U.S. patent application number 12/625815 was filed with the patent office on 2010-08-12 for method and composition for treating immune complex associated disorders.
This patent application is currently assigned to Trustees of Boston University. Invention is credited to Elizabeth A. Leadbetter, Ann Marshak-Rothstein, Ian R. Rifkin, Mark J. Shlomchik.
Application Number | 20100203053 12/625815 |
Document ID | / |
Family ID | 26981316 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100203053 |
Kind Code |
A1 |
Marshak-Rothstein; Ann ; et
al. |
August 12, 2010 |
METHOD AND COMPOSITION FOR TREATING IMMUNE COMPLEX ASSOCIATED
DISORDERS
Abstract
The present invention provides methods and compositions for
treating immune complex associated diseases (ICAD), such as SLE,
rheumatoid arthritis, and hepatitis-C related immune complex
disease (e.g., cryoglobulinemia) in a subject having an ICAD or at
risk for developing ICAD. The invention is based upon the
surprising finding that chromatin-containing immune complexes
activate autoreactive B cells and dendritic cells by a dual
receptor engagement process which, in both cell types, involves a
Toll-like receptor (TLR). The methods of treating ICAD comprise
administering a compound to an individual in need thereof that
either 1) inhibits formation of the immune complex either by
preventing formation and/or binding to the TLR, or 2) interferes
with binding of an autoantigen-containing immune complex (or the
antigenic component thereof) to the TLR, or 3) inhibits signaling
pathways initiated by dual engagement of BCR and TLR (in B cells)
or FcR and TLR (in dendritic cells) via immune complexed or
uncomplexed autoantigens.
Inventors: |
Marshak-Rothstein; Ann;
(Newton, MA) ; Leadbetter; Elizabeth A.;
(Roslindale, MA) ; Rifkin; Ian R.; (Boston,
MA) ; Shlomchik; Mark J.; (Woodbridge, CT) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Trustees of Boston
University
Boston
MA
Yale University
New Haven
CT
|
Family ID: |
26981316 |
Appl. No.: |
12/625815 |
Filed: |
November 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10487885 |
Sep 27, 2004 |
7709451 |
|
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PCT/US02/28708 |
Sep 9, 2002 |
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12625815 |
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60318096 |
Sep 7, 2001 |
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60367578 |
Mar 26, 2002 |
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Current U.S.
Class: |
424/135.1 ;
435/7.1; 435/7.24; 514/1.1; 514/44A |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/44 20130101; G01N 33/5047 20130101; A61P 37/06 20180101;
A61P 37/02 20180101; A61P 43/00 20180101; A61P 1/16 20180101; A61K
2039/505 20130101; A61P 19/02 20180101; G01N 2500/00 20130101; G01N
2500/02 20130101; A61P 29/00 20180101; C07K 2317/21 20130101; G01N
33/564 20130101; G01N 2800/102 20130101; C07K 16/28 20130101; A61P
37/00 20180101 |
Class at
Publication: |
424/135.1 ;
514/44.A; 514/12; 435/7.1; 435/7.24 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088; A61K 38/16
20060101 A61K038/16; G01N 33/53 20060101 G01N033/53; A61P 37/06
20060101 A61P037/06 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government Support under
Contract Nos. RO1 AR-35230 and K08 DK-02597 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1. A method of treating a patient having or at risk of having an
Immune Complex Associated Disease (ICAD) or a systemic autoimmune
disease, comprising administering to a patient having or at risk of
having an ICAD or a systemic autoimmune disease an effective amount
of a compound that inhibits immune complexes or autoantigens from
binding to or activating a Toll-like receptor (TLR), wherein the
Toll-like receptor is chosen from Toll-like receptor 9 (TLR9) and
Toll-like receptor 3 (TLR3) and wherein said immune complex
comprises an autoantibody and an autoantigen bound to a cell
receptor, to treat the ICAD or systemic autoimmune disease.
2. The method of claim 1, wherein the ICAD is systemic lupus
erythematosus.
3. The method of claim 1, wherein the ICAD is rheumatoid
arthritis.
4. The method of claim 1, wherein the ICAD is hepatitis-C related
immune complex disease.
5. The method of claim 1 wherein the Toll-like receptor is
TLR9.
6. The method of claim 1 wherein the Toll-like receptor is
TLR3.
7. The method of claim 1, wherein the compound is selected from the
group consisting of (a) compounds that bind components of the
immune complex and prevent its formation or binding to the
Toll-like receptor; (b) Toll-like receptor decoys; (c) compounds
that inhibit the activity of MyD88 or other components of a
TLR-initiated signaling cascade; (d) compounds that inhibit
production of immune complex components; and (e) Toll-like receptor
antagonists.
8. The method of claim 7, wherein the Toll-like receptor antagonist
is an inhibitory oligonucleotide.
9. The method of claim 7, wherein the Toll-like receptor antagonist
is a dominant negative Toll-like receptor.
10. The method of claim 1, wherein the compound is an antibody that
binds a Toll-like receptor.
11. The method of claim 10, wherein the antibody is a single chain
antibody.
12. The method of claim 1, wherein the compound comprises a
cocktail of compounds that inhibit binding to a combination of at
least two of Toll-like receptor 2 (TLR2), TLR3, and TLR9.
13. A method for screening for compounds that inhibit immune
complex formation or binding to a Toll-like receptor, comprising
contacting immune complex components with a compound being screened
and with a TLR chosen from Toll-like receptor 3 (TLR3) and
Toll-like receptor 9 (TLR9); measuring binding of an antigenic
fragment of an immune complex to the TLR, wherein said immune
complex comprises an autoantibody and an autoantigen; and
identifying the compound being screened as an inhibitor of immune
complex formation or of binding to a Toll-like receptor when the
binding with the compound is reduced compared to binding without
the compound.
14. The method of claim 13, wherein the TLR is TLR9.
15. The method of claim 13, wherein the binding with the compound
is reduced at least 50 percent compared to binding without the
compound.
16. A method for screening for compounds that inhibit immune
complex formation or binding to a Toll-like receptor, comprising
contacting immune complex components with a compound being screened
and with a dendritic cell that expresses a TLR chosen from
Toll-like receptor 3 (TLR3) and Toll-like receptor 9 (TLR9);
measuring activation of the dendritic cell; and identifying the
compound being screened as an inhibitor of immune complex formation
or of binding to a Toll-like receptor when activation of the
dendritic cell with the compound is reduced compared to activation
of the dendritic cell without the compound.
