. And
further, the teachings provided herein disclose all that is
necessary to identify, make and use operatively equivalent
cell-containing compositions. At bottom, all that is required is
that equivalent cell-containing compositions are effective to
modulate an immune response in accordance with the methods
disclosed herein. As will be appreciated by the skilled
practitioner, equivalent embodiments of the present composition can
be identified using only routine experimentation together with the
teachings provided herein.
[0074] In certain preferred embodiments, endothelial cells used in
the implantable material of the present invention are isolated from
the aorta of human cadaver donors. Each lot of cells is derived
from a single or multiple donors, tested extensively for
endothelial cell purity, biological function, the presence of
bacteria, fungi, known human pathogens and other adventitious
agents. The cells are cryopreserved and banked using well-known
techniques for later expansion in culture for subsequent
formulation in biocompatible implantable materials. In other
embodiments, living cells can be harvested from a donor or from the
patient for whom the implantable material is intended.
[0075] Cell Preparation.
[0076] As stated above, suitable cells can be obtained from a
variety of tissue types and cell types. In certain preferred
embodiments, human aortic endothelial cells used in the implantable
material are isolated from the aorta of cadaver donors. In other
embodiments, porcine aortic endothelial cells (Cell Applications,
San Diego, Calif.) are isolated from normal porcine aorta by a
similar procedure used to isolate human aortic endothelial cells.
Each lot of cells is derived from a single or multiple donors,
tested extensively for endothelial cell viability, purity,
biological function, the presence of mycoplasma, bacteria, fungi,
yeast, known human pathogens and other adventitious agents. The
cells are further expanded, characterized and cryopreserved to form
a working cell bank at the third to sixth passage using well-known
techniques for later expansion in culture and for subsequent
formulation as biocompatible implantable material.
[0077] The following is an exemplary protocol for preparing
endothelial cells suitable for use with the present invention.
Human or porcine aortic endothelial cells are prepared in T-75
flasks pre-treated by the addition of approximately 15 ml of
endothelial cell growth media per flask. Human aortic endothelial
cells are prepared in Endothelial Growth Media (EGM-2, Cambrex
Biosciences, East Rutherford, N.J.). EGM-2 consists of Endothelial
Cell Basal Media (EBM-2, Cambrex Biosciences) supplemented with
EGM-2 which contain 2% FBS. Porcine cells are prepared in EBM-2
supplemented with 5% FBS and 50 .mu.g/ml gentamicin. The flasks are
placed in an incubator maintained at approximately 37.degree. C.
and 5% CO.sub.2/95% air, 90% humidity for a minimum of 30 minutes.
One or two vials of the cells are removed from the -160.degree.
C.-140.degree. C. freezer and thawed at approximately 37.degree. C.
Each vial of thawed cells is seeded into two T-75 flasks at a
density of approximately 3.times.10.sup.3 cells per cm3,
preferably, but no less than 1.0.times.10.sup.3 and no more than
7.0.times.10.sup.3; and the flasks containing the cells are
returned to the incubator. After about 8-24 hours, the spent media
is removed and replaced with fresh media. The media is changed
every two to three days, thereafter, until the cells reach
approximately 85-100% confluence preferably, but no less than 60%
and no more than 100%. When the implantable material is intended
for clinical application, only antibiotic-free media is used in the
post-thaw culture of human aortic endothelial cells and manufacture
of the implantable material of the present invention.
[0078] The endothelial cell growth media is then removed, and the
monolayer of cells is rinsed with 10 ml of HEPES buffered saline
(HEPES). The HEPES is removed, and 2 ml of trypsin is added to
detach the cells from the surface of the T-75 flask. Once
detachment has occurred, 3 ml of trypsin neutralizing solution
(TNS) is added to stop the enzymatic reaction. An additional 5 ml
of HEPES is added, and the cells are enumerated using a
hemocytometer. The cell suspension is centrifuged and adjusted to a
density of, in the case of human cells, approximately
1.75.times.10.sup.6 cells/ml using EGM-2 without antibiotics, or in
the case of porcine cells, approximately 1.50.times.10.sup.6
cells/ml using EBM-2 supplemented with 5% FBS and 50 .mu.g/ml
gentamicin.
[0079] Biocompatible Matrix.
[0080] According to the present invention, the implantable material
comprises a biocompatible matrix. The matrix is permissive for cell
growth, and cell anchoring to and/or embedding within the matrix. A
particularly preferred matrix is one characterized by a
three-dimensional configuration such that anchored and/or embedded
cells can create and occupy a multi-dimensional habitat. Porous
matrices are preferred. The matrix can be a solid or a non-solid.
Certain non-solid matrices are flowable and suitable for
administration via injection-type or infusion-type methods. In
certain embodiments, the matrix is flexible and conformable. The
matrix also can be in the form of a flexible planar form. The
matrix also can be in the form of a gel, a foam, a suspension, a
particle, a microcarrier, a microcapsule, or a fibrous structure.
In certain preferred embodiments, non-solid forms of matrix to
which cells are anchored and/or in which cells are embedded can be
injected or infused when administered.
[0081] One currently preferred matrix is Gelfoam.RTM. (Pfizer, New
York, N.Y.), an absorbable gelatin sponge (hereinafter "Gelfoam
matrix"). Gelfoam matrix is a to porous and flexible sponge-like
matrix prepared from a specially treated, purified porcine dermal
gelatin solution.
[0082] According to another embodiment, the biocompatible matrix
material can be a modified matrix material. Modifications to the
matrix material can be selected to optimize and/or to control
function of the cells, including the cells' phenotype (e.g., the
immunomodulatory phenotype) as described elsewhere herein, when the
cells are associated with the matrix. According to one embodiment,
modifications to the matrix material include coating the matrix
with attachment factors or adhesion peptides. Exemplary attachment
factors include, for example, fibronectin, fibrin gel, and
covalently attached cell adhesion ligands (including for example
RGD) utilizing standard aqueous carbodiimide chemistry. Additional
cell adhesion ligands include peptides having cell adhesion
recognition sequences, including but not limited to: RGDY, REDVY,
GRGDF, GPDSGR, GRGDY and REDV.
[0083] According to another embodiment, the matrix is a matrix
other than Gelfoam. Additional exemplary matrix materials include,
for example, fibrin gel, alginate, polystyrene sodium sulfonate
microcarriers, collagen coated dextran microcarriers, cellulose,
PLA/PGA and pHEMA/MMA copolymers (with polymer ratios ranging from
1-100% for each copolymer). According to a preferred embodiment,
these additional matrices are modified to include attachment
factors, as recited and described above.
[0084] According to another embodiment, the biocompatible matrix
material is physically modified to improve cell attachment to the
matrix. According to one embodiment, the matrix is cross linked to
enhance its mechanical properties and to improve its cell
attachment and growth properties. According to a preferred
embodiment, an alginate matrix is first cross linked using calcium
sulfate followed by a second cross linking step using calcium
chloride and routine protocols.
[0085] According to yet another embodiment, the pore size of the
biocompatible matrix is modified. A currently preferred matrix pore
size is about 25 .mu.m to about 100 .mu.m; preferably about 25
.mu.m to 50 .mu.m; more preferably about 50 .mu.m to 75 .mu.m; even
more preferably about 75 .mu.m to 100 .mu.m. Other preferred pore
sizes include pore sizes below about 25 .mu.m and above about 100
.mu.m. According to one embodiment, the pore size is modified using
a salt leaching technique. Sodium chloride is mixed in a solution
of the matrix material and a solvent, the solution is poured into a
mold, and the solvent is allowed to evaporate. The matrix/salt
block is then immersed in water and the salt leached out leaving a
porous structure. The solvent is chosen so that the matrix is in
the solution but the salt is not. One exemplary solution includes
PLA and methylene chloride.
[0086] According to an alternative embodiment, carbon dioxide gas
bubbles are incorporated into a non-solid form of the matrix and
then stabilized with an appropriate surfactant. The gas bubbles are
subsequently removed using a vacuum, leaving a porous
structure.
[0087] According to another embodiment, a freeze-drying technique
is employed to control the pore size of the matrix, using the
freezing rate of the ice microparticles to form pores of different
sizes. For example, a gelatin solution of about 0.1-2% porcine or
bovine gelatin can be poured into a mold or dish and pre-frozen at
a variety of different temperatures and then lyophilized for a
period of time. The material can then be cross-linked by using,
preferably, ultraviolet light (254 nm) or by adding gluteraldehyde
(formaldehyde). Variations in pre-freezing temperature (for example
-20.degree. C., -80.degree. C. or -180.degree. C.), lyophilizing
temperature (freeze dry at about -50.degree. C.), and gelatin
concentration (0.1% to 2.0%; pore size is generally inversely
proportional to the concentration of gelatin in the solution) can
all affect the resulting pore size of the matrix material and can
be modified to create a preferred material. The skilled artisan
will appreciate that a suitable pore size is that which promotes
and sustains optimal cell populations having the phenotypes
described elsewhere herein.
[0088] Cell Seeding of Biocompatible Matrix.
[0089] The following is a description of one exemplary
configuration of a biocompatible matrix. As stated elsewhere,
preferred matrices are solid or non-solid, and can be formulated
for implantation, injection or infusion.
[0090] Pre-cut pieces of a suitable biocompatible matrix or an
aliquot of suitable biocompatible flowable matrix are re-hydrated
by the addition of EGM-2 without antibiotics at approximately
37.degree. C. and 5% CO.sub.2/95% air for 12 to 24 hours. The
implantable material is then removed from their re-hydration
containers and placed in individual tissue culture dishes.
Biocompatible matrix is seeded at a preferred density of
approximately 1.5-2.0.times.10.sup.5 cells
(1.25-1.66.times.10.sup.5 cells/cm.sup.3 of matrix) and placed in
an incubator maintained at approximately 37.degree. C. and 5%
CO.sub.2/95% air, 90% humidity for 3-4 hours to facilitate cell
attachment. The seeded matrix is then placed into individual
containers (Evergreen, Los Angeles, Calif.) tubes, each fitted with
a cap containing a 0.2 .mu.m filter with EGM-2 and incubated at
approximately 37.degree. C. and 5% CO.sub.2/95% air. The media is
changed every two to three days, thereafter, until the cells have
reached confluence. The cells in one preferred embodiment are
preferably passage 6, but cells of fewer or more passages can be
used.
[0091] Cell Growth.
[0092] A sample of implantable material is removed on or around
days 3 or 4, 6 or 7, 9 or 10, and 12 or 13, the cells are counted
and assessed for viability, and a growth curve is constructed and
evaluated in order to assess the growth characteristics and to
determine whether confluence, near-confluence or post-confluence
has been achieved. Generally, one of ordinary skill will appreciate
the indicia of acceptable cell growth at early, mid- and late time
points, such as observation of an exponential increase in cell
number at early time points (for example, between about days 2-6
when using porcine aortic endothelial cells), followed by a near
confluent phase (for example, between about days 6-8), followed by
a plateau in cell number once the cells have reached confluence
(for example, between about days 8-10) and maintenance of the cell
number when the cells are post-confluent (for example, between
about days 10-14).
[0093] Cell counts are achieved by complete digestion of the
aliquot of implantable material with a solution of 0.5 mg/ml
collagenase in a HEPES/Ca.sup.++ solution. After measuring the
volume of the digested implantable material, a known volume of the
cell suspension is diluted with 0.4% trypan blue (4:1 cells to
trypan blue) and viability assessed by trypan blue exclusion.
Viable, non-viable and total cells are enumerated using a
hemocytometer. Growth curves are constructed by plotting the number
of viable cells versus the number of days in culture.
[0094] For purposes of the present invention, confluence is defined
as the presence of at least about 4.times.10.sup.5 cells/cm.sup.3
when in an exemplary flexible planar form of the implantable
material (1.0.times.4.0.times.0.3 cm), and preferably about
7.times.10.sup.5 to 1.times.10.sup.6 total cells per aliquot (50-70
mg) when in an injectable or infusible composition. For both, cell
viability is at least about 90% preferably but no less than
80%.
[0095] An exemplary embodiment of the present invention comprises a
biocompatible matrix and cells suitable for use with any one of the
various clinical indications or treatment paradigms described
herein. Specifically, in one preferred embodiment, the implantable
material comprises a biocompatible matrix and endothelial cells,
endothelial-like cells, or analogs of either of the foregoing. In
one currently preferred embodiment, the implantable material is in
a flexible planar form and comprises endothelial cells, preferably
vascular endothelial cells such as but not limited to human aortic
endothelial cells and the biocompatible matrix Gelfoam.RTM. gelatin
sponge (Pfizer, New York, N.Y., hereinafter "Gelfoam matrix").
[0096] Implantable material of the present invention comprises
cells anchored to and/or embedded within a biocompatible matrix.
Anchored to and/or embedded within means securely attached via cell
to cell and/or cell to matrix interactions such that the cells
withstand the rigors of the preparatory manipulations disclosed
herein. As explained elsewhere herein, an operative embodiment of
implantable material comprises a near-confluent, confluent or
post-confluent cell population having a preferred phenotype. It is
understood that embodiments of implantable material likely shed
cells during preparatory manipulations and/or that certain cells
are not as securely attached as are other cells. All that is
required is that implantable material comprise cells that meet the
functional or phenotypical criteria set forth elsewhere herein.
[0097] The implantable material of the present invention was
developed on the principals of tissue engineering and represents a
novel approach to addressing the herein-described clinical needs.
The implantable material of the present invention is unique in that
the viable cells anchored to and/or embedded within the
biocompatible matrix are able to supply to the site of
administration multiple cell-based products in physiological
proportions under physiological feed-back control. As described
elsewhere herein, the cells suitable for use with the implantable
material are endothelial, endothelial-like cells, or analogs of
each of the foregoing. Local delivery of multiple compounds by
these cells and physiologically-dynamic dosing provide more
effective regulation of the processes responsible for modulating an
immune response. The implantable material of the present invention
can provide an environment which mimics supportive physiology and
is conducive to modulation of an immune response.