17. The method of claim 16, wherein the TLR is TLR9.
18. The method of claim 16, wherein the measuring activation of the
dendritic cell comprises measuring production of a cytokine chosen
from TNF-.alpha., interferon-.alpha., and BAFF.
19. The method of claim 16, wherein the measuring activation of the
dendritic cell comprises measuring upregulated expression on the
dendritic cell of a costimulatory molecule chosen from CD80, CD86,
and MHC class II.
20. The method of claim 16, wherein the activation of the dendritic
cell with the compound is reduced at least 50 percent compared to
activation of the dendritic cell without the compound.
21. The method of claim 16, wherein the immune complex components
comprise an autoantibody and an autoantigen.
22. A method for screening for compounds that inhibit immune
complex formation or binding to a Toll-like receptor, comprising
contacting immune complex components with a compound being screened
and with a B cell that expresses a TLR chosen from Toll-like
receptor 3 (TLR3) and Toll-like receptor 9 (TLR9); measuring
activation and/or proliferation of the B cell; and identifying the
compound being screened as an inhibitor of immune complex formation
or of binding to a Toll-like receptor when activation and/or
proliferation of the B cell with the compound is reduced compared
to activation and/or proliferation of the B cell without the
compound.
23. The method of claim 22, wherein the TLR is TLR9.
24. The method of claim 22, wherein the measuring activation of the
B cell comprises measuring upregulated expression on the B cell of
a costimulatory molecule chosen from CD80, CD86, and MHC class
II.
25. The method of claim 22, wherein the activation and/or
proliferation of the B cell with the compound is reduced at least
50 percent compared to activation and/or proliferation of the B
cell without the compound.
26. The method of claim 22, wherein the immune complex components
comprise an autoantibody and an autoantigen.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/487,885, having a 371(c) date of Sep. 27,
2004, which is a national stage of PCT/US02/28708, filed Sep. 9,
2002, which claims benefit of U.S. Patent Application No.
60/367,578, filed Mar. 26, 2002, and U.S. Patent Application No.
60/318,096, filed Sep. 7, 2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods and compositions
for treating immune complex associated diseases, preferably
systemic lupus erythematosus (SLE), and other systemic autoimmune
diseases associated with the subject having aberrant Toll-like
receptor (TLR)/B cell receptor (BCR) dual engagement (in B cells)
or TLR/Fc gamma receptor dual engagement (in dendritic cells and/or
macrophages).
[0005] 2. Background
[0006] Autoimmune diseases are a fairly common but poorly
understood group of diseases in which an individual's immune system
either 1) begins recognizing self antigens as foreign and starts
destroying tissues expressing such antigens thereby causing a
disease, or 2) forms immune complexes with these antigens which
then deposit in tissues and cause inflammatory pathology. Such
autoimmune diseases include, for example, diabetes wherein the
immune system turns against and destroys insulin producing
pancreatic islet cells; multiple sclerosis, wherein the target
antigen is the myelin sheath protecting neurons leading to
destruction of function of motor neurons; psoriasis, where the
target of the immune system is skin; rheumatoid arthritis, where
the target organ is cartilage; and systemic lupus erythematosus
(SLE), which presents itself as targeting a variety of tissues with
no apparent specificity or selectivity although the target antigens
themselves are extremely consistent and characteristic. Because the
mechanisms leading to the development of autoimmune diseases in
general are mostly unknown, their treatment is often directed to
generally suppressing the immune system. Such general
immunosuppressive therapies often cause a variety of undesirable
side effects including cancer, infertility, and increased
susceptibility to infections by viruses, fungi, yeast, and
bacteria. Therefore, it would be desirable to understand the
mechanisms that cause the immune system to turn against self
antigens to enable development of more specific therapies for the
treatment of the autoimmune diseases.
[0007] An example of a poorly understood autoimmune disease is
systemic lupus erythematosus (SLE), commonly known as Lupus. SLE is
characterized by dysregulation of the immune system resulting in
the production of antinuclear antibodies, the generation of
circulating immune complexes, and the activation of the complement
system. The immune complexes build up in the tissues and joints
causing inflammation, and degradation to both joints and tissues.
While the word "systemic" correctly suggests that the disease
affects the entire body and most organ systems, the disease most
often involves inflammation and consequent injury to the joints,
skin, kidney, brain, the membranes in body cavities, lung, heart,
and gastrointestinal tract. An individual with SLE often
experiences unpredictable acute episodes or "outbreaks" and equally
unexpected remissions. The pathologic hallmark of the disease is
recurrent, widespread, and diverse vascular lesions resembling a
rash or changes on the surface of the skin.
[0008] The prevalence of SLE in the United States is an issue of
some debate. Estimates of prevalence range from 250,000 to
2,000,000 persons. Although reported in both the extremely old and
the extremely young, the disease mainly affects women of
childbearing age. Among children SLE is three times more common in
females than in males. In the 60% of SLE patients who experience
the onset of this disease between puberty and the fourth decade of
life, the female to male ratio is 9:1. Thereafter, the female
preponderance again falls to that observed in prepubescent children
(i.e. 3:1). In addition, the disorder appears to be three times
more common in persons of African and Asian descent than in persons
of Caucasian descent.
[0009] The etiology of SLE remains unknown. A genetic
predisposition, the systemic proliferation of sex hormones, and
various environmental triggers, such as viral infections have been
suggested to play a role in triggering the aberrant immune
responses that typify the disease. A role for genetics is suggested
by the increased percentage of two histocompatibility antigens,
HLA-DR2 and HLA-DR3, in patients with SLE. In addition, there is an
increased frequency of the extended haplotypes HLA-A1, B8, and DR3
in affected individuals. The role of heredity is further supported
by the concordance for this illness among monozygotic twins. The
polygenic nature, however, of this genetic predisposition as well
as the contribution of environmental factors is suggested by the
concordance rate, which is only moderate and reported to be between
25% and 60%.