[0098] Evaluation of Functionality.
[0099] For purposes of the invention described herein, the
implantable material is tested for indicia of functionality prior
to delivery to a recipient. For example, as one determination of
suitability, conditioned media are collected during the culture
period to ascertain levels of heparan sulfate or transforming
growth factor-.beta.1 (TGF-.beta.1) or basic fibroblast growth
factor (b-FGF) or nitric oxide which are produced by the cultured
endothelial cells. In certain preferred embodiments, the
implantable material can be used for the purposes described herein
when total cell number is at least about 1, preferably about 2,
more preferably at least about 4.times.10.sup.5 cells/cm.sup.3 of
flexible planar form; percentage of viable cells is at least about
80-90%, preferably .gtoreq.90%, most preferably at least about 90%;
heparan sulfate in conditioned media is at least about 0.1-0.5
preferably at least about 0.23 microg/mL/day. If other indicia are
desired, then TGF-.beta.1 in conditioned media is at least about
200-300, preferably at least about 300 picog/ml/day; b-FGF in
conditioned media is below about 200 picog/ml, preferably no more
than about 400 picog/ml.
[0100] Heparan sulfate levels can be quantitated using a routine
dimethylmethylene blue-chondroitinase ABC digestion
spectrophotometric assay. Total sulfated glycosaminoglycan (GAG)
levels are determined using a dimethylmethylene blue (DMB) dye
binding assay in which unknown samples are compared to a standard
curve generated using known quantities of purified chondroitin
sulfate diluted in collection media. Additional samples of
conditioned medium are mixed with chondroitinase ABC to digest
chondroitin and dermatan sulfates prior to the addition of the DMB
color reagent. All absorbances are determined at the maximum
wavelength absorbance of the DMB dye mixed with the GAG standard,
generally around 515-525 nm. The concentration of heparan sulfate
per day is calculated by subtracting the concentration of
chondroitin and dermatan sulfate from the total sulfated
glycosaminoglycan concentration in conditioned medium samples.
Chondroitinase ABC activity is confirmed by digesting a sample of
purified chondroitin sulfate. Conditioned medium samples are
corrected appropriately if less than 100% of the purified
chondroitin sulfate is digested. Heparan sulfate levels may also be
quantitated using an ELISA assay employing monoclonal
antibodies.
[0101] If desired, TGF-.beta.1 and b-FGF levels can be quantitated
using an ELISA assay employing monoclonal or polyclonal antibodies,
preferably polyclonal. Control collection media can also be
quantitated using an ELISA assay and the samples corrected
appropriately for TGF-.beta.1 and b-FGF levels present in control
media. Nitric oxide (NO) levels can be quantitated using a standard
Griess Reaction assay. The transient and volatile nature of nitric
oxide makes it unsuitable for most detection methods. However, two
stable breakdown products of nitric oxide, nitrate (NO.sub.3) and
nitrite (NO.sub.2), can be detected using routine photometric
methods. The Griess Reaction assay enzymatically converts nitrate
to nitrite in the presence of nitrate reductase. Nitrite is
detected colorimetrically as a colored azo dye product, absorbing
visible light in the range of about 540 nm. The level of nitric
oxide present in the system is determined by converting all nitrate
into nitrite, determining the total concentration of nitrite in the
unknown samples, and then comparing the resulting concentration of
nitrite to a standard curve generated using known quantities of
nitrate converted to nitrite.
[0102] Also, any one or more of the foregoing assays can be used
alone or in combination as a screening assay for identifying a cell
as suitable for use with the implantable material of the present
invention.
[0103] While the earlier-described preferred immunomodulatory
phenotype can be assessed using one or more of the optional
quantitative heparin sulfate, TGF-.beta.1, NO and/or b-FGF
functional assays described above, implantable material can be
evaluated for the presence of one or more preferred
immunomodulatory phenotypes as follows. For purposes of the present
invention, one such preferred, readily identifiable phenotype
typical of cells of the present invention is an altered immunogenic
phenotype as measured by the in vitro assays described below.
Another readily identifiable phenotype typical of cells of the
present invention is an ability to block or interfere with
dendritic cell maturation as measured by the in vitro assays
described below. Each phenotype is referred to herein as an
immunomodulatory phenotype and cells exhibiting such a phenotype
have immunomodulatory functionality.
[0104] Evaluation of Immunomodulatory Functionality:
[0105] For purposes of the invention described herein, the
immunomodulatory functionality of implantable material can be
tested as follows. For example, samples of the implantable material
are evaluated to ascertain their ability to reduce expression of
MHC class II molecules, to reduce expression of co-stimulatory
molecules, to inhibit the maturation of co-cultured dendritic
cells, and to reduce the proliferation of T cells. In certain
preferred embodiments, the implantable material can be used for the
purposes described herein when the material is able to reduce
expression of MHC class II molecules by at least about 25-80%,
preferably 50-80%, most preferably at least about 80%; to reduce
expression of co-stimulatory molecules by at least about 25-80%,
preferably 50-80%, most preferably at least about 80%; inhibit
maturation of co-cultured dendritic cells by at least about 25-95%,
preferably 50-95%, most preferably at least about 95%; and/or
reduce proliferation of lymphocytes by at least about 25-90%,
preferably 50-90%, most preferably at least about 90%.
[0106] Levels of expression of MHC class II molecules and
co-stimulatory molecules can be quantitated using routine flow
cytometry and FACS-analysis, described in detail below.
Proliferation of lymphocytes can be quantitated can be quantitated
by in-vitro coculturing .sup.3[H]-thymidine-labeled
CD3+-lymphocytes with the implantable composition via
scintillation-counting as described below in detail. Inhibition of
dendritic cell maturation can be quantitated by either co-culturing
the implantable material with dendritic cells and evaluating
surface expression of various markers on the dendritic cells by
flow cytometry and FACS analysis, or by measuring dendritic cell
uptake of FITC-conjugated dextran by flow cytometry. Each of these
methods is described in detail below.
[0107] Also, any one or more of the foregoing assays can be used
alone or in combination as a screening assay for identifying a cell
as suitable for use with the implantable material of the present
invention.
[0108] Methods of Use and Clinical Indications:
[0109] This invention is directed generally to materials and
methods for modulating an immunologically adverse response,
including an inflammatory reaction, to an exogenous immunogen or
stimulus as well as an endogenous immunogen or stimulus. The
invention is also directed to a cell-, tissue-, or organ-associated
immunogen. For example, the present invention can modulate an
adverse immune response to non-syngeneic or syngeneic cells,
tissues or organs and/or ameliorate a pre-existing immune condition
such as but not limited to an autoimmune condition. This discussion
of implantable materials and methods of use for suitable clinical
indications will make reference to the following terms and
concepts.
[0110] An early phase immune response depends on innate immunity.
During an innate immune response, a variety of innate immune
mechanisms recognize and respond to the presence of immunogen.
Innate immunity is present in all individuals at all times and
principally discriminates between self, altered self and non-self.
For example, a type of innate immune cell is the Natural Killer
(NK) cell, the dendritic cell and the monocyte. The innate immune
response is followed by an adaptive immune response, mediated by
clonal selection of specific lymphocytes and resulting in a more
tailored and long-lasting immune response against the recognized
antigen.
[0111] The adaptive immune response, or adaptive immunity, is the
response of antigen-specific lymphocytes to antigen, including the
development of immunological memory. Adaptive immune responses are
generated by clonal selection of lymphocytes. Adaptive immune
responses are distinct from innate and non-adaptive phases of
immunity, which are not mediated by clonal selection of
antigen-specific lymphocytes. The adaptive immune response includes
both cell-mediated immunity and humoral immunity. For example, an
adaptive immune cell is a B-cell or T-cell lymphocyte.
[0112] One of the hallmarks of an adaptive immune response is
establishment of immunological memory. Immunological memory is the
ability of the immune system to respond more rapidly and
effectively to immunogens been encountered previously, and reflects
the pre-existence of a clonally expanded population of
antigen-specific lymphocytes.
[0113] Protective immunity can be either cell-mediated immunity or
humoral immunity. Humoral immunity is specific immunity mediated by
antibodies made in a humoral immune response. Cell-mediated
immunity describes any adaptive immune response in which antigen
specific T cells play a main role.
[0114] Autoimmune diseases are mediated by sustained adaptive
immune responses specific for self antigens. Tissue injury results
because the antigen is an intrinsic component of the body and
consequently effector mechanisms of the immune system are directed
at self tissues. Also, since the offending autoantigen can not be
removed from the body, the immune response persists, and there is a
constant supply of new autoantigen, which amplifies the
response.
[0115] Although some syngeneic grafts or transplants may be
accepted long-term, even syngeneic grafts can be problematic for a
recipient. In fact, even when autologous cells are harvested,
manipulated ex vivo and returned to the original donor,
non-acceptance may occur to some extent. Typically, grafts
differing at the MHC or at other genetic loci are rejected in the
short term by a recipient T-cell response. When donor and recipient
differ at the MHC, for example, the immune response is directed at
the non-self MHC molecule or other surface molecules expressed by
the graft. Acceptance or rejection of a graft or transplant invokes
immune events such as antigen recognition, T-cell activation,
T-helper cell recruitment and ultimately graft destruction.
[0116] An inflammatory reaction is initiated by a local immune
response. Acute inflammation is an early transient episode, while
chronic inflammation persists such as during autoimmune responses.
Inflammation reflects the effects of cytokines on local blood
vessels. Cytokines have important effects on the adherent
properties of the blood vessel endothelium, causing circulating
leukocytes to stick to the endothelial cells of the blood vessel
wall and migrate through the wall. Later-stage inflammatory
responses also involve lymphocytes of the adaptive immune response
which have been activated by immunogen.
[0117] Exemplary methods of treatment and clinical indications are
discussed below. This is not intended to be an exhaustive
discussion. The present invention contemplates any clinical
indication suitable for treatment with the present invention,
including any clinical indication typified by or otherwise
associated with an immunological event having adverse clinical
consequences for a patient.
[0118] Syngeneic and Non-syngeneic Transplants:
[0119] The present invention can be used to reduce or diminish a
transplant recipient's adverse response to a cell, tissue and/or
organ transplant, whether it be a syngeneic or a non-syngeneic
transplant. The present invention can also be used to stabilize or
maintain a transplant recipient's acceptance of a cell, tissue or
organ transplant, whether it be a syngeneic or a non-syngeneic
transplant. As taught herein, modulation of an adverse immune
response occurs when implantable material is used as a
pre-transplant treatment, coincident treatment or post-transplant
treatment. For example, it is contemplated that a pre-treatment can
acclimate a recipient's immune system which facilitates later
acceptance of the transplant. Similarly, coincident treatment can
shorten the time course of physiological events which ultimately
result in acceptance and ameliorate any adverse immunological
events provoked by the transplant. Post-transplant treatments,
whether single or multiple, can perpetuate a state of acceptance
and keep adverse immunological events in check if/when such events
occur. Clinically, typical indications suitable for treatment with
the implantable material of present invention include, but are not
limited to, allorejection, xenorejection, ischemia-reperfusion
injury associated with transplanted tissues or organs, and
repetitive treatment courses. Repetitive treatment courses include,
for example, recurrent atherosclerosis at different vessel sites
requiring repetitive intervention and repetitive replenishing
injections of pancreas islet cells. For purposes of the present
invention, blood is a type of tissue and blood transfusion
recipients can benefit from treatment with the present invention
for all the foregoing reasons. Similarly, immunological-based
diseases associated with cell, tissue and/or organ transplants
benefit from the treatment paradigms set forth above.
[0120] Complement Dependent Cytotoxicity:
[0121] In addition to reducing, modulating or eliminating the
innate immune response and/or the adaptive immune response, as
outlined above, the implantable material of the present invention
can also reduce, modulate or eliminate the severity of the
complement cascade and the inflammatory side effects of complement
activation. For example, attenuation of the complement cascade
using the implantable material or the present invention reduces
complement mediated cell lysis of a transplanted tissue or organ,
thereby ameliorating transplant dysfunction and extending the
duration of successful treatment.
[0122] Interventional Therapies:
[0123] As taught herein, the present invention can modulate the
severity or robustness of an already-existing immune response as
well as a future response provoked by subsequent earlier
exposure(s) to an immunogen. Under such circumstances, implantable
material can intervene by blocking escalation of an adverse immune
response or diminishing onset of hypersensitivity, respectively.
Suppression of a memory response can avoid further physiological
insult which can jeopardize a patient's organ health, for example.
In the case of an already-existing condition, such as an
auto-immune condition, the present invention can quell the
devastating effects of unabated immunological assaults on a
patient's tissues or organs. In essence, such patients are
continuously exposed to offending immunogen and their immune
response escalates out-of-control resulting in serious, often
fatal, disease sequalae.
[0124] While an auto-immune condition can be likened to serial
challenges with an offending immunogen, other clinical indications
can be considered similarly. For example, as suggested above, a
recipient of a syngeneic or non-syngeneic transplant is subject to
serial challenges. Replenishment of a transplant, such as kidney
islet cells which deteriorate over time, constitutes a serial
challenge. Secondary infarctions or secondary vascular injuries can
be considered serial challenges. Another example is a disease such
as but not limited to vasculitis. Any of the foregoing can be
effectively managed using the materials and methods of the present
invention.
[0125] Supplanting Immunosuppressive Agents:
[0126] As explained elsewhere herein, it is contemplated that
administration of the implantable material of the present invention
inhibits sufficiently at least T cell activation such that the need
to administer harmful immunosuppressive agents is eliminated or
significantly reduced. However, it is also contemplated that a
certain class of patients, such as a patient pre-disposed to highly
exacerbated immune responses, can be treated with both implantable
material and an immunosuppressive agent. The implantable material
of the present invention, when administered prior to or coincident
with transplantation of syngeneic or non-syngeneic tissue, can
permit reduced dosages of immunosuppressive agent, if one is
necessary, to manage a potential graft rejection response.
[0127] Potent immunosuppressive agents, for example, cyclosporin A,
tacrolimus (FK-506), sirolimus (rapamycin), mycophenolate mofetil,
leflunomide, glucocorticoids, cytostatics, azathioprine, and
prednisone, are administered to a transplant recipient to inhibit T
cell activation and increase the probability of graft survival.
However, administration of potent immunosuppressive agents
increases the risk of cancer and infection and contributes to the
risk of other side effects including hypertension, dyslipidemia,
hyperglycemia, peptic ulcers, and liver and kidney injuries. The
present invention can permit more prudent and less risky dosing
regimens of such agents. Additionally, immunosuppressants which are
typically administered to an organ recipient can be administered
prior to, coincident with and/or subsequent to administration of
the implantable material of the present invention. For example,
implantable materials can amplify the beneficial effects of
immunosuppressants while minimizing the risks of such agents in
recipients whose immune system is overstimulated or
over-sensitized, perhaps reducing the time in which
immunomodulation is actually achieved. It is further contemplated
that dosages of immunosuppressants, in certain embodiments, are
less than those typically administered in the absence of
implantable material, thereby exposing a recipient to less toxic
doses of immunosuppressants.
[0128] Altering the Time Course of an Immune Response:
[0129] In a preferred embodiment of the invention, matrix-anchored
and/or embedded endothelial cells are administered to diminish or
delay an immune or inflammatory response. It is not necessary that
the implantable material completely eliminate an immune or
inflammatory response to be considered effective. Rather, the
material need only alter the time course of a response, such as by
reducing the duration of an immune or inflammatory response or by
reducing an acute inflammatory response to a chronic inflammatory
response. Delaying an immune or inflammatory response allows a
coincident or later administered therapy to effectively treat a
recipient in the absence to of an immune or inflammatory response
and/or to increase the duration of transplant acceptance. Thus any
delay or reduction in the severity of an adverse immune response is
beneficial clinically to a patient.
[0130] Furthermore, the implantable material of the present
invention can also be used to manage or reduce an immune response
and inflammatory reaction associated with any exogenous foreign
body or foreign material introduced to a patient, or any form of
exogenous stimulus. The present invention contemplates exogenous
immunogens which are naturally-occurring. The present invention
also contemplates exogenous immunogens, including but not limited
to pharmaceutical agents, toxins, surgical implants, infectious
agents and chemicals. For purposes of the present invention, an
exogenous immunogen can be an exogenous stimulus such as, but not
limited to, environmental stress, injury, exposure or any stimulus
which provokes an adverse immune response or inflammatory
reaction.
[0131] For example, synthetic graft materials, such as a synthetic
PTFE.RTM. arteriovenous graft, or other synthetic surgical
materials or prosthetic devices, can induce a foreign body reaction
in the host. This type of immune or inflammatory response can also
be reduced or eliminated by administering the implantable material
of the present invention to the patient prior to or at the time of
implanting the synthetic material. Administration subsequent to
implantation is also effective. Reducing any foreign body reaction
in the host improves the overall function and/or outcome of the
treatment.
[0132] General Considerations.
[0133] In certain embodiments of the invention, additional
therapeutic agents are administered prior to, coincident with
and/or following administration of the implantable material. For
example, cytokines or growth factors can also be incorporated into
the implantable material, depending on the clinical indication
necessitating the implant, including agents which can mute an
immune-related humoral or cellular event, or tissue-associated
biochemical cascade. Other types of therapeutic agents include
those which can promote the longevity of cells anchored to and/or
embedded within the implantable material and/or agents which can
delay the bioerosion of an erodible biocompatible matrix post
implantation. Any of the foregoing can be administered locally or
systemically; if locally, certain agents can be contained within
the implantable material or contributed by the cells per se.
[0134] Administration Considerations.
[0135] As contemplated herein, the implantable material of the
present invention can be delivered to or situated at any compatible
anatomical site provided that conditions at the site do not cause
mechanical-type or physical-type disruption or untimely
disintegration of the implantable material, or otherwise compromise
the physical integrity or the functionality of the implantable
material. For example, the present invention can be situated
subcutaneously, perivascularly, or intraperitoneally. One preferred
site is a skin pouch. Other preferred sites can be perivascular or
non-perivascular. The implantable material can be situated adjacent
to or in contact with an organ or a tubular anatomical structure
which can be a vascular or non-vascular structure. The present
invention can be delivered to any compatible site for purposes of
either systemic modulation of a humoral or cellular immune
response, or for purposes of local modulation of an inflammatory
reaction, or both. Certain preferred embodiments of implantable
material can reside at an implantation site for at least about
56-84 days, preferably about at least 7 days, more preferably about
at least 14 days, even more preferably about at least 28 days, and
most preferably more than about 28 days before it bioerodes.
[0136] When ready for delivery to a recipient, the implantable
material when in an exemplary flexible planar form, is a
1.times.4.times.0.3 cm (1.2 cm.sup.3) sterile piece with preferably
approximately 5-8.times.10.sup.5 preferably at least about
4.times.10.sup.5 cells/cm.sup.3 and at least about 90% viable
cells, for example, human aortic endothelial cells derived from a
single cadaver donor source, per cubic centimeter in approximately
45-60 ml, preferably about 50 ml, endothelial growth medium (for
example, endothelial growth medium (EGM-2) containing no phenol red
and no antibiotics. When porcine aortic endothelial cells are used,
the growth medium is also EBM-2 containing no phenol red, but
supplemented with 5% FBS and 50 .mu.g/ml gentamicin.
[0137] In certain embodiments contemplated herein, the implantable
material of the present invention is a flowable composition
comprising a particulate biocompatible matrix which can be in the
form of a gel, a foam, a suspension, a particle, a microcarrier, a
microcapsule, or other flowable material. Any non-solid flowable
composition for use with an injection-type or infusion-type
delivery device is contemplated herein. In certain embodiments, the
flowable composition is preferably a shape-retaining composition.
An implantable material comprising cells in, on or within a
flowable-type particulate matrix as contemplated herein can be
formulated for use with any injection-type delivery device ranging
in internal diameter from about 22 gauge to about 26 gauge and
capable of delivering about 50 mg of flowable composition
comprising particulate material containing preferably about 1
million cells in about 1 to about 3 ml.
[0138] According to a currently preferred embodiment, the flowable
composition comprises a biocompatible particulate matrix such as
Gelfoam.RTM. particles, Gelfoam.RTM. powder, or pulverized
Gelfoam.RTM. (Pfizer Inc., New York, N.Y.) (hereinafter "Gelfoam
particles"), a product derived from porcine dermal gelatin.
According to another embodiment, the particulate matrix is
Cytodex-3 (Amersham Biosciences, Piscataway, N.J.) microcarriers,
comprised of denatured collagen coupled to a matrix of cross-linked
dextran.
[0139] Endovascular Administration.
[0140] The flowable composition can also be administered via an
intraluminal or endovascular route even though the final deposition
site is not intraluminal. For example, the composition can be
delivered by any device able to be inserted within the blood
vessel. Endoscopic guidance systems may be used to locate the
delivery device at the site of administration, including, for
example, intravascular ultrasound (IVUS), color Doppler ultrasound,
duplex ultrasound, other routine ultrasound, angiography, magnetic
resonance angiography (MRA), magnetic resonance imaging (MRI), CT
scanning, fluoroscopy to identify the location of a stent and/or
other endoscopic guidance systems known in the field. Additionally,
the site of administration may be located using tactile
palpation.
[0141] In one instance, the intraluminal delivery device is
equipped with a traversing or penetrating device which penetrates
the luminal wall of a blood vessel to reach a non-luminal surface
of a blood vessel. The flowable composition is then deposited on
the non-luminal surface. It is contemplated herein that a
non-luminal, also termed an extraluminal, surface can include any
site exterior to a blood vessel or any perivascular surface of a
vessel, or can be within the adventitia, media, or intima of a
blood vessel, for example. For purposes of this invention,
non-luminal or extraluminal means any surface except an interior
surface of the lumen. It is also contemplated that deposition
within the perivascular space can be accomplished via an
intraluminal delivery device and does not require contact with the
extraluminal surface of the traversed vessel.
[0142] The penetrating devices contemplated herein can permit, for
example, a single point of delivery or a plurality of delivery
points arranged in a desired geometric configuration to accomplish
delivery of the flowable composition to a non-luminal surface of a
blood vessel without disrupting an injured or diseased target site.
A plurality of delivery points can be arranged, for example, in a
circle, a bulls-eye, or a linear array arrangement to name but a
few. The penetrating device can also be in the form of a stent
perforator, such as but not limited to, a balloon stent including a
plurality of delivery points.
[0143] Percutaneous Administration. Flowable composition can be
delivered via a percutaneous route using a needle, catheter or
other suitable delivery device. The flowable composition can be
delivered percutaneously coincident with use of a guidance method
to facilitate delivery to the site in need of treatment. The
guidance step is optional. Endoscopic guidance systems can be used
to locate a site of extraluminal administration, for example,
including intravascular ultrasound (IVUS), color Doppler
ultrasound, duplex ultrasound, other routine ultrasound, to
angiography, magnetic resonance angiography (MRA), magnetic
resonance imaging (MRI), CT scanning fluoroscopy. Additionally, the
site of administration can be located using tactile palpation. Upon
entry into the perivascular or peritoneal space, for example, the
clinician can deposit the flowable composition on any non-luminal
surface or at any non-luminal site. The guiding or identifying step
is optionally performed and not required to practice the methods of
the present invention. In another embodiment, the implantable
flowable composition is delivered locally to a surgically-exposed
extraluminal site.
[0144] Also contemplated is administration by infusion. Infusion
can be accomplished as a bolus-type dose or a slower-type, gradual
dose. The skilled clinician will recognize the advantages of each
and will recognize the circumstances in which to employ one or the
other modes of administration. All that is required is routine
clinical infusion apparatus.
Experimental Materials and Procedures
[0145] Material Preparation and Evaluation.
[0146] As described in greater detail elsewhere herein, porcine
aortic endothelial cells and human aortic endothelial cells were
individually isolated and cultured. The cultured cells were then
seeded on a three-dimensional biocompatible matrix, such as
Gelfoam, and incubated until the cells reached confluence. The
functionality of the endothelial cells anchored to and/or embedded
within the matrix was evaluated according to the previously
discussed protocols.
[0147] Endothelial Cell-Induced Immune Reaction in Rats.
[0148] Fifty-four Sprague-Dawley rats received 5.times.10.sup.5
porcine aortic endothelial cell transplants in the subcutaneous
dorsal space as Gelfoam-embedded cells, saline-suspended cell
pellets, or as pellets adjacent to empty Gelfoam. After dorsal
incision, a small subcutaneous cavity was built in blunt technique
and Gelfoam-embedded cells carefully inserted or cells injected.
Empty control Gelfoam matrices were incubated in complete DMEM
prior to implantation. Sera were collected serially from 0 to 56
days, aliquoted and stored at -70.degree. C.
[0149] Endothelial Cell-Induced Immune Reaction in Mice.
[0150] Thirty-six B6-mice received 5.times.10.sup.5 porcine aortic
endothelial cell implants in the subcutaneous dorsal space as
Gelfoam-embedded cells, saline-suspended cell pellets, or as
pellets adjacent to empty Gelfoam. Empty control Gelfoam matrices
were incubated in complete DMEM prior to implantation. To evaluate
the impact of matrix-embedding on immunological memory the same
groups of mice were rechallenged with the identical treatment on
day 100. Sera were collected serially from 0 to 90 days after each
implantation-procedure, aliquoted and stored at -70.degree. C. Four
mice of each group were sacrificed on day 28 and day 128
respectively for splenocyte isolation.
[0151] Endothelial Cell-Induced Immune Reaction in Serially
Challenged Mice.
[0152] Porcine aortic endothelial cells (PAE) isolated from
LargeWhite swine aorta were either seeded on Gelfoam as previously
described or grown to confluence on polystyrene plates. B6-mice
received injections in the subcutaneous dorsal space on days 0, 21,
35 of 5.times.10.sup.5 PAE (n=24, pre-sensitized mice) or saline
(n=24, naive mice). On day 42, 12 mice from each group received
5.times.10.sup.5 matrix-embedded or free PAE. Host immune reactions
and lytic damage of endothelial cells were studied for the
following 90 days. Sera were collected serially from days 42 to
132, aliquoted and stored at -70.degree. C. Six mice of each group
were sacrificed on day 70, the remaining on day 132 for splenocyte
isolation.
EXPERIMENTS
Endothelial Cells Embedded in a Three-Dimensional Matrix Grow in a
Three-Dimensional Pattern
[0153] Scanning electron microscopy was performed to evaluate the
growing pattern of endothelial cells grown within a biocompatible
matrix. Implantable material comprising endothelial cell anchored
to and/or embedded within a Gelfoam matrix were rinsed with PBS,
divided into 0.5 cm specimens, fixed with 3% glutaraldehyde (Sigma
Chemicals; St. Louis, Mo.) 90 min, and transferred to distilled
water. After incubation in 1% OsO4, specimens were rinsed with
distilled water and dehydrated in serial solutions of ethanol (30,
50, 75, 80, 85, 90, 95, and 100%) at 15 min intervals, and
hexamethyldisilazane (Sigma) (50%, 100%) at 30 min intervals.
Specimens were evaporated overnight in 100% HMDS and thereafter
coated with gold in a plasma coater (Edwards Coating System, U.K.).
Scanning electron microphotographs were obtained at 5-kV
acceleration voltage (Stereoscan 240, Cambridge Instruments,
U.K.).
[0154] Scanning electron microscopy revealed a 3-D growing pattern
of porcine aortic endothelial cells along the interstices of the
Gelfoam-matrix. Cell viability remained at 95% over the 2-week
culture course.