[0010] The precise initiating etiology of SLE is unknown. However,
it is generally accepted that most of the clinical manifestations
of the disease are caused either directly or indirectly by
autoantibody production and the subsequent formation of pathogenic
immune complexes. These autoantibodies, which are produced by
dysregulated B lymphocytes, have distinct specificities recognizing
discrete nuclear autoantigens including, among others, DNA,
nucleosomes and subnucleosomes. Certain RNA/protein complexes
including the Sm antigen and small nuclear ribonucleoproteins
(snRNP) are additional characteristic autoantigenic specificities.
The pathogenic immune complexes are formed by binding of the
autoantibodies to their respective nuclear autoantigens.
[0011] Autoantibodies in SLE often circulate as immune complexes
(IC) bound with their respective autoantigens. Chromatin or
chromatin fragments such as DNA, nucleosomes or subnucleosome
particles are especially common autoantigenic specificities in both
mice and humans (Tan, E. Adv. Immunol. 44, 93-151 (1989); Monestier
and Novick, Mol. Immunol. 33: 89-99., 1996)
[0012] The central goals in the treatment of SLE, therefore, are
either to attempt to suppress the dysfunctional B lymphocytes
thereby decreasing the production of autoantibody or, to attempt to
diminish the pathogenicity of the immune complexes once they have
formed. At present these goals can only be achieved, and often
incompletely so, by the use of intensive systemic immunosuppressive
drug therapy using drugs such as cortisone, azathioprine,
hydroxychloroquine and cyclophosphamide. These therapies are
associated with many serious and undesirable side-effects including
infections, infertility, retinopathy and cancer. Therefore, new
treatments for SLE, and other autoimmune diseases, would be
desirable.
SUMMARY OF THE INVENTION
[0013] It is therefore the purpose of the present invention to
provide methods and compositions for treating immune complex
associated diseases (ICAD), such as SLE, rheumatoid arthritis, and
hepatitis-C related immune complex disease (e.g., cryoglobulinemia)
in a subject having an ICAD or at risk for developing ICAD.
[0014] We have discovered that chromatin-containing immune
complexes activate autoreactive B cells and dendritic cells by a
dual receptor engagement process. In both cell types a Toll-like
receptor (TLR) is involved. TLR9, located in a cytoplasmic
compartment, is the essential second receptor required for cell
activation. In the case of the B cell, the B cell antigen receptor
located on the cell surface is the essential first receptor
required for cell activation. In the case of the dendritic cell, a
stimulatory Fc gamma receptor located on the cell surface is the
essential first receptor required for cell activation. We have
found a method of treating ICAD by administering a compound to an
individual in need thereof that either 1) inhibits formation of the
immune complex (i.e., autoantibody and nuclear autoantigen) either
by preventing formation and/or binding to the Toll-like receptor
(TLR), or 2) interferes with binding of an autoantigen-containing
immune complex (or the antigenic component thereof) to the TLR, or
3) inhibits signaling pathways initiated by dual engagement of BCR
and TLR (in B cells) or FcR and TLR (in dendritic cells) via immune
complexed or uncomplexed autoantigens. The compound is administered
in a pharmaceutically acceptable carrier.
[0015] Preferably, the ICAD is SLE, rheumatoid arthritis or
hepatitis-C related immune complex disease (e.g.,
cryoglobulinemia). In an other embodiment, the ICAD is related to
an immune reaction in a host after organ transplantation.
[0016] Although not working to be bound by theory, we believe that
immune complexes (IC) containing an autoantigen, such as chromatin,
but not IC containing a foreign antigen, are able to activate
autoreactive B cells and that this activation is absolutely
dependent on the ability of the autoantigen-containing IC to
sequentially engage either the B cell receptor (BCR) in B cells or
Fc.gamma.R in dendritic cells, and a second receptor, a Toll-like
receptor. This finding establishes a novel link between the innate
and adaptive immune systems and suggests a general mechanism
whereby autoreactive B cells or dendritic cells specific for
protein/nucleic acid autoantigens are activated.
[0017] According to one aspect of the invention a method is
provided for treating a patient having an ICAD or at risk for an
ICAD by identifying an individual with ICAD and administering an
effective amount of a compound capable of inhibiting the
autoantigen or autoantigen/immune complex from forming and/or from
activating B cells or dendritic cells.
[0018] A person at risk for ICAD or systemic autoimmune disease
includes individuals having at least a parent, grandparent or
sibling who has an ICAD or systemic autoimmune disease.
[0019] The compound is selected from a group consisting of
compounds that bind components of the immune complex and either
prevent its formation or prevent the autoantigen from activating a
Toll-like receptor (TLR). Such compounds include Toll-like receptor
decoys, compounds that inhibit the activity of MyD88, compounds
that inhibit production of immune complex components (e.g.,
antisense nucleotides), dominant-negative TLR, a Toll-like receptor
antagonist, and compounds that inhibit signaling pathways activated
by the interaction or binding of the immune complex or autoantigen
to the TLR. Preferably, the compound binds and/or inhibits function
of Toll-like receptors TLR2, TLR3, and TLR9 or functional domains
thereof. More preferably, the TLR is TLR3 or TLR9 or a functional
fragment thereof.
[0020] Compounds that bind components of the immune complex and
prevent its formation or prevent binding of the complex to the
Toll-like receptor and compounds that inhibit MyD88 signaling
include polyclonal and monoclonal antibodies, dominant negative
proteins that can block wildtype TLR activity or block components
of the TLR-mediated signaling cascade, inhibitory
oligodeoxynucleotides (ODN), such as S-ODN 2088 (Lenart, et al.,
Antisense Nucleic Acid Drug Dev. 4, 247-256 (2001)), antisense
nucleotides including RNA and modified nucleotides, or other
pathway specific kinase inhibitors. The compound is a compound
other than chloroquine.
[0021] In one embodiment the invention provides methods for
screening compounds or agents that inhibit immune complex formation
or binding to the Toll-like receptor and/or inhibit B
cell/dendritic cell activation. The methods comprise contacting
immune complex components with a test agent and measuring B cell or
dendritic cell activation and/or proliferation and/or binding of
the complex to the Toll-like receptor.