[0155] Experimental data indicate that the in-vivo immunoacceptance
of Gelfoam-embedded cells is an effect of the three-dimensional
growing pattern of endothelial cells in the matrix rather than from
the presence of the biocompatible matrix alone. Typically,
implanted cells or proteins combined within tissue-engineered
biomaterials serve as a source of antigens immuno-stimulating. Yet,
the Gelfoam matrix is immunoneutral and itself has no immune
protective effect since injection of porcine aortic endothelial
cells adjacent to Gelfoam matrix alone evoked the same immune
response as free injected endothelial cells. The nature of
endothelial cells contributes to this unique form of
immunomodulation observed with matrix-embedded cell preparations.
In particular, these cells have a sidedness: a basal surface that
interacts with basement membrane and superior surface that
interacts with flowing blood and cellular elements. Data suggest
that endothelial cell function is anchorage- and density-dependent.
Systemic diseases like hypertension, alterations in lipid and
glucose metabolism or exposure to toxins alter anchorage-dependent
regulation and the amplitude and nature of immune responses against
the endothelium and phenotypic transformation of intact endothelial
cells from matrix-adherent to free contributes to initiation of
vascular disease.
Modulation of Surface Molecules Including Co-Stimulatory and
Adhesion Molecules.
[0156] Expression levels of costimulatory and adhesion molecules on
endothelial cells in vitro were quantified by flow cytometry. FITC-
and PE-labeled antibodies were used and included mouse anti-porcine
P-selectin antibody, mouse anti-porcine CD31 (clone LCI-4),
anti-human CD54 (clone 15.2), anti-human CD62E (clone 1.2B6),
anti-human CD58 (clone 1C3), anti-human CD80 (clone BB1),
anti-human CD86 (clone 2331), anti-human 4-1BB-ligand (PE-labeled,
clone C65-485), rat anti-mouse IgG.sub.1 (clone A85-1), and
anti-mouse IgM (clone R6-60.2), rabbit anti-rat IgG, rabbit
anti-human CD40, goat anti-rabbit IgG, mouse anti-human CD106
(clone 1.G11B1), mouse anti-human HLA-DP, DQ, DR (clone CR3/43),
mouse anti class I MHC (IgG.sub.2a), rat anti-mouse IgG.sub.2a,
mouse anti-human ox40-ligand, mouse anti-human Programmed Death
Ligand 1 (PD-L1, clone MIH1), anti-human PD-L2 (clone MIH18), and
anti-human inducible costimulator ligand (ICOS-ligand, clone
MIH12).
[0157] Endothelial cell monolayers or endothelial cells embedded in
Gelfoam were harvested after culture in complete medium (CD31,
CD58, PD-L2, ox40-ligand, MHC-I), stimulated with 100 U/ml
TNF-.alpha. (CD54, CD80, CD86, CD106, E-selectin, P-selectin) or
200 U/ml TNF-.alpha. (ICOS-L) for 24 hours, 10 .mu.g/ml LPS for 24
hours (4-1BB-ligand), 1000 U/ml IFN-.gamma. (MHC-11, CD40), or 100
U/ml IFN-.gamma. and 25 ng/ml TNF-.alpha. (PD-L1) for 48 hours.
Media were aspirated and cells were washed with PBS. Monolayers
incubated in 1.0 mM PBS/EDTA for 5 min, and disrupted by gentle
shaking. Gelfoam were digested with collagenase type I, shown to
have no effect on expression of surface molecules. Cell-suspensions
were washed and 3.times.10.sup.5 cells were resuspended in FACS
buffer (PBS containing 0.1% BSA and 0.1% sodium azide, Sigma
Chemicals; St. Louis, Mo.). Endothelial cells were incubated with
primary antibodies for 30 min at 4.degree. C. If necessary, cells
were resuspended in FACS buffer and stained with a secondary
antibody for 30 min at 4.degree. C. Cells were then washed, fixed
in 1% paraformaldehyde, and 10.sup.4 cells were analyzed by flow
cytometry using a FACScalibur instrument and CellQuest software
(Becton Dickinson, San Diego, Calif.).
[0158] Embedding porcine aortic endothelial cells in a
three-dimensional biocompatible matrix altered the expression of
surface molecules. Constitutive expression of CD58 was
significantly reduced in porcine aortic endothelial cells embedded
in Gelfoam compared to CD58 expression of porcine aortic
endothelial cells grown on tissue culture polystyrene plates
(-60.4%, p<0.002). There was also a significant reduction in
upregulation of costimulatory and adhesion molecules, and MHC class
II on matrix-embedded porcine aortic endothelial cells compared to
porcine aortic endothelial cells grown on polystyrene plates under
FACS-analysis (CD80: -64.9%, p<0.002; CD86: -65.4%, p<0.001;
CD40: -53.8%, p<0.005; ICAM-1: -68.7%, p<0.001; VCAM-1:
-53.9%, p<0.005; E-selectin: -71.8%, p<0.0005; P-selectin:
-79.9%, p<0.0002; MHC II: -78.3%, p<0.0002). There were no
significant differences in surface expression of MHC class I and
CD31.
[0159] Similarly, embedding human aortic endothelial cells in a
three-dimensional biocompatible matrix altered the expression of
surface molecules. Human aortic endothelial cells grown in a 3D
matrix exhibited a significantly reduced expression profile of CD58
and showed a significant lack in upregulation of costimulatory and
adhesion molecules. However, there were no significant differences
in ICAM-1, E-selectin, MHC I, and CD31 expression levels between
human aortic endothelial cells embedded in Gelfoam and human aortic
endothelial cells grown on tissue culture polystyrene plates.
Furthermore, there were no significant differences in constitutive
expression of PD-L2 (100%, p=0.73) and in upregulation of PD-L1
(86%, p=0.09).
[0160] Thus, embedding endothelial cells in a three-dimensional
biocompatible matrix reduces costimulatory and adhesion molecules.
Matrix embedded porcine aortic endothelial cells and human aortic
endothelial cells exhibited significantly lower expression levels
of costimulatory and adhesion molecules on activated endothelial
cells.
[0161] Expression of CD31, MHC-11, CD58, ICAM-1 and E-selectin was
also analyzed in the implants in vitro by confocal microscopy and
in rats in vivo by immunohistochemical analysis. Endothelial cells
were seeded on cover slips or embedded in Gelfoam-matrices. After
washing with PBS and fixation with 3% paraformaldehyde for 20 min
(cover slips) or overnight (Gelfoam), endothelial cells were
blocked with rat serum (Bethyl Laboratories, TX) for 30 min. Before
staining with antibodies, Gelfoam matrices were cut into 2 mm thick
slices. Endothelial cells were stained with the appropriate amount
of antibodies for 1 (cover slips) or 2 hours (Gelfoam) and analyzed
on a Zeiss LSM510 Laser scanning confocal microscope. Staining
intensity was quantified with ImageJ software (National Institute
of Health, Bethesda, Md.) and normalized against CD31
expression.
[0162] Confocal microscopy revealed reduced expression-levels of
CD58, ICAM-1, E-selectin, and MHC-II on matrix embedded porcine
aortic endothelial cells whereas CD31 expression remained unchanged
(p<0.02). Cell-substrate anchoring had no effect on MHC-I
expression but markedly muted the expected upregulation of MHC-II
molecules. Porcine aortic endothelial cells embedded in Gelfoam
evoked only a modest proliferation of xenogeneic CD4.sup.+ T cells
in-vitro similar to the response seen with blockade of MHC-II
binding in free porcine aortic endothelial cells.
Modulation of the Immune Response In Vivo
[0163] Matrix embedded porcine aortic endothelial cells showed a
lower stimulation of the initial event in the recruitment of
leukocytes which involve P- and E-selectin, and of VCAM-1 which is
closely associated with T cell recruitment at sites of immune
inflammation. The full panel of general and species specific
costimulatory molecules was down regulated by matrix embedding,
including the first report of endothelial cell-expression and
suppression of 4-1BB-ligand. At the same time, expression and
upregulation of PD-L1 and PD-L2, members of the B7-family that act
as countervailing inhibitory molecules, remained intact after
matrix embedding. These in-vitro findings translated into a
significantly muted immune reaction in rats after implantation of
matrix-embedded porcine aortic endothelial cells.
[0164] The cellular response to implantation was also evaluated
immunohistochemically in six rats from each group on day 28 post
implantation. Five-micrometer paraffin sections were cut and
antigen retrieval performed by microwave heating for 10 minutes in
a 0.01 mol/L citrate buffer, pH 6.0. Leukocytes, T and B
lymphocytes were identified by an avidin-biotin peroxidase complex
method. The primary antibodies were mouse anti-rat CD45R0, to
identify leukocytes (Research Diagnostics; 1:50 dilution), mouse
anti-rat CD4, to identify CD4.sup.+-T cells (Pharmingen; 1:10
dilution), and mouse anti-rat CD8, to identify CD8.sup.+-T cells
(Pharmingen; 1:50 dilution). Rat spleen was used as a positive
control, and mouse IgG as negative controls. Primary antibodies
were applied for 1 hour at room temperature, and all sections were
counterstained with Mayer's hematoxylin solution (Sigma). Six
nonoverlapping fields (x600) were examined The results for each
group were averaged.
[0165] Embedding endothelial cells in a three-dimensional
biocompatible matrix, as compared to injected free PAE or PAE
injected adjacent to a three-dimensional biocompatible matrix, also
reduced the immune response in rats in vivo. Porcine aortic
endothelial cells embedding in Gelfoam significantly reduced
formation of porcine aortic endothelial cell-specific IgG in vivo.
Serum cytokines (MCP-1, IL-6, to TNF-.alpha.) rose, peaking five
days after implantation, in rats receiving free porcine aortic
endothelial cells and injections of porcine aortic endothelial
cells adjacent to Gelfoam. In contrast, cytokine levels did not
increase above background in animals with matrix-embedded porcine
aortic endothelial cells.
[0166] Immunohistological studies revealed evidence of cellular
infiltration into and around the implants/injection site at 28
days. After injection of free porcine aortic endothelial cells and
injection of porcine aortic endothelial cells adjacent to Gelfoam,
T cells were abundant within the implant/injection side, whereas
large numbers of CD45R0 positive leukocytes were also found at the
periphery of the graft. In contrast, the tissue surrounding the
implant and Gelfoam-porcine aortic endothelial cells itself were
infiltrated with 4.5 fold fewer leukocytes and CD4.sup.+-T cells,
and 3.3 fold fewer CD8.sup.+ T cells than the other cell
implantation groups.
[0167] Circulating rat immunoglobulins specific for the implanted
porcine aortic endothelial cells were also measured by flow
cytometry. 2.times.10.sup.5 porcine aortic endothelial cells were
detached from tissue culture polystyrene plates with 0.25%
trypsin/0.04% EDTA, pelleted, washed, resuspended in FACS buffer
and incubated with serum from recipient rats for 30 min at
4.degree. C. (diluted 1:10 in FACS buffer). After washing twice
with cold FACS buffer, cells were incubated with FITC-conjugated
anti-rat IgG. Following 30 min incubation at 4.degree. C. in the
dark, the samples were again washed twice with cold FACS buffer,
fixed in 1% paraformaldehyde, and 10.sup.4 cells were analyzed by
flow cytometry using a FACScalibur instrument and CellQuest
software. Rat IL-6 (R&D Systems, MN, detection limit 21 pg/ml),
rat TNF-.alpha. (R&D Systems, detection limit <5 pg/ml), and
rat MCP-1 (Amersham, detection limit <5 pg/ml)
serum-concentrations were to quantified by ELISA on days 0, 5, 12,
and 28 post implantation. Measurements were performed at the same
time by the same ELISA to avoid variations of assay conditions.
[0168] The levels of immunoglobulins specific for the implanted
porcine aortic endothelial cells in serum of the experimental mice
were also measured by flow cytometry. 2.times.10.sup.5 porcine
aortic endothelial cells, from the same strain as the implanted
cells, were detached from cell culture plates with 0.25%
trypsin/0.04% EDTA, pelleted, washed, and resuspended in FACS
buffer (PBS, 1% FCS, 0.1% sodium azide). These cells were then
incubated with serum from recipient mice for 60 min at 4.degree. C.
(diluted 1:10 in FACS buffer). After washing twice with FACS
buffer, cells were incubated with FITC-conjugated rat anti-mouse
IgG.sub.2a (Southern biotechnology, AL), IgG.sub.1 (clone A85-1),
or IgM (clone R6-60.2, BD Pharmingen, Calif.) respectively.
Following 30 min incubation at 4.degree. C. in the dark, the
samples were again washed twice with cold FACS buffer, fixed in
0.25 ml 1% paraformaldehyde, and 10.sup.4 cells were analyzed by
flow cytometry using a FACScalibur instrument and CellQuest
software.
[0169] Embedded endothelial cells in a three-dimensional
biocompatible matrix, as compared to injected free PAE and PAE
injected adjacent to a three-dimensional biocompatible matrix,
reduced the Th2-driven immune response in mice in vivo. To
characterize the magnitude and nature of the porcine aortic
endothelial cell-specific antibody response, serum was collected
from mice after implantation of porcine aortic endothelial cells in
the subcutaneous dorsal space as Gelfoam-embedded cells,
saline-suspended cell pellets, or as pellets adjacent to empty
Gelfoam. Post-implantation anti-porcine aortic endothelial cell
IgG.sub.1 and IgM levels were similar and significantly higher in
mice receiving porcine aortic endothelial cell pellets or porcine
aortic endothelial cell pellets adjacent to empty Gelfoam compared
to recipients of porcine aortic endothelial cells embedded in
Gelfoam (FIGS. 1A and 1B). There was a transient and minor
elevation in anti-porcine aortic endothelial cell IgG.sub.2a 12
days after implantation (p<0.005) after implantation of
matrix-embedded porcine aortic endothelial cell mice which was not
seen in mice receiving pelleted porcine aortic endothelial cells or
implants of empty Gelfoam with injection of pelleted porcine aortic
endothelial cells (FIG. 1C).
[0170] FIGS. 1A, 1B and 1C graphically depict circulating
PAE-specific IgG in mice after subcutaneous injection of free PAE,
of Gelfoam-grown endothelial cells, or after concomitant injection
of PAE adjacent to Gelfoam alone as determined via flow-cytometry.
Graphic depiction of results from all mice (n=18 per group to day
28, n=12 per group day 56-100 post-implantation) demonstrates a
statistically significant difference between the matrix-embedded
and other forms of PAE implantation for IgG.sub.1 (FIG. 1A) and IgM
(FIG. 1B). There was a transient and minor elevation in anti-PAE
IgG.sub.2a 12 days after implantation of matrix-embedded PAE (FIG.
1C).
[0171] Compared to unstimulated HAE grown on tissue culture plates,
matrix-embedded HAE expressed significantly higher levels of the
inhibitory signaling molecules suppressor-of-cytokine-signaling
(SOCS).sub.3 (0.007.+-.0.001 vs. 0.003.+-.0.0003 RU, p<0.001).
Hence stimulation with IFN-.gamma. resulted in significantly lower
expression of MHC II on matrix-embedded HAE (37.+-.5 vs. 68.+-.4%,
p<0.001). Despite unchanged IFN-.gamma.-receptor expression
levels (p=0.39) substrate adherence reduced IFN-.gamma.-induced
phosphorylation of Janus kinase 1 and 2 and
signal-transducer-and-activator-of-transcription-1. This was
followed by diminished expression of interferon-regulatory
factor-1, CIITA (0.01.+-.0.004 vs. 0.03.+-.0.004 RU, p<0.005),
and HLA-DR (0.17.+-.0.04 vs. 0.27.+-.0.05 RU, p<0.02) in
matrix-embedded HAE. Reduced MHC II expression on matrix-embedded
HAE resulted in muted ability to induce proliferation of allogeneic
T cells (4152.+-.255 vs. 19619.+-.327 cpm, p<0.001).
[0172] Interestingly, embedding endothelial cells in a three
dimensional matrix nearly completely diminishes the observed
Th2-driven immune response, mutes lytic activity and attenuates
differentiation of naive T cells into effector cells. In accordance
with previous results, these data suggest that Gelfoam embedding of
cells provides immune protection by immune activation at the T-cell
level via reduced expression levels of MHC class II molecules as
well as costimulatory and adhesion molecules.
Modulation of Lymphocyte Proliferation and Lytic Activity.
[0173] Porcine aortic endothelial cells grown on polystyrene wells
or embedded in Gelfoam were seeded in 96 well plates at
5.times.10.sup.4 cells/well and stimulated with 40 ng/ml porcine
INF-.gamma. for 48 hours, followed by mitomycin C treatment (Sigma,
50 .mu.g/ml for 30 min) to prevent background proliferation. Human
CD4.sup.+ lymphocytes were purified by negative selection with a
CD4.sup.+ T cell isolation kit II (Miltenyi Biotec, Germany)
according to the manufacturer's instructions and added at
2.times.10.sup.5 cells/well. In some experiments a murine antibody
directed against HLA-DP, DQ, DR blocked activation via MHC class II
molecules. .sup.3[H]-thymidine incorporation was measured on day 6
by 16 h pulse (1 .mu.Ci/ml, Amersham). Thymidine uptake of
mitomycin-treated porcine aortic endothelial cells, medium or T
cells alone was used as negative controls.
[0174] To evaluate lymphocyte lytic activity in mice in vivo,
splenocyte isolation and evaluation was performed. Spleens of 4
mice from each group were isolated aseptically in a laminar flow
hood on day 28 after porcine aortic endothelial cell-implantation.
Organs were cut in several pieces. Clumps were further dispersed by
drawing and expelling the suspension several times through a
sterile syringe with a 19-Gauge needle. Afterwards, the suspension
was expelled through a 200 .mu.m mesh nylon screen. Cells were
washed twice with RPMI (containing 2 mM L-glutamine, 0.1 M HEPES,
200 U/ml Penicillin G, 200 .mu.g/ml streptomycin and 5%
heat-inactivated calf serum, Life Technologies) and immediately
used.
[0175] To further evaluate lymphocyte lytic activity in mice in
vivo, a Calcein-AM release assay was performed. Porcine aortic
endothelial cells from the same strain of injected cells were
resuspended in complete medium at a final concentration of
2.times.10.sup.4/well and incubated with 15 .mu.M calcein-AM
(Molecular Probes) for 40 min at 37.degree. C. with occasional
agitation. After two washes with complete medium, splenocytes as
effector cells were added at a final concentration of
5.times.10.sup.5/well. Spontaneous and maximum release were
examined as controls in six replicate wells that contained only
target cells in complete medium and six wells with target cells in
medium plus 2% Triton X-100 for the last 20 minutes. After 3 hour
incubation at 37.degree. C./5% CO.sub.2 samples were measured using
a Fluoroskan Ascent FL dual-scanning microplate luminofluorimeter
(Thermo Electron Corporation, TX). Data were expressed as arbitrary
fluorescent units (AFU). Specific lysis was calculated according to
the formula [(test release-spontaneous release)/(maximum
release-spontaneous release)].times.100.
[0176] Embedding endothelial cells in a three-dimensional
biocompatible matrix reduced lymphocyte proliferation. The
proliferative response of isolated human CD4.sup.+ T cells to
untreated and INF-.gamma. treated porcine aortic endothelial cells
(40 ng/ml. 48 hours) grown in tissue culture plates or embedded in
Gelfoam was assayed by thymidine incorporation. The strong
CD4.sup.+ T cell proliferation noted after exposure to porcine
aortic endothelial cells pretreated with INF-.gamma. was nearly
eliminated when porcine aortic endothelial cells were
matrix-embedded (17087.2.+-.3749.75 vs. 5367.8.+-.1976.3 cpm,
p<0.01). The presence of MHC II antibody blocked lymphocyte
proliferation in response to INF-.gamma.-treated porcine aortic
endothelial cells by 65% to a level comparable to matrix embedded
porcine aortic endothelial cells. Mitomycin-treated porcine aortic
endothelial cells did not show a significant proliferation after 6
day culture (61.+-.13 cpm) as well as culture of isolated CD4.sup.+
T cells alone (83.+-.27 cpm).
[0177] Similarly, embedding endothelial cells in a
three-dimensional biocompatible matrix, as compared to injected
free PAE and PAE injected adjacent to a three-dimensional
biocompatible matrix, reduced lymphocyte lytic activity in mice in
vivo. Lymphocytes from mice spleens from the three different
treatment groups were isolated 28 days after porcine aortic
endothelial cell implantation. Donor porcine aortic endothelial
cells were labeled with Calcein-AM and endothelial cell-lysis was
measured by a calcein fluorescence release assay after coincubation
with lymphocytes. Lymphocytes from mice after pure porcine aortic
endothelial cell-injection (36.8.+-.3.9%) and after concomitant
porcine aortic endothelial cell-injection (33.9.+-.4.7%) showed the
highest lytic activity as compared to lymphocytes isolated from
mice after implantation of porcine aortic endothelial cell-Gelfoam
constructs (22.4.+-.4.2%, p<0.05; FIG. 2).
[0178] FIG. 2 graphically depicts splenocytes from mice receiving
free PAE and shows significantly increased lytic activity when
compared to matrix-embedded PAE. Rechallenge of mice with free PAE
significantly increased xenogeneic lytic activity of isolated
splenocytes.
[0179] Modulation of Th2 Cytokine-Producing Cells and
Cytokines.
[0180] Immunospot plates (Millipore, Bedford, Mass.) were coated
with 5 .mu.g/ml of anti-mouse interferon (IFN)-.gamma., anti-mouse
interleukin (IL)-2, anti-mouse IL-4, or anti-mouse IL-10 mAb (all
BD Pharmingen) in sterile PBS overnight. The plates were then
blocked for two hours with complete RPMI-medium without phenol red,
containing 10% heat-inactivated calf serum. Splenocytes
(0.5.times.10.sup.6 in 100 .mu.l complete RPMI-medium) and the same
strain of porcine aortic endothelial cells used for implantation
(0.5.times.10.sup.6 in 100 .mu.l complete RPMI-medium) were then
placed in each well and cultured for 48 hours at 37.degree. C. in
5% CO.sub.2. After washing with deionized water followed by washing
with PBS containing 0.05% Tween (PBST), 2 .mu.g/ml of biotinylated
anti-mouse IFN-.gamma., anti-mouse IL-2, anti-mouse IL-4, or
anti-mouse IL-10 mAb (all BD Pharmingen) were added overnight
respectively. The plates were then washed three times in PBST,
followed by one hour of incubation with horseradish
peroxidase-conjugated streptavidin (BD Pharmingen). After washing
four times with PBST followed by PBS, the plates were developed
using 3-amino-9-ethyl-carbazole (BD Pharmingen). The resulting
spots were counted on a computer-assisted enzyme-linked immunospot
image analyzer (Cellular Technology Ltd., ORT). The number of spots
in the wells with medium, splenocytes or porcine aortic endothelial
cells alone was subtracted from xenoresponses to account for
background in data analysis.
[0181] Embedding endothelial cells in a three-dimensional
biocompatible matrix, as compared to injected free PAE and PAE
injected adjacent to a three-dimensional biocompatible matrix,
reduced Th2 cytokine-producing cells in mice in vivo. The frequency
of Th1 cytokine (IFN-.gamma., IL-2) and Th2 cytokine (IL-4,
IL-10)-producing cells was measured by ELISPOT assay in splenocytes
recovered from animals after implantation of different forms of
porcine aortic endothelial cells. At day 28 postimplantation, the
frequency of Th2 cytokine-producing cells was significantly lower
in splenocytes isolated from mice receiving matrix embedded porcine
aortic endothelial cells compared with those isolated from mice
receiving free porcine aortic endothelial cells or porcine aortic
endothelial cells adjacent to empty Gelfoam ((IL-4: p<0.0001,
IL-10 <0.001; FIG. 3A). In contrast, there were no significant
differences in the frequency of Th1 cytokine-producing cells in
splenocytes isolated from the three groups (FIG. 3B).
[0182] FIG. 3A graphically depicts the frequencies of
xenoantigen-specific cytokine-producing cells in recipients after
implantation of mice with free PAE, matrix-embedded PAE, or PAE
injection adjacent to empty Gelfoam. There were no significant
differences in frequency of xenoreactive INF-.gamma. and IL-2
producing T-cells between the three groups on day 28. However,
rechallenge with matrix-embedded PAE evokes a significant increase
in xenoantigen-specific INF-.gamma. and IL-2 producing T-cells.
[0183] FIG. 3B depicts representative ELISPOT wells for one mouse
of each treatment group 28 days after first implantation and second
implantation respectively. IL-4 production in response to PAE was
measured. The number of IL-4 spots in each well was determined by
computer-assisted image analysis.
[0184] FIG. 3C graphically depicts that recipients of free PAE
exhibited a significant increased frequency of xenoreactive IL-4
and IL-10 producing T-cells compared to recipients of
matrix-embedded PAE on day 28. Rechallenge with free PAE or PAE
injection adjacent to empty Gelfoam matrices significantly
increased frequency of xenoantigen-specific IL-4 and IL-10
producing T-cells on day 128.
[0185] Modulation of Effector Cells.
[0186] Splenocytes recovered from the recipients were resuspended
in FACS buffer at a concentration of 2.times.10.sup.6/ml. Cells
were stained with anti-CD4 FITC (clone L3T4), antiCD8 FITC (clone
Ly-2), anti-CD44 R-PE (clone Ly-24), and anti-CD 62L
allophycocyanin (clone Ly-22), and isotype controls (all BD
PharMingen). CD4.sup.+ and CD8.sup.+ effector cells expressing
CD44.sup.high and CD62L.sup.low were enumerated, as previously
described.
[0187] Embedding endothelial cells in a three-dimensional
biocompatible matrix prevented xenorejection in mice in vivo. To
determine the effect of matrix embedding on the generation and
function of xenoreactive CD4.sup.+ and CD8.sup.+ T cells, we
measured the number of CD62L.sup.low CD44.sup.high found in the
spleens of mice treated after implantation of matrix-embedded
porcine aortic endothelial cells, implantation of saline-suspended
cell pellets, or as pellets adjacent to empty Gelfoam 28 days
following implantation (FIG. 4). CD4.sup.+ and 8.sup.+ effector
cells have been reliably identified as CD62L.sup.lowCD44.sup.high
cells. The percentage of CD62L.sup.lowCD44.sup.high cells increased
significantly in free porcine aortic endothelial cell-recipients
and mice receiving porcine aortic endothelial cells adjacent to
empty Gelfoam compared with mice receiving matrix-embedded porcine
aortic endothelial cells; the frequency of
CD4.sup.+CD62L.sup.lowCD44.sup.high T cells outnumbered CD8.sup.+
effector cells in all groups (ratio 1.7-2.3).
[0188] FIG. 4A graphically plots significantly increased CD4.sup.+
and CD8.sup.+ effector cells in mice receiving free PAE. CD4.sup.+
effector cells outnumbered CD8.sup.+ T-cells on days 28 and 128.
CD4.sup.+ splenocytes recovered from mice were analyzed by flow
cytometry using CD62L and CD44 as markers for effector T cells.
Representative plots from mice receiving free (a), matrix-embedded
(b), or PAE adjacent to Gelfoam (c) 28 days after implantation.
[0189] FIG. 4B graphically depicts expansion of effector cells
increases after rechallenge in mice receiving free PAE but not
matrix-embedded PAE.
Modulation of Xenorejection and Immunological Memory.
[0190] Embedding endothelial cells in a three-dimensional
biocompatible matrix produced immunological memory after
implantation of non-vascularized xenogeneic tissue. Th1 cytokines
play critical roles in the prevention of xenorejection by
down-regulating the Th2-driven humoral responses. In this regard,
the data demonstrate to that tissue engineered endothelial cells
can evoke a significant increase of porcine aortic endothelial
cell-specific IgG.sub.2a antibodies and a significant increase in
xenoreactive Th1 producing splenocytes after rechallenge.