[0022] In an other embodiment, a method of diagnosing an ICAD is
provided. The method comprises taking a biological sample
comprising IgG of an individual suspected of having ICAD,
incubating the biological sample together with RF+ B cells or
dendritic cells, and measuring the activation of the RF+ B cells or
dendritic cells, wherein a change in activity in the RF+ B cell or
dendritic cell cultures exposed to the biological sample from the
individual suspected of having ICAD relative to the RF+ B cells or
dendritic cells exposed to a biological sample comprising IgG from
a control individual is indicative of ICAD. Preferably, the change
is an increase in activity. Activation of the B cells can be
measured, for example by measuring proliferation or upregulation of
co-stimulatory molecules such as CD80 and CD86 as well as
upregulation of MHC class II molecules. Activation of dendritic
cells can be assessed by measuring the expression of co-stimulatory
molecules, production of cytokines, e.g., TNF-.alpha., or changes
in the dendritic cell phenotype.
[0023] In yet another embodiment, the invention provides an in vivo
model system for evaluating a compound or an agent for its efficacy
in treating ICAD. The model system comprises administering a test
agent to an ICAD model animal including mouse and rat models, and
measuring B cell or dendritic cell activation and/or proliferation
and/or binding of the complex to the Toll-like receptor in such
animal, wherein decreased activation is indicative of an agent
which is capable of treating ICAD. Alternatively, one can use cell
lines expressing Toll-like receptors and screen for compounds that
modulate, preferably inhibit or block, such receptors.
[0024] Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention and, together with the description, serve to explain
and illustrate the objects, advantages, and principles of the
invention. In the drawings,
[0026] FIG. 1 shows that anti-nucleosome antibody (PL2-3)
stimulation of AM14 RF+ B cells is DNase sensitive. The IgG2a
anti-nucleosome antibody binds to chromatin released from the B
cells in culture to form an immune complex which is recognized by
the B cell via it's IgG2a-specific antigen receptor. AM14 RF+
spleen cells were pre-incubated with various concentrations of
DNase I (0, 5, 15, or 50 .mu.g/ml) for 15 minutes prior to the
addition of goat anti-mouse IgM F(ab').sub.2, PL2-3 (IgG2a.sup.j
anti-nucleosome mAb), or LPS. Results are expressed as the
percentage of the maximum response to each ligand in the absence of
DNase.
[0027] FIGS. 2A-2B show that anti-TNP/TNP-BSA IC fail to
efficiently stimulate AM14 RF+ B cell proliferation. The anti-TNP
mAbs Hy1.2 (IgG2a.sup.a) and C4010 (IgG2a.sup.b) were mixed with
varying concentrations of TNP-BSA [50 .mu.g/ml (circle), 12.5
.mu.g/ml (square), 3.1 .mu.g/ml (up-triangle)] to form IC with
antibody/protein ratios of 1:1, 4:1, and 16:1 respectively. In FIG.
2A, IC formation was confirmed by a shift in Clq binding activity
relative to uncomplexed antibodies (down-triangle). In FIG. 2B the
ability of the anti-TNP/TNP-BSA IC described above to stimulate RF+
B cell proliferation was compared to the stimulation induced by the
anti-nucleosome mAb PR1-3, uncomplexed Hy1.2, C4010, or 50 .mu.g/ml
TNP-BSA.
[0028] FIG. 3 shows that autoantibody/autoantigen-IC stimulation of
RF+ B cells is not complement receptor dependent. Spleen cells from
each of two WT control mice (gray), RF+CR+/+ mice (white), and
RF+CR-/- mice (hatched) were stimulated with goat anti-mouse IgM
F(ab').sub.2, CpG S-ODN 1826, the anti-nucleosome mAbs PR1-3
(IgG2a.sup.j), PL2-3 (IgG2a.sup.j), PL2-8 (IgG2b), or 3% serum from
representative autoimmune mice (lpr/gld and gld).
[0029] FIGS. 4A-4C show that autoAb/autoAg-IC stimulation of RF+ B
cells is MyD88-dependent. In FIG. 4A, T-depleted spleen cells from
wildtype (WT) and RF+ MyD88-/- mice were stained with B220 and an
idiotype specific antibody, 4G7. In FIG. 4B, MyD88 WT or knock-out
mice were identified by PCR. In FIG. 4C spleen cells from each of
two WT (gray), RF+ MyD88+/+ (white), RF+ MyD88-/- (hatched) mice
were stimulated with anti-IgM F(ab').sub.2, CpG S-ODN 1826, LPS,
anti-nucleosome mAbs PR1-3, PL2-3, PL2-8, or 3% lpr/gld serum.
Total cpm for the anti-IgM stimulated cultures were 83,414/39,049
(WT); 93,126/61,315 (RF+ MyD88+/+); and 39,826/57,484 (RF+
MyD88-/-). Results are expressed as the percentage of the anti-IgM
response and are representative of four separate experiments.
[0030] FIGS. 5A-5C demonstrate that autoAb/autoAg-IC stimulation of
RF+ B cells can be blocked by inhibitors of the TLR9 signaling
pathway. In FIG. 5A, RF+ B cells were preincubated for 15 minutes
with 1 or 2 .mu.g/ml chloroquine (a), preincubated for 2 hrs with
concanamycin B (b), or preincubated for 30 minutes with inhibitory
CpG S-ODN 2088 (c), prior to the addition of the stimulatory
ligands anti-IgM F(ab').sub.2, 5-50 .mu.g/ml PL2-3, 0.3-2.0
.mu.g/ml CpG S-ODN 1826, LPS, lipopeptide, or porin B. In FIG. 5B,
RF+ B cells were preincubated for 30 min with 12 .mu.g/ml CpG S-ODN
2088 prior to the addition of stimulatory ligands. Results are
expressed as a percentage of the anti-IgM response in the absence
of inhibitor. The data in (FIG. 5A) and (FIG. 5C) is representative
of 2-4 experiments, while (FIG. 5B) is the mean of two
experiments.
[0031] FIGS. 6A-B show the results of an experiment where
MyD88-deficient (MyD88-/-) Fas-sufficient mice were bred with
MyD88-sufficient (MyD88+/+) Fas-deficient (lprlpr) autoimmune mice
to generate MyD88-/- lpr/lpr mice, and appropriate control groups.