[0191] One hundred days after the first implantation, the remaining
mice in each group were rechallenged with porcine aortic
endothelial cells identical to their first encounter. Mice
receiving saline-suspended cell pellets or pellets adjacent to
empty Gelfoam showed a significant IgG.sub.1-driven porcine aortic
endothelial cell-specific antibody response exceeding the response
observed after the first course of implantation (FIG. 5A). Only a
weak IgM-antibody release was seen (FIG. 5B). In marked contrast,
mice receiving matrix-embedded porcine aortic endothelial cells did
not show an increase in porcine aortic endothelial cell-specific
anti-IgG.sub.1 and IgM levels but exhibited a significant release
of porcine aortic endothelial cell-specific IgG.sub.2a antibodies
that was absent in the other two mice groups (FIG. 5C).
[0192] FIGS. 5A, 5B and 5C graphically depict rechallenge mice
(n=12 per group to day 128, n=6 per group day 156-190
post-implantation) with free PAE or PAE adjacent to Gelfoam
significantly increased formation of PAE-specific
IgG.sub.1-antibodies compared to rechallenge with matrix-embedded
PAE (FIG. 5A). Rechallenge has no influence on PAE-specific
IgM-formation (FIG. 5B) and there were no significant differences
of PAE-specific IgG.sub.2a-antibodies between the three groups
(FIG. 5C).
[0193] In line with these results, isolated splenocytes from mice
receiving free porcine aortic endothelial cells or porcine aortic
endothelial cell-injections adjacent to empty Gelfoam showed
significantly increased capability to lyse porcine aortic
endothelial cells 28 days after rechallenge, whereas
lysing-capability of splenocytes from mice receiving a second
implant of matrix-embedded porcine aortic endothelial cells was
significantly weaker than after the first implantation (FIG. 6).
The frequency of xenoreactive IL-4 and IL-10 producing T cells
increased significantly in mice after reimplantation of free
porcine aortic endothelial cells, the frequency of Th2 producing
splenocytes after rechallenge with matrix-embedded porcine aortic
endothelial cells was unchanged. However, rechallenge with
matrix-embedded porcine aortic endothelial cells induced a higher
frequency of xenoreactive INF-.gamma. and IL-2 producing
splenocytes than after the first course of implantation.
[0194] FIG. 6 graphically depicts matrix embedding or MHC II
blockade restore proliferation of mice splenocytes exposed to PAE
to unstimulated levels. Mice splenocytes proliferate in response to
INF-.gamma. stimulated PAE. Matrix-embedding endothelial cells or
presence of MHC II antibody blocked splenocyte proliferation in
response to INF-.gamma. treated PAE by .about.79%. Each value
represents mean.+-.SD.
[0195] Furthermore, 28 days after rechallenge the percentage of
CD4.sup.+ effector cells further increased in mice receiving free
porcine aortic endothelial cells and increased significantly in
mice receiving porcine aortic endothelial cells adjacent to empty
Gelfoam implants but remained unchanged in mice receiving matrix
embedded porcine aortic endothelial cells. The same pattern was
obvious for CD8.sup.+ effector T cells.
[0196] In vitro stimulation of naive mice splenocytes with PAE
revealed a significantly muted proliferative response of
splenocytes when incubated with INF-.gamma. stimulated
matrix-embedded endothelial cells compared to free endothelial
cells. The presence of MHC II antibody blocked splenocyte
proliferation in response to INF-.gamma.-treated PAE by 79% to a
level comparable to matrix embedded PAE.
[0197] Overall, the spleen size in mice receiving matrix-embedded
porcine aortic endothelial cells appeared smaller than in the other
groups at the end of the study period (62.9.+-.9.6, 112.7.+-.16.9,
102.5.+-.18.8 mm.sup.3; p<0.05).
[0198] Thus, cognate interactions between naive T cells and resting
endothelial cells can lead to tolerance in vitro and in vivo. These
data document formation of immunological memory after implantation
of non-vascularized xenogeneic tissue. Immunological memory was
characterized by a significant increase in antigen-specific
IgG.sub.1 and IgM levels, lytic activity of splenocytes and
tendency towards increased differentiation into effector T cells.
In contrast, rechallenging mice with matrix embedding of
endothelial cells led to a reduced lytic ability of splenocytes,
frequency of effector CD4.sup.+ and CD8.sup.+ T cells was
unchanged. Whereas rechallenge with matrix-embedded porcine aortic
endothelial cells had no influence on generation of anti-PAE
IgG.sub.1 and IgM, IgG.sub.2a levels increased significantly.
Modulation of Fractalkine Expression.
[0199] Chemokines and adhesion molecules are critical in recruiting
circulating immune cells into the vessel wall. Fractalkine has both
chemoattractive and adhesive functions and is involved in the
pathogenesis of atherosclerosis, cardiac allograft rejection,
glomerulonephritis, and rheumatoid arthritis. We compared
expression and secretion of fractalkine between free and
matrix-embedded human aortic endothelial cells (HAE) via RT-PCR,
Western blot, flow-cytometry and ELISA. Adhesion assays were
conducted with cytokine-stimulated HAE and .sup.51Cr labeled
natural killer (NK) cells.
[0200] HAE were stimulated with 100 U TNF.alpha./ml (Sigma) and 100
U IFN-.gamma./ml (Roche) at 37.degree. C. in a humidified air
atmosphere containing 5% CO.sub.2, conditions demonstrated to
result in maximal fractalkine levels in cultured endothelial
cells.
[0201] Flow Cytometry:
[0202] Endothelial cell monolayers or endothelial cells
matrix-embedded in Gelfoam were harvested after stimulation with
TNF.alpha. and IFN-.gamma. for indicated time periods. Media were
aspirated and cells were washed with PBS. Monolayers were incubated
in 1 mM PBS/EDTA for 5 min, and disrupted by gentle shaking.
Gelfoam-grown cells were digested with collagenase type I
(Worthington Biochemical, N.J.), which was shown to have no effect
on CX3CL1-expression. Cell-suspensions were washed and
3.times.10.sup.5 cells fixed in 4% paraformaldehyde for 10 min.
After two washing steps, cells were resuspended in saponin-buffer
(0.1% saponin, 0.05% NaN.sub.3 in Hanks' Balanced Salt Solution),
centrifuged and the supernatant decanted. HAE were then incubated
with FITC-conjugated mouse anti-human CX3CL1 (IgG.sub.1, clone
51637, R&D Systems, Minneapolis, Minn.) or a matched isotype
control (clone MOPC-31C, Pharmingen) for 45 min at 4.degree. C.
Cells were then washed and 10.sup.4 cells were analyzed by flow
cytometry using a FACScalibur instrument and CellQuest
software.
[0203] Western Blot Analysis:
[0204] Cell monolayers or cells digested from Gelfoam matrices by
collagenase-treatment were washed in PBS buffer and cell lysates
were prepared by incubation with lysis buffer (20 mM Tris, 150 mM
NaCl, pH 7.5, 1% Triton X-100, 1% deoxycholate, 0.1% SDS and
protease inhibitor; Roche). Samples were separated on 4-20% Ready
Tris-HCl gels (BioRad Laboratories, Hercules, Calif.). A positive
control for fractalkine detection was used, consisting of an 85- to
90-kDa form of recombinant human fractalkine lacking the
carboxy-terminal 57 amino acids (R&D Systems). Proteins were
then transferred onto PVDF membranes (Millipore, Billerica, Mass.)
by using glycin-Tris transfer buffer. Blot membranes were blocked
in Starting Block blocking buffer (Pierce, Rockford, Ill.) for 1
hour. For fractalkine-detection, blocked membranes were incubated
with goat anti-human fractalkine polyclonal antibody (R&D
Systems) at a dilution of 1:200 in blocking buffer overnight at
4.degree. C. Membranes were then washed three times at room
temperature with wash buffer consisting of PBS with 0.05% Tween 20
and then incubated with secondary antibody, a rabbit anti-goat IgG
conjugated to horseradish peroxidase (Santa Cruz Biotechnology,
Santa Cruz, Calif.) at a 1:3.000 dilution in blocking buffer for 2
hours at room temperature followed by washing in five changes of
wash buffer. For detection of fractalkine bands, the blot was
incubated with chemiluminescence substrate (Western Lightning
Chemiluminescence Reagent Plus kit, Perkin-Elmer, Boston, Mass.)
according to the manufacturer's instructions followed by exposure
to X-ray film (Kodak X-Omat Blue XB-1).
[0205] ELISA:
[0206] Conditioned medium from endothelial cell monolayers or
endothelial cells embedded in Gelfoam after cytokine stimulation
was harvested for indicated time periods. Secreted fractalkine was
detected with a commercially available enzyme-linked immunosorbent
assay (ELISA) detection kit (R&D Systems). Briefly 96-well
Immulon plates (Fisher Scientific, Pittsburgh, Pa.) were coated
overnight at room temperature with 100 .mu.l of 4 .mu.g/ml of mouse
anti-human fractalkine capture antibody in PBS. After three washes
with wash buffer (PBS-0.05% Tween-20) plates were blocked for 3 h
in 1% bovine serum albumin-5% sucrose in PBS. 100 .mu.l of
standards (420 ng/ml of recombinant human fractalkine (provided
with kit) was used diluted as twofold serial dilutions in diluent
buffer) or conditioned medium were added, followed by incubation
overnight at room temperature. After three washing steps the plate
was incubated with 100 .mu.l of 500 ng/ml mouse anti-human
fractalkine detection antibody in PBS for 2 hours at room
temperature followed by incubation with 100 .mu.l of streptavidin
conjugated to horseradish-peroxidase for 30 min at room
temperature. Color was then developed by adding 100 .mu.l hydrogen
peroxide solution mixed with tetramethylbenzidine (R&D
Systems). The optical density was then read at a wavelength of 450
nm.
[0207] NK cell-endothelial cell binding assays: HAE were grown to
confluence in 6-well plates (6.times.10.sup.5 cell/well) or
embedded in Gelfoam matrices and activated with 100 U TNF.alpha./ml
and 100 U IFN-.gamma./ml for 20 hours at 37.degree. C. in a
humidified air atmosphere containing 5% CO.sub.2 and washed once
with PBS. Gelfoam matrices were digested with collagenase type I,
cells counted and plated at a concentration of 6.times.10.sup.5
cells/well in 6-well plates for 1 hour to allow adherence. Isolated
NK cells were incubated with 10 .mu.Ci of .sup.51Cr/10.sup.6 NK
cells, washed in PBS and then resuspended (5 10.sup.5/well) in 400
.mu.l of medium alone or medium containing anti-CX3CR1 antibody at
20 .mu.g/ml for 20 min. The NK cell suspension was added to the
endothelial monolayer under gentle rocking conditions (10
cycles/min). After 30 min the medium was decanted and the wells
were gently washed. Adherent cells were lysed by treating with 1%
Triton in PBS. Total binding was determined by measuring individual
well-associated counts per minutes using a gamma counter. The
analyses illustrated were representative of at least three
independent experiments.
[0208] Matrix-embedding repressed induction of fractalkine mRNA.
Whereas resting endothelial cells grown on tissue culture
polystyrene plates or within a three-dimensional matrix did not
express fractalkine, stimulation of HAE grown on tissue culture
polystyrene plates with TNF.alpha. and IFN-.gamma. induced
fractalkine mRNA expression in a time dependent manner. Fractalkine
mRNA in HAE grown on tissue culture polystyrene plates expression
peaked at 12 hours stimulation with cells still expressing
significant amounts of mRNA after stimulation for 24 hours. In
contrast, induction of fractalkine mRNA expression was
significantly reduced in matrix-embedded endothelial cells at all
time points studied. The maximum was also reached after 12 hours
cytokine stimulation but was only .about.10% of expression levels
in endothelial cells grown to confluence on tissue culture
polystyrene plates (p<0.0001).
[0209] Matrix-embedding inhibited fractalkine protein expression in
HAE. Western blot analysis revealed lower protein expression levels
of fractalkine in HAE embedded within Gelfoam matrices compared to
endothelial cells grown on tissue culture polystyrene plates. There
was no fractalkine-specific protein band detectable in unstimulated
endothelial cells and in endothelial cells stimulated for 4 hours.
Endothelial cells grown on tissue culture polystyrene plates
expressed fractalkine after 8 hours of stimulation and exhibited
maximal expression from 16 to 24 hours of stimulation with
TNF.alpha. and IFN-.gamma.. Protein-expression in matrix-embedded
HAE was detectable later (12 hours), weaker and disappeared within
24 hours of cytokine stimulation.
[0210] In analogy to Western blot results, flow cytometry analysis
revealed significant higher fractalkine protein expression level in
HAE grown on tissue culture polystyrene plates. Whereas maximal
expression on matrix-embedded endothelial cells was reached after
16 hours of cytokine stimulation (22.8.+-.5.7%), endothelial cells
grown on tissue culture polystyrene plates reached a maximal and
significant increased fractalkine expression after 20 hours
stimulation with TNF.alpha. and IFN-.gamma. (76.5.+-.8.6%;
p<0.0001).
[0211] Experimental data indicate a reduced secretion of
fractalkine from cytokine stimulated matrix-embedded endothelial
cells. Fractalkine levels were also measured as cumulative levels
of soluble fractalkine released into the endothelial culture
supernatants by ELISA. Levels of soluble fractalkine paralleled
those in Western blot and flow cytometry analysis: fractalkine
secreted from HAE grown on tissue culture polystyrene plates
significantly exceeded levels secreted by matrix-embedded HAE
(32.2.+-.2.4 vs. 13.8.+-.1.7 pg/ml after 24 hours of culture;
p<0.0002).
[0212] Experimental data indicate a reduced adhesion of NK cells to
matrix-embedded endothelial cells. To study the functional
relevance of our finding, an adhesion assay with .sup.51Cr labeled
NK cell and cytokine-stimulated HAE grown on tissue culture
polystyrene plates or matrix-embedded was performed next. As
revealed by gamma-counting, significantly more NK-cells adhered to
allogeneic HAE grown on tissue culture polystyrene plates than
embedded within Gelfoam (6335.+-.420 vs. 1735.+-.135 cpm;
p<0.0002; FIG. 5). The importance of fractalkine expression for
NK cell adhesion to activated endothelial cells could be
demonstrated as addition of 20 .mu.g/ml anti-CX3CL1 significantly
augmented adhesion of NK cells to cytokine stimulated HAE by
.about.74% (p<0.005 vs. without anti-CX3CL1). NK cells did not
adhere to tissue culture polystyrene plates or Gelfoam alone.
Modulation of the Immune Response in Heightened Immune Reactivity
Mice.
[0213] Endothelial cell injections induced antibody formation in
mice. In naive B6 mice three serial subcutaneous injections of
5.times.10.sup.5 PAE raised circulating endothelial cell-specific
IgG.sub.1 (2210.+-.341 vs. 53.+-.12 mean fluorescence intensity
(MFI); p<0.0001) and IgM antibodies compared to saline
injections (136.+-.39 vs. 49.+-.14 MFI; p<0.02). There were no
PAE-specific IgG.sub.2a antibodies detectable in serum of either
mouse groups (data not shown) 42 days after first injection of
PAE.
[0214] Matrix-embedded endothelial cells prevented humoral immune
reactivity. Implantation of matrix-embedded xenogeneic endothelial
cells, in marked contrast to implantation of free cells, failed to
induce a significant humoral immune response in naive mice (d 42,
IgG.sub.1: 210.+-.102 vs. 735.+-.327 MFI; p<0.001; IgM: 60.+-.11
vs. 299.+-.51 MFI; p<0.001; FIGS. 7A and 7B). Injection of free
PAE in pre-sensitized serially challenged mice resulted in an
elevated humoral immune response with a pronounced increase in
IgG.sub.1 antibody-levels (3795.+-.448 MFI; p<0.0002 vs. naive
mice) and slight increase in PAE-specific IgM (164.+-.28 MFI). In
marked contrast, implantation of matrix-embedded PAE in
pre-sensitized serially challenged mice did not increase
PAE-specific antibodies: moreover antibody-levels specific for the
injected PAE slowly decreased with time (IgG.sub.1: 1578.+-.334
MFI; p<0.0005 vs. free PAE; IgM: 69.+-.5 MFI; p<0.01 vs. free
PAE; FIGS. 7A and 7B). There was no increase in PAE-specific
IgG.sub.2a-antibodies in the four treatment groups (data not shown)
supporting previous reports of a dominating Th2 response in
xenografting.
[0215] FIGS. 7A and 7B graphically depict circulating PAE-specific
IgG.sub.1 (FIG. 7A) and IgM (FIG. 7B) in naive and pre-sensitized
serially challenged mice after subcutaneous implantation of
non-embedded or matrix-embedded PAE. Graphic depiction of results
from all mice (n=12/group to day 70, n=6/group day 71-132
post-implantation) demonstrates significant differences between
matrix-embedded and free PAE implantation. Antibody-levels after
implantation of matrix-embedded PAE slowly diminish. Data are
expressed as mean values.+-.SD.
[0216] Matrix-embedded endothelial cells are poor inducers of
cellular immunity. ELISPOT-analysis revealed a high frequency of
xenogeneic T-helper cell (Th)2-cytokine (IL-4, IL-10) producing
splenocytes in naive and pre-sensitized serially challenged mice 90
days after implantation of free but not after implantation of
matrix-embedded PAE. The frequency of xenoreactive splenocytes in
pre-sensitized serially challenged mice exceeded xenoreactive
splenocyte activation and differentiation in naive mice receiving
free PAE (IL-4: 907.+-.59 vs. 680.+-.129; p<0.02; IL-10:
1096.+-.94 vs. 888.+-.151 number of spots; p<0.02; FIGS. 8A and
8B). Yet, compared to implantation of matrix-embedded PAE in naive
mice, implantation of matrix-embedded PAE in pre-sensitized
serially challenged mice elicited only a slight increase in IL-4
(322.+-.75 vs. 199.+-.99 number of spots; p<0.05; p<0.0005
vs. free PAE; FIG. 8A) but not in IL-10 producing xenoreactive
splenocytes (403.+-.142 vs. 451.+-.135 number of spots; p=0.27;
p<0.001 vs. free PAE; FIG. 8B). Significantly fewer Th2-cytosine
producing splenocytes were present in pre-sensitized serially
challenged mice receiving matrix-embedded PAE compared to naive
mice receiving free PAE (p<0.001). The frequency of Th1-cytokine
(IFN-.gamma. and IL-2) producing splenocytes did not differ
significantly between the four treatment groups again supporting a
predominant Th2-role in xenoreactivity (data not shown).
[0217] FIGS. 8A and 8B graphically depict the frequencies of
xenoreactive cytokine-producing cells in recipients after
implantation of free PAE or matrix-embedded PAE in naive and
pre-sensitized serially challenged mice. Data are expressed as mean
values.+-.SD. Naive and pre-sensitized serially challenged
recipients of free PAE exhibited significant increased frequencies
of IL-4 (FIG. 8A) and IL-10 (FIG. 8B) producing splenocytes
compared to recipients of matrix-embedded PAE.
[0218] The increase in cytokine-producing splenocytes in mice
receiving non-embedded PAE was paralleled by an increase of
CD4.sup.+ and CD8.sup.+ effector T cells over time (CD4.sup.+:
44.+-.2 naive mice, 54.+-.4% pre-sensitized mice, p<0.05;
CD8.sup.+: 20.+-.2; 21.+-.2%; FIGS. 9A and 9B). Accordingly,
differentiation of T cells into CD44.sup.high/CD62L.sup.low T cells
was significantly muted in naive and pre-sensitized serially
challenged mice exposed to matrix-embedded PAE (CD4.sup.+: 22.+-.2
naive mice, 21.+-.3% pre-sensitized mice; p<0.01 vs. free PAE;
CD8.sup.+: 12.+-.2; 14.+-.3%; p<0.02 vs. free PAE; FIGS. 9A and
9B). CD4.sup.+ outnumbered CD8.sup.+ effector T cells 1.7-2.6 in
all treatment groups. A strong correlation was noted between the
frequency of Th2-cytokine producing splenocytes and extent of T
cell differentiation cells into
CD4.sup.+CD44.sup.high/CD62L.sup.low (IL-4: r=0.81; p<0.0001;
IL-10 r=0.88; p<0.0001; FIG. 10) and
CD8.sup.+CD44.sup.high/CD62L.sup.low effector cells (IL-4: r=0.79;
p<0.0001; IL-10 r=0.86; p<0.0001) across all treatment groups
on day 132.
[0219] FIGS. 9A and 9B graphically depict significantly increased
CD4.sup.+ (Figure 9A) and CD8.sup.+ (FIG. 9B) effector cells in
mice receiving free PAE. Splenocytes recovered from mice were
analyzed by flow-cytometry using CD62L and CD44 as markers for
effector T cells. No difference between naive and pre-sensitized
serially challenged mice when endothelial cells are
matrix-embedded. Data are expressed as mean values.+-.SD.
[0220] FIGS. 10A and 10B are Spearman correlations of the
frequencies of Th2-cytokine producing splenocytes and the extent of
T cell differentiation into effector cells. FIG. 10A graphically
depicts the frequency of IL-2 cytokines. FIG. 10B graphically
depicts the frequency of IL-10 cytokines. The correlations suggest
that cytokine levels correlate linearly with effector T cell
induction. Area of the density ellipse represents the 95%
confidence interval.
[0221] Matrix-embedded endothelial cells are shielded from lytic
damage. The ability of host lymphocytes to damage xenogeneic
endothelial cells was characterized on day 70 and day 132. Calcein
release plateaued at effector: target ratios of 25:1. For this
ratio, endothelial cell damage was 1.6 fold higher in naive mice
and 1.7 fold higher in pre-sensitized mice when receiving
non-embedded in place of matrix-embedded PAE on d70 (p<0.001).
These ratios increased to 1.9 and 2.3 respectively after 132 days
(p<0.0005; FIG. 11). Of note, the extent of endothelial damage
in pre-sensitized mice receiving matrix-embedded PAE was
significant lower when compared to naive mice receiving free PAE
(20.9.+-.2.3 vs. 37.1.+-.3.4% AFU; p<0.001; FIG. 11).
[0222] The ability of host lymphocytes to damage xenogeneic
endothelial cells was characterized on day 70 and day 132. FIG. 11
graphically depicts the degree of damage to endothelial cells in
naive and pre-sensitized mice when the endothelial cells are free
or matrix embedded. Endothelial damage via lysis is significantly
reduced in naive and pre-sensitized mice receiving matrix-embedded
compared to free PAE. 2.times.10.sup.4 PAE were labeled with
calcein and incubated with 5.times.10.sup.5 splenocytes isolated
after 70 and 132 days respectively.
[0223] Calcein release plateaued at effector: target ratios of
25:1. For this ratio, endothelial cell damage was 1.6 fold higher
in naive mice and 1.7 fold higher in pre-sensitized mice when
receiving non-embedded in place of matrix-embedded PAE on day 70
(p<0.001). These ratios increased to 1.9 and 2.3 respectively
after 132 days (p<0.0005; FIG. 11). Of note, the extent of
endothelial damage in pre-sensitized mice receiving matrix-embedded
PAE was significant lower when compared to naive mice receiving
free PAE (20.9.+-.2.3 vs. 37.1.+-.3.4% AFU; p<0.001; FIG.
11).
Modulation of Dendritic Cell Maturation.
[0224] Dendritic cells are antigen-presented cells that have the
unique ability to both initiate and regulate immune responses.
Mature dendritic cells promote T cell differentiation into effector
and memory cells whereas immature dendritic cells present
(self-)antigens in a tolerogenic fashion. Dendritic cells are
implicated in a variety of endothelial-mediated diseases, and
activated endothelial cells induce their maturation. Because
dendritic cells are critical in immune reactivity, it follows that
endothelial cell-driven dendritic cell maturation is dependent on
endothelial cell-matrix contact.
[0225] Preparation, culture and maturation of dendritic cells:
Peripheral blood was collected from healthy volunteers and
fractionated over Ficoll-Paque (Sigma Chemicals, St. Louis, Mo.) by
a standard procedure. To derive dendriti cells, total peripheral
blood monocytic cells (PBMC) were cultured at 2.times.10.sup.6
cells/ml in complete media (RPMI 1640, 10% heat-inactivated calf
serum, 0.1 mM sodium pyruvate (Life Technologies)) for 1.5 hours in
tissue culture flasks. Following incubation, nonadherent cells were
removed by extensive washing with a 1.times. solution of HBSS (Life
Technologies). The remaining adherent cells were then cultured in
complete media containing 20 ng/ml interleukin (IL)-4 and 20 ng/ml
GM-CSF (Peprotech, Rocky Hill, N.J.) for 5 days in a CO.sub.2
incubator at 37.degree. C. The resulting cells were semi- to
nonadherent and MHC II.sup.low/CD14.sup.-/low/CD83.sup.- (data not
shown).
[0226] For further maturation, adherent and nonadherent dendritic
cells were harvested, extensively washed, counted and
5.times.10.sup.5 dendritic cells were stimulated with a cytokine
cocktail (10 ng/ml IL-1.beta., 1000 U/ml IL-6, 20 ng/ml IL-4,
GM-CSF, and TNF-.alpha.; all Preprotech), 1.5.times.10.sup.5 HAE or
1.5.times.10.sup.5 PAE for 48 hours. Endothelial cells were either
presented as suspensions after grown to confluence on tissue
culture plates or surface adherent embedded within Gelfoam
matrices. Every assay was repeated at least four times. After
maturation, dendritic cells were isolated from any contaminating
endothelial cells with magnetic bead-labeled CD1a antibodies
(Miltenyi, Bergisch-Gladbach, Germany). Flow cytometry analysis
revealed 98% purity of the isolated DC (data not shown).
[0227] Real-time PCR: Total RNA was extracted from isolated
dendritic cells and the remaining endothelial cells using the
RNeasy Mini Kit (Qiagen, Valencia, Calif.) according to the
manufacturer's instructions. Complementary DNA was synthesized
using TaqMan reverse transcription reagents from Applied Biosystems
(Foster City, Calif.). Real-time PCR analysis was performed with an
Opticon Real Time PCR Machine (MJ Research, Waltham, Mass.) using
SYBR Green PCR Master Mix (Applied Biosystems) and selected
primers. Data from the reaction were collected and analyzed by the
complementary Opticon computer software. Relative quantification of
gene expression were calculated with standard curves and normalized
to GAPDH.
[0228] Flow Cytometry:
[0229] Dendritic cell or endothelial cell suspensions were washed
and 3.times.10.sup.5 cells were resuspended in FACS buffer (PBS
containing 0.1% BSA and 0.1% sodium azide; Sigma Chemicals).
Standard flow cytometric analysis assessed surface expression of
various markers. The following monoclonal antibodies directly
conjugated with phycoerythrin (PE) or fluorescein isothiocyanate
(FITC) were used in single-color flow cytometric analysis: PE-CD1a
(clone HI149, IgG.sub.1), FITC-CD3 (clone UCHT1, IgG.sub.1),
PE-CD14 (clone TUK4, IgG.sub.2a), PE-CD31 (clone WM59, IgG.sub.1),
FITC-CD40 (clone 5C3, IgG.sub.1), FITC-CD54 (clone 15.2,
IgG.sub.1), FITC-CD80 (clone BB1, IgM), FITC-CD83 (clone HB15e,
IgG.sub.1), FITC-CD86 (clone 2331, IgG.sub.1), FITC-CD106 (clone
51-10C9, IgG.sub.1), FITC-HLA-DP, DQ, DR (clone CR3/43, IgG.sub.1),
FITC-Toll-like receptor (TLR).sub.2 (clone TL2.3, IgG.sub.2a), and
FITC-TLR4 (clone HTA125, IgG.sub.2a). Appropriate isotype control
antibodies (mouse PE-IgG.sub.1, PE-IgG2a, FITC-IgG.sub.1,
FITC-IgG.sub.2a, FITC-IgM) were used respectively. Antibodies were
purchased from DakoCytomation (Carpinteria, Calif.), Serotec
(Raleigh, N.C.) or PharMingen (San Diego, Calif.). After staining,
cells were washed and fixed in 1% paraformaldehyde before analysis
on a FACScalibur instrument and CellQuest software (Becton
Dickinson, Mountain View, Calif.).