Anti-nuclear-antibodies (ANA) in the sera of age-matched MyD88+/+
lprlpr (FIG. 6A) and MyD88-/- lpr/lpr (FIG. 6B) littermates (12-13
weeks of age) were detected by indirect immunofluorescence using
HEp-2 cells as substrate as previously described (Rifkin et al.,
Journal of Immunology 161: 5164-5170, 1998). Serum from a diseased
autoimmune MRL-lpr/lpr mouse served as the positive control and
serum from a non-autoimmune BALB/c mouse as the negative control.
This in vivo experiment demonstrates that, in a standard and widely
used lupus mouse strain, autoantibody production requires signaling
through MyD88. This strongly suggests that TLR activation is
required for the development of lupus in this model.
[0032] FIGS. 7A-7B show that B cells can be activated through TLR9
and that this activation can be blocked by TLR9 inhibition with ODN
2088. B cells were purified from the spleen of MRL+/+ mice and
either pre-incubated, or not pre-incubated, for 30 minutes with
varying doses of ODN 2088, an inhibitory oligodeoxynucleotide that
specifically blocks signaling through TLR9. Anti-IgM F(ab').sub.2
(Anti-IgM), that activates through the B cell receptor (7A), or the
stimulatory ODN 1826, that specifically activates through TLR9
(7B), was then added to the cultures. After 20-24 hours, .sup.3[H]
thymidine was added to the cultures and, after an additional 14-18
hours, proliferation was determined by measuring thymidine
incorporation using an LKB Wallac 1212 Rackbeta counter. Only
stimulation through TLR9 is blocked by ODN 2088 demonstrating the
specificity of the inhibitory effect: stimulation induced by B cell
receptor cross-linking is not inhibited.
[0033] FIGS. 8A-8C show that dendritic cells can be specifically
activated through TLR9 and this activation can be blocked by TLR9
inhibition with ODN 2088.
[0034] FIG. 9 shows the inhibitory effects of ODN 2088 on
chromatin-containing immune complex mediated B cell proliferation
are long-lasting. B cells were purified from the spleen of MRL+/+
mice and either pre-incubated, or not pre-incubated, for 16-20
hours with 12 .mu.g/ml ODN 2088. After this pre-incubation, the
cells were washed to remove ODN 2088 from the cultures and then the
B cells were cultured with either medium alone or with fresh ODN
2088 at 12 .mu.g/ml. Thirty minutes later the anti-chromatin
monoclonal antibody PL2-3 (50 .mu.g/ml) or LPS (10 .mu.g/ml) were
added to the cultures. After 20-24 hours, .sup.3[H] thymidine was
added to the cultures and, after an additional 14-18 hours,
proliferation was determined by measuring thymidine incorporation
using an LKB Wallac 1212 Rackbeta counter.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention provides methods and compositions for
treating and/or preventing immune complex associated diseases
(ICAD), in a subject having an ICAD or at risk for the ICAD.
[0036] For the purposes of this invention the term "immune complex
associated diseases" or "ICAD" refers to diseases including, but
not limited to systemic lupus erythematosus (SLE) and related
connective tissue diseases, rheumatoid arthritis, hepatitis-C and
hepatitis B related immune complex disease (e.g.,
cryoglobulinemia), Behcets disease, autoimmune
glomerulonephritides, and vasculopathy associated with the presence
of LDL/anti-LDL immune complexes.
[0037] We have discovered that immune complexes (IC) containing an
autoantigen, such as chromatin, but not IC containing a foreign
antigen, are able to activate autoreactive B cells and that this
activation is dependent on the ability of the
autoantigen-containing IC to sequentially engage both the B cell
receptor and a second receptor, a Toll-like receptor. This finding
establishes a novel link between the innate and adaptive immune
systems and consequently a general mechanism whereby autoreactive B
cells specific for protein/nucleic acid autoantigens are
activated.
[0038] The Toll-like receptors (TLRs) are a family of membrane
bound receptor proteins that are critically involved in innate
immune responses and recognize pathogen associated molecular
patterns or determinants that appear unique to microorganisms and
are involved in activating immune cells against the source of these
microbial particles.
[0039] The Toll-like family of receptors have leucine-rich repeats
in their extracellular domains and the Toll/IL-1 receptor homology
domain in their cytoplasmic domains. The Toll family is remarkably
conserved in evolution. The first member discovered was the product
of the toll gene which is part of the signaling pathway responsible
for the specification of dorsal-ventral polarity in the early
development of the fruit fly Drosophila melanogaster.
[0040] Currently ten mammalian homologues have been identified,
called TLR1 through TLR10. Both the IL-1 receptor and the TLRs
share similar downstream effects, such as the activation of immune
response genes, and all these receptors work through signaling
cascades that include the adaptor protein MyD88 (Mussio et al.,
Science 278:1612; Wesche et al., Immunity 7:837) which has been
previously described as a myeloid differentiation protein (Lord et
al., Oncogene 5:1095). In general, the different TLRs are thought
to be activated by different types of microbial particles (Hemmi et
al., Nature 408:740-745 (2000); Underhill et al., Nature 402:39-43
(1999); Aliprantis et al., EMBO J., 19:3325-3336 (2000)). However,
there is also accumulating evidence that in addition to microbial
particles, mammalian TLRs can also recognize certain self
(mammalian) antigens, in particular cytoplasmic components that are
released from cells as a result of cell death (Akira et al., Nat.
Immunol., 2: 675-680 (2000).
[0041] The term "Toll-like receptor" is herein meant to include an
intact Toll-like receptor, for example a receptor that has been
described in the Online Mendelian Inheritance in Man under access
numbers 601194 TOLL-LIKE RECEPTOR 1, TLR1; 603028 TOLL-LIKE
RECEPTOR 2, TLR2; 603029 TOLL-LIKE RECEPTOR 3, TLR3; 603030
TOLL-LIKE RECEPTOR 4, TLR4; 603031 TOLL-LIKE RECEPTOR 5, TLR5;
605403 TOLL-LIKE RECEPTOR 6, TLR6; 300365 TOLL-LIKE RECEPTOR 7,
TLR7; 300366 TOLL-LIKE RECEPTOR 8, TLR8; 605474 TOLL-LIKE RECEPTOR
9, TLR9; and 606270 TOLL-LIKE RECEPTOR 10, TLR10 or a functional
fragment thereof such as, for example, a soluble form of the
Toll-like receptor, i.e. where the membrane binding domain has been
deleted or altered, in some embodiments the cytoplasmic domain is
also not present, or a MyD88 binding or interacting fragment of the
Toll-like receptor or a homolog of the Toll-like receptor capable
of binding to or interacting with MyD88. More preferably the TLR is
TLR9.
[0042] Our experiments to this point have identified the importance
of the interaction between TLR9 and the chromatin component of the
chromatin-containing immune complexes. However, in SLE and related
diseases immune complexes commonly form with autoantibodies and
distinct RNA/protein antigenic complexes including the Sm antigen
and small nuclear ribonucleoproteins (snRNP) (Tan, E. Adv. Immunol.
44, 93-151 (1989)). In addition, in immune complex associated
diseases such as hepatitis C, pathogenic immune complexes form
between antibodies and the RNA containing viruses. TLR3 has been
identified as a specific receptor for double-stranded RNA
(Alexopoulou, Nature 413, 732-738 (2001). Thus, it is reasonable to
anticipate that TLR3 engagement will be important in the activation
process elicited by these RNA-containing immune complexes.
[0043] Preferably the TLR is TLR3 or TLR 9, or a functional
fragment thereof or a fragment that is homologous to TLR 3 or TLR 9
and capable of binding or interacting with MyD88.
[0044] The Toll-like receptor useful according to the present
invention can also be a fusion receptor wherein the Toll-like
receptor is fused with another protein such as a Myc-tag. The
preferred Toll-like receptors include Toll-like receptor-2 or
Toll/Interleukin-1 receptor-like 4 (Chaudhary, et al., Blood 91:
4020-4027, 1998), Toll-like receptor-3 (Rock, et al., Proc. Nat.
Acad. Sci. 95: 588-593, 1998), and Toll-like receptor-9 (Chuang and
Ulevitch, Europ. Cytokine Netw. 11: 372-378, 2000; Du, et al.,
Europ. Cytokine Netw. 11: 362-371, 2000) or functional fragments
thereof or homologs that have a similar function, such as for
example, binding MyD88.
[0045] As used herein, the terms "treatment" or "treating" include:
(1) preventing such disease from occurring in a subject who may be
predisposed to these diseases but who has not yet been diagnosed as
having them; (2) inhibiting these diseases, i.e., arresting their
development; or (3) ameliorating or relieving the symptoms of these
diseases, i.e., causing regression of the disease states.
[0046] The compounds preferably inhibit activation of B cells (BC)
or dendritic cells (DC) by at least about 50% in an in vitro or in
vivo assays discussed below. More preferably the compounds inhibit
autoantigenic activation of BCs or DCs via the Toll-like receptor
by 75%, most preferably 95%. Additional compounds are identified
and tested in the screening assays discussed in more detail
below.
[0047] The compound useful according to the present invention is
selected from a group consisting of compounds that bind components
of the immune complex or Toll-like receptors. These compounds
either prevent formation of the immune complex; prevent the
autoantigen or the autoantigenic fragment of the immune complex
from activating a Toll-like receptor (TLR) or prevent the
downstream molecular signaling, such as that mediated through
MyD88, from the Toll-like receptor. Such compounds include
Toll-like receptor decoys, compounds that inhibit the activity of
MyD88, compounds that inhibit production of immune complex
components (e.g., antisense nucleotides)), dominant-negative TLR, a
Toll-like receptor antagonist or blocker (such as ODN 2088 or
antibodies against the TLRs), and compounds that inhibit signaling
pathways activated by the interaction or binding of the
autoantigenic fragment of the immune complex to the TLR.
Preferably, the compound binds and/or inhibits function of
Toll-like receptors TLR2, TLR3, and TLR9 or functional domains
thereof. More preferably, the TLR is TLR3 or TLR9 or a functional
fragment thereof.
[0048] The invention further provides efficient screening methods
to identify pharmacological agents or lead compounds for agents
which inhibit binding of the immune complex (or the autoantigenic
component thereof) to the TLR, preferably TLR2, TLR3, TLR9, or any
combination thereof, most preferably TLR3 or TLR9. The methods are
amenable to automated, cost-effective high throughput drug
screening and have immediate application in a broad range of
pharmaceutical drug development programs.
[0049] Generally, these screening methods involve assaying for
compounds that modulate, for example, the autoantigen interaction
with a TLR, preferably TLR2, TLR3, TLR9, or any combination
thereof, most preferably TLR3 or TLR 9. Still, more preferably the
compound modulates interaction with TLR 9. A wide variety of assays
for binding agents are provided including labeled in vitro
protein-protein binding assays, immunoassays, cell based assays,
etc.
[0050] In vitro binding assays employ a mixture of components
including a TLR polypeptide, preferably TLR2, TLR3, TLR9, or any
combination thereof, most preferably TLR3 or TLR9, still more
preferably TLR9 which may be part of a fusion product with another
peptide or polypeptide, e.g. a tag for detection or anchoring, etc.
The assay mixtures may also comprise a natural intracellular TLR
binding target, such as MyD88. While native full-length binding
targets may be used, it is frequently preferred to use portions
(e.g. peptides) thereof so long as the portion provides binding
affinity and avidity to the subject MyD88 polypeptide conveniently
measurable in the assay. The assay mixture also comprises a
candidate pharmacological agent. Candidate agents encompass
numerous chemical classes, though typically they are organic
compounds; preferably small organic compounds and are obtained from
a wide variety of sources including libraries of synthetic or
natural compounds. A variety of other reagents may also be included
in the mixture. These include reagents like salts, buffers, neutral
proteins, e.g. albumin, detergents, protease inhibitors, nuclease
inhibitors, antimicrobial agents, etc. may be used.
[0051] The resultant mixture is incubated under conditions whereby,
but for the presence of the candidate pharmacological agent, the
TLR polypeptide specifically binds the cellular binding target,
portion or analog with a reference binding affinity. The mixture
components can be added in any order that provides for the
requisite bindings and incubations may be performed at any
temperature that facilitates optimal binding. Incubation periods
are likewise selected for optimal binding but also minimized to
facilitate rapid, high-throughput screening.