[0230] Endocytic Activity:
[0231] Endocytic activity of dendritic cells was measured by uptake
of FITC-conjugated dextran (MW 40.000; Molecular Probes, Eugene,
Oreg.) as previously described. Briefly, dendritic cells at various
states of maturation were incubated in complete media with 1 mg/ml
FITC-conjugated dextran for 1 hour at 37.degree. C. to measure
specific uptake, or at 4.degree. C. to measure nonspecific binding.
Cells were then washed extensively and analyzed by flow cytometry
as described above.
[0232] Mixed lymphocyte reaction assay: CD3.sup.+ T-cells from an
unrelated donor were prepared from total PBMC by negative selection
using antibody depletion and magnetic beads according to the
manufacturer's instruction (Dynal Biotech, Lake Success, N.Y.). The
nonmagnetic fraction contained greater than 95% CD3.sup.+ T-cells,
as assessed by flow cytometry. 2.times.10.sup.5 CD3.sup.+
T-cells/well were seeded in 96-well round-bottom plates. Purified
cytokine- or endothelial cell-matured dendritic cells were
.gamma.-irradiated (3000 rad from a .sup.137Cs source) and added to
T-cells at 1.times.10.sup.4, 4.times.10.sup.3, or 2.times.10.sup.3
cells/well to give final ratios of 1:20, 1:50, or 1:100 DC:T-cells.
On day 5, 1 .mu.Ci of .sup.3H-thymidine (Perkin-Elmer, Boston,
Mass.) was added to each well. Cells were harvested 18 hours later
and .sup.3H-thymidine uptake quantified using a Packard TopCount
.gamma.-counter (GMI, Ramsey MI).
[0233] Western Blot:
[0234] After separation from dendritic cells, endothelial cells
were washed in PBS buffer and cell lysates were prepared by
incubation with lysis buffer (20 mM Tris, 150 mM NaCl, pH 7.5, 1%
Triton X-100, 1% deoxycholate, 0.1% SDS and protease inhibitor;
Roche, Indianapolis, Ind.). Samples were separated on 4-20% Ready
Tris-HCl gels (BioRad Laboratories, Hercules, Calif.). Proteins
were then transferred onto PVDF membranes (Millipore, Billerica,
Mass.) using glycin-Tris transfer buffer. Jurkat (TLR2) or HL-60
whole cell lysates (TLR4, both Santa Cruz Biotechnologies, Santa
Cruz, Calif.) served as controls. Membranes were blocked in
Starting Block blocking buffer (Pierce, Rockford, Ill.) for 1 hour.
Blocked membranes were incubated with rabbit anti-human TLR2
(dilution 1:250 in blocking buffer) or TLR4 antibodies (dilution
1:200, both Santa Cruz Biotechnologies) overnight at 4.degree. C.
Membranes were then washed three times at room temperature with
wash buffer consisting of PBS with 0.05% Tween 20 and then
incubated with secondary antibody, a goat anti-rabbit IgG
conjugated to horseradish peroxidase (Santa Cruz Biotechnology,
Santa Cruz, Calif.) at a 1:1.000 dilution in blocking buffer for 2
hours at room temperature followed by washing in five changes of
wash buffer. For detection of TLR bands, the blot was incubated
with chemiluminescence substrate (Western Lightning
Chemiluminescence Reagent Plus kit; Perkin-Elmer) according to the
manufacturer's instructions followed by exposure and analysis on a
Fluor Chem SP (Alpha Innotech, San Leandro, Calif.).
[0235] Non-adherent endothelial cells directed maturation of
monocyte-derived dendritic cells. In line with previous
observations, monocytes differentiated into immature dendritic
cells after 5 days of culture in GM-CSF and IL-4 (data not shown).
Prolonged cytokine-stimulation with IL-1.beta., TNF-.alpha., and
IL-6 for 48 hours upregulated costimulatory (CD40: 2.3 fold
compared to immature dendritic cells, CD80:1.9 fold, CD86: 1.6
fold) and HLA-DR molecules (1.5 fold) together with expression of
CD83 (2.2 fold) as an established dendritic cells-maturation
marker. Exposure to saline suspensions of allo- and xenogeneic
endothelial cells after growth to confluence in tissue culture
plates induced full maturation of monocyte-derived dendritic cells
to a similar degree as prolonged treatment with a cytokine
cocktail. HAE or PAE alone induced dendritic cell costimulatory
molecule expression with increases in CD40 (HAE: 2.1 fold, PAE: 2.5
fold compared to immature dendritic cells), CD80 (2.1 fold, 2.3
fold; p<0.05 vs. cytokine-stimulation), CD86 (1.6 fold, 1.7),
HLA-DR (1.7 fold, 2.2 fold; p<0.05 vs. HAE, p<0.002 vs.
cytokine-stimulation), and CD83 (2.6 fold; p<0.05 vs. cytokine
stimulation, 3.2 fold; p<0.02 vs. HAE, p<0.001 vs.
cytokine-stimulation).
[0236] In a similar fashion, dendritic cell TLR2 and 4 expression
were upregulated upon exposure to saline suspensions of HAE (1.5
and 2.5 fold respectively) to a similar or greater extent than
cytokine stimulation (1.5 fold for both TLR compared to immature
dendritic cells). This effect was even more pronounced after
co-incubation of dendritic cells with non-adherent xenogeneic PAE
(TLR2: 2.4 fold; p<0.05 vs. cytokine- and HAE-stimulated, TLR4:
3.0 fold; p<0.05 vs. HAE, p<0.001 vs. cytokine-stimulation).
Similar results could be obtained for mRNA transcript levels.
Additionally, dendritic cells matured with cytokines or
non-adherent endothelial cells displayed significant upregulation
of IL12 p40 mRNA (immature: 0.03.+-.0.02 relative units (RU),
cytokine-stimulated: 0.23.+-.0.03 RU, p<0.002, HAE-stimulated:
0.31.+-.0.05 RU, p<0.001, PAE-stimulated: 0.28.+-.0.03,
p<0.002).
[0237] Incubation with substrate-adherent endothelial cells
resulted in incomplete dendritic cell-maturation and sustains
endocytic activity. In marked contrast to co-culture with
non-adherent endothelial cells, co-culture of dendritic cells with
substrate-adherent HAE and PAE embedded within a three-dimensional
matrix restricted dendritic cell maturation: these dendritic cells
displayed only weak upregulation of CD40 (substrate-adherent HAE:
1.5 fold compared to immature DC, p<0.02 vs. non-adherent HAE,
substrate-adherent PAE: 1.3 fold, p<0.002 vs. non-adherent PAE),
CD80 (substrate-adherent HAE and PAE: 1.3 fold, p<0.005 vs.
non-adherent EC), CD86 (substrate-adherent HAE: 1.1 fold, PAE: 1.2
fold, both p<0.005 vs. non-adherent EC), CD83
(substrate-adherent HAE: 1.5 fold, p<0.001 vs. non-adherent HAE,
PAE: 1.4 fold, p<0.0002 vs. non-adherent PAE), and TLR4
(substrate-adherent HAE: 1.5 fold, PAE: 1.3 fold, both p<0.005
vs. non-adherent endothelial cells). Co-incubation with
substrate-adherent endothelial cells failed to induce HLA-DR and
TLR2 expression on dendritic cells at all (p<0.005). Incubation
with empty Gelfoam matrices alone had no effect on maturation of
monocyte-derived dendritic cells (data not shown). Real-time PCR
analysis revealed the same pattern of incomplete maturation when
dendritic cells were exposed to substrate-adherent allo- and
xenogeneic endothelial cells. Induction of IL12 p40 was similarly
significantly weaker when dendritic cells had been matured with
substrate-adherent endothelial cells (HAE-stimulated: 0.06.+-.0.01,
p<0.005, PAE-stimulated 0.07.+-.0.02, p<0.02).
[0238] Immature dendritic cells efficiently captured antigen and
exhibited a high level of endocytosis. FITC-conjugated dextran
uptake increased when monocytes were cultured for 3 and 5 days in
GM-CSF and IL-4 (423.3.+-.121.8 mean fluorescence intensity (MFI),
239.8.+-.42.8 MFI, p<0.0001). Maturation is typically
accompanied by concomitant increase in antigen presenting function
and reduced capacity for antigen capture via endocytic activity.
Dextran uptake typically decreases with continued
cytokine-stimulation (89.7.+-.14.7 MFI, p<0.0001 vs. d5) and
with co-incubation with non-adherent HAE (92.+-.20.3 MFI) or PAE
(82.4.+-.16.5 MFI). In marked contrast, dendritic cells retained
their endocytic activity when endothelial cells were presented in a
substrate-adherent three-dimensional state and dextran uptake was
markedly increased (substrate-adherent HAE: 203.2.+-.11.3 MFI,
p<0.05 vs. d5, p<0.0001 vs. non-adherent HAE;
substrate-adherent PAE: 254.3.+-.32 MFI, p<0.0001 vs.
non-adherent PAE).
[0239] Dendritic cells exhibited reduced T-cell proliferation
activity after cultivation with substrate-adherent endothelial
cells. The ability to promote T cell differentiation into effector
and memory cells is an important functional marker for the
maturation grade of dendritic cells. Whereas cytokine-treated and
non-adherent endothelial cell exposed dendritic cells induced
T-cell proliferation over the full spectrum of dendritic cell:
T-cell ratios tested (74789.+-.1777, HAE: 97522.+-.1630, and PAE:
101616.+-.4302 cpm) this ability was significantly muted in
dendritic cells co-incubated with substrate-adherent HAE
(18320.+-.1000 cpm, p<0.002) and PAE (20080.+-.683 cpm,
p<0.0001).
[0240] Activation of substrate-adherent endothelial cells was
reduced when co-cultured with dendritic cells. Real-time PC,
flow-cytometry and Western blot analysis revealed reduced
activation of HAE and PAE after co-culture for 2 days with
dendritic cells. After magnetic-bead based isolation of dendritic
cells, the remaining cells were greater than 95% pure for the
endothelial-cell specific marker CD31 (data not shown). Real-time
PCR analysis demonstrated reduced mRNA expression levels for
adhesion molecules, CD58, HLA-DR and TLR-molecules on
substrate-adherent HAE when compared to their non-adherent
counterparts. Reduced mRNA-expression levels translated into
reduced surface and intracellular expression with 3.6 fold lower
expression of ICAM-1 on substrate-adherent when compared to
non-adherent HAE (1.3 fold decrease for PAE), 4.9 fold decrease of
VCAM-1 for HAE (PAE: 2.7 fold), and 16 fold decrease of HLA-DR for
HAE (PAE: 23 fold decrease). Densitometry analysis of Western blots
revealed increased TLR2 (HAE: 1.5 fold increase, PAE: 1.6 fold
increase; p<0.05) and TLR4 expression (HAE: 2.3 fold increase,
PAE: 2 fold increase; p<0.01) in non-adherent endothelial cells
when compared to substrate-adherent endothelial cells after
co-incubation with dendritic cells for 48 hours.
[0241] Thus, whereas non-adherent endothelial cells induced
maturation of monocyte-derived dendritic cells to an extent similar
to that seen with a cytokine-cocktail, co-incubation with
substrate-adherent endothelial cells induced only minor
upregulation of mRNA transcript and protein levels of adhesion,
costimulatory and HLA-DR molecules on dendritic cells. Dendritic
cells co-incubated with substrate-adherent endothelial cells also
lacked upregulation of IL12 mRNA and CD83 expression that serve as
direct maturation markers. The immature state of dendritic cells
after co-cultivation with substrate-adherent endothelial cells was
mirrored by sustained ability to uptake dextran. Functionally,
whereas dendritic cells exposed to non-adherent endothelial cells
displayed enhanced T-cell stimulatory activity in mixed lymphocyte
reactions, T-cell proliferation after exposure to
substrate-adherent endothelial cell-matured dendritic cells was
significantly weaker.
[0242] Further Experiments: Effects on Immune Response
[0243] Treatment of Transplantation Rejection:
[0244] A population of normal (not immune compromised) organ
transplant recipients will be identified. The population will be
divided into three groups, one of which will receive an effective
amount of the implantable material of the present invention prior
to receipt of a transplant organ. A second group will receive an
effective amount of implantable material of the present invention
coincident with receipt of a transplant organ. A third group will
not receive the implantable material of the present invention, but
will receive a transplant organ. Reduction of and/or amelioration
of an immune response and/or an inflammatory response will be
monitored over time by evaluating the proliferation of T-cell
lymphocytes and B-cell lymphocytes in serum samples and by
monitoring the duration of transplant organ acceptance. It is
expected that candidates receiving an effective amount of the
implantable material of the present invention will display a
reduction in proliferation of lymphocytes and/or an increase in the
duration of transplant organ acceptance.
[0245] Treatment of Autoimmune Disease:
[0246] A population of patients diagnosed with an autoimmune
disease will be identified. The population will be divided into two
groups, one of which will receive an effective amount of the
implantable material of the present invention. Reduction of and/or
amelioration of an autoimmune response and/or an inflammatory
response will be monitored over time by evaluating the
proliferation of T-cell lymphocytes and B-cell lymphocytes in serum
samples and by monitoring the intensity and duration of symptoms
associated with the autoimmune disease. It is expected that
candidates receiving an effective amount of the implantable
material of the present invention will display a reduction in
proliferation of lymphocytes and/or a reduction in the frequency
and/or intensity of symptoms.
* * * * *