[0052] After incubation, the agent-biased binding between the TLR
polypeptide and one or more binding targets is detected by any
convenient way. A difference in the binding affinity of the TLR
polypeptide to the target in the absence of the agent as compared
with the binding affinity in the presence of the agent indicates
that the agent modulates the binding of the TLR polypeptide to the
TLR binding target. Analogously, in the cell-based assay also
described below, a difference in TLR-dependent transcriptional
activation in the presence and absence of an agent indicates the
agent modulates TLR function, for example, binding to MyD88. A
difference, as used herein, is statistically significant and
preferably represents at least a 50%, more preferably at least a
75%, still more preferably at least a 90% difference.
[0053] The preferred assay to test a pharmaceutical agent for
potency of interrupting the activation of B cells or dendritic
cells comprises RF+ B cells or dendritic cells which are incubated
with a test agent and a TLR stimulatory agent, such as PR1-3,
PL2-3, or a CpG S-ODN 1826 (see, e.g. Leadbetter et al., Nature,
416:603-607, 2002) in a suitable cell culture medium. Incubation
with the test agent can be performed prior to or simultaneously
with the addition of the stimulatory agent.
[0054] The activation of the B cells can be observed using a number
of techniques well known to one skilled in the art. For example,
the .sup.3[H] thymidine incorporation assay can be performed to
measure proliferation of B cells. Alternatively, activation of B
cells can be analyzed using fluorescence activated cell sorting
(FACS) to measure cell surface expression of, for example, CD80,
CD86 or MHC class II molecules. Activation of dendritic cells can
be observed by measuring production of cytokines, such as
TNF-.alpha., interferon-.alpha., IL-12 and BAFF, or co-stimulatory
molecules such as CD80, CD86 or MHC class II molecules (Banchereau,
et al., Annu. Rev. Immunol. 18: 767-811, 2000; Mackay and Browning,
Nat. Rev. Immunol. 2: 465-475).
[0055] Alternatively, dendritic cell activation can be observed by
measuring morphologic changes in the DC (Banchereau, et al., Annu.
Rev. Immunol. 18: 767-811, 2000).
[0056] If the stimulation of the expression of, for example a
co-stimulatory molecule is decreased as a result of adding the test
agent, the test agent is considered to be a potential agent to
treat ICAD. The expression is considered decreased if it is at
least about 50%, more preferably at least about 60-75%, most
preferably at least about 90% decreased as compared to a cell
sample where no test agent has been added. More detailed examples
are given in the Examples in the end of this section.
[0057] A preferred assay mixture of the invention is set forth in
the Examples. An assay mixture of the invention also comprises a
candidate pharmacological agent. Generally, a plurality of assay
mixtures are run in parallel with different candidate agent
concentrations to obtain a differential response to the various
concentrations. Typically, one of these assay mixtures serves as a
negative control, i.e. at zero concentration or below the limits of
assay detection. Candidate agents encompass numerous chemical
classes, though typically they are organic compounds and preferably
small organic compounds. Small organic compounds suitably may have
e.g. a molecular weight of more than about 50 yet less than about
2,500. Candidate agents may comprise functional chemical groups
that interact with proteins and/or DNA.
[0058] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and peptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Libraries can be composed of small polypeptides
(see, for instance, Lam et al., Nature, 354: 82, 1991 and WO
92/00091; Geysen et al., J Immunol Meth, 102: 259, 1987: Houghten
et al., Nature, 354: 84, 1991 and WO 92/09300 and Lebl et al., Int
J Pept Prot Res, 41, 201, 1993). Alternatively, libraries of small
non-peptide molecules can be based upon a common template or core
structure (see, for instance, Ellman and Bunin, J Amer Chem Soc,
114:10997, 1992 for benzodiazepine template; WO 95/32184 for
oxazolone and aminidine template; WO 95/30642 for dihydrobenzopyran
template and WO 95/35278 for pyrrolidine template.
[0059] Additionally, natural and synthetically produced libraries
and compounds are readily modified through conventional chemical,
physical, and biochemical means. In addition, known pharmacological
agents may be subject to directed or random chemical modifications,
such as acylation, alkylation, esterification, amidification,
etc.
[0060] Antibodies and binding fragments thereof or aptamers
(available from, for example SomaLogic Inc., Boulder, Colo.) that
bind immune complex components and prevent formation or TLR binding
are also useful. Examples of antibodies useful according to the
invention include monoclonal antibodies against TLRs such as
anti-TLR3 monoclonal antibody (Matsumoto et al., Biochem. Biophys.
Res. Commun., 293:1364-1369, 2002) anti-TLR-9 (Toll-like receptor
9, Imgenex, San Diego, Calif.) or humanized versions thereof.
[0061] The term dominant negative Toll-like receptor as used herein
refers to Toll-like receptors, that have been engineered to have a
defect, such as deletion of the domain required for downstream
signaling but which can bind the antigenic portion of the immune
complex.
[0062] The antibodies and binding fragments thereof can be either
polyclonal or monoclonal, but preferably are monoclonal. If
polyclonal, the antibodies can be in the form of antiserum or
monospecific antibodies, such as purified antiserum which has been
produced by immunizing animals with purified protein. Preferably,
however, the antibodies are monoclonal antibodies so as to minimize
the administration of extraneous proteins to an individual.
Monoclonal antibodies can be prepared according to well known
protocols. See. e.g., Skare et al., J. Biol. Chem. 268: 16302-16308
(1993); and U.S. Pat. Nos. 4,918,163 and 5,057,598, which are
incorporated herein by reference. The antibodies can be whole,
Fab's, single chain, single domain heavy chain, etc. Single chain
antibodies are preferable. Methods for the production of single
chain binding polypeptides are described in detail in, e.g., U.S.
Pat. No. 4,946,778, which is incorporated herein by reference.
[0063] When an antibody or other protein or peptide is used the
peptide is preferably conjugated to a carrier such as biotin or a
poly(alkaline oxide), for example polyethylene glycol (PEG).
Polymeric substances such as dextran, polyvinyl pyrrolidones,
polysaccharides, starches, polyvinyl alcohols, polyacryl amides or
other similar polymers can be used. The poly(alkaline oxide) can
include monomethoxy polyethylene glycol, polypropylene glycol,
block copolymers of polyethylene glycol and the like. Polyethylene
glycol as poly(alkylene oxide) is preferred. The polymers can also
be distally capped with C.sub.1-4 alkyls instead of monomethoxy
groups.
[0064] For administration to humans, e.g., as a component of a
composition for in vivo treatment, the monoclonal antibodies are
preferably substantially human or humanized to minimize
immunogenicity, and are in substantially pure form. By
"substantially human" is meant that the immunoglobulin portion of
the composition generally contains at least about 70% human
antibody sequence, preferably at least about 80% human, and most
preferably at least about 90-95% or more of a human antibody
sequence.
[0065] For therapeutic applications, the compounds may be suitably
administered to a subject such as a mammal, particularly a human,
alone or as part of a pharmaceutical composition, comprising the
compounds together with one or more acceptable carriers thereof and
optionally other therapeutic ingredients. The carrier(s) must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0066] The pharmaceutical compositions of the invention include
those suitable for oral, rectal, nasal, topical (including buccal
and sublingual), vaginal, parenteral (including subcutaneous,
intramuscular, intravenous and intradermal), ocular using eye
drops, transpulmonary using aerosolubilized or nebulized drug
administration. The formulations may conveniently be presented in
unit dosage form, e.g., tablets and sustained release capsules, and
in liposomes, and may be prepared by any methods well know in the
art of pharmacy. (See, for example, Remington: The Science and
Practice of Pharmacy by Alfonso R. Gennaro (Ed.) 20th edition, Dec.
15, 2000, Lippincott, Williams & Wilkins; ISBN:
0683306472.)
[0067] Such preparative methods include the step of bringing into
association with the molecule to be administered ingredients such
as the carrier which constitutes one or more accessory ingredients.
In general, the compositions are prepared by uniformly and
intimately bringing into association the active ingredients with
liquid carriers, liposomes or finely divided solid carriers or
both, and then if necessary shaping the product.
[0068] Compositions of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or
packed in liposomes and as a bolus, etc.
[0069] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets optionally may
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein.
[0070] Compositions suitable for topical administration include
lozenges comprising the ingredients in a flavored basis, usually
sucrose and acacia or tragacanth; and pastilles comprising the
active ingredient in an inert basis such as gelatin and glycerin,
or sucrose and acacia.
[0071] Compositions suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0072] It will be appreciated that actual preferred amounts of a
given compound used in a given therapy will vary according to the
particular compound being utilized, the particular compositions
formulated, the mode of application, the particular site of
administration, the patient's weight, general health, sex, etc.,
the particular indication being treated, etc. and other such
factors that are recognized by those skilled in the art including
the attendant physician or veterinarian. Optimal administration
rates for a given protocol of administration can be readily
determined by those skilled in the art using conventional dosage
determination tests.
[0073] The present invention also provides methods for diagnosing
ICAD in an individual. The method comprises of measuring the
Toll-like receptor mediated activation of a B cell or dendritic
cell induced by a biological sample containing an immune
complex(es) consisting of autoantigen(s) and autoantibody(ies).
Activation of a Toll-like receptor in B cells can be measured
indirectly, for example by measuring proliferation of the B cells,
expression of various co-stimulatory molecules such as CD80, CD86
or upregulation of MHC class II molecules. Activation of Toll-like
receptor in dendritic cells can be measured by observing
morphologic changes of the dendritic cells either with or without
specific staining, or by measuring expression of co-stimulatory
molecules such as CD80 and CD86, or by measuring upregulation of
activation markers such as CD69, or by measuring upregulation of
chemokine receptors such as CCR7, or by measuring the production of
cytokines such as TNF-.alpha. using a number of different
techniques including assaying mRNA or protein levels.
[0074] For example, change in the expression of TNF-.alpha. can be
measured using RNA isolated from dendritic cells. RNA quantitation
methods include polymerase chain reaction (PCR) based methods such
as, for example, a TaqMan.RTM. system (Applied Biosystems),
Brilliant Quantitative PCR (Stratagene), Platinum.RTM. quantitative
PCR (Resgen, Inc.). RNA is isolated from a biological sample, for
example blood sample, which is taken from an individual suspected
of having ICAD. The amount of Toll-like receptor mRNA is quantified
using, for example, techniques listed above and compared to a
sample from a control individual.
[0075] Preferably, assays such as FACS analysis are used to measure
protein expression of the co-stimulatory molecules. If the
expression of these co-stimulatory molecules is increased, it
indicates that the individual is affected with an ICAD. The
activity is considered increased if the assay shows at least about
5% increase in the amount of the co-stimulatory molecule compared
to the control sample.
[0076] Alternatively, serum from a test individual suspected of
having ICAD can be injected to a cell culture expressing a
functional Toll-like receptor pathway. The activity of the
Toll-like receptor is consequently measured from the cell culture
treated, in parallel, with control and test subject serum. If the
activity of the Toll-like receptor is increased in the cells
treated with the test individual's serum compared to the cells
treated with the control serum, it indicates that the individual is
affected with an ICAD. The expression is considered increased if it
is at least about 5% higher than in the control sample.
[0077] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof that the foregoing description as well as the examples that
follow are intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modification within the
scope of the invention will be apparent to those skilled in the art
to which the invention pertains.
EXAMPLE 1
[0078] Autoreactive B cells are present in the lymphoid tissues of
healthy individuals, but typically remain quiescent. When this
homeostasis is perturbed, the formation of self-reactive antibodies
can have serious pathological consequences. B cells expressing an
antigen-receptor specific for self-IgG make a class of
autoantibodies known as rheumatoid factor (RF). Here we show that
effective activation of RF+ B cells is mediated by IgG2a/chromatin
immune complexes and requires the sequential engagement of the
antigen-receptor and a MyD88-dependent Toll-like receptor family
member. These data establish a critical link between the innate and
adaptive immune systems in the development of systemic autoimmune
disease and explain the preponderance of autoantibodies reactive
with nucleic acid/protein particles. The unique features of this
dual-engagement pathway should facilitate the development of
therapies that specifically target autoreactive B cells.
[0079] We have now developed a model in vitro system for analyzing
the factors involved in the activation of autoreactive B cells and
dendritic cells in autoimmune disease. One of the prevalent
autoantibody specificities in lupus-prone lpr mice is rheumatoid
factor (RF). RFs are antibodies that are reactive with self IgG. RF
B cells become activated, expand in number, and differentiate into
antibody secreting cells in autoimmune strains of mice, but not in
mice of a non-autoimmune background. In order to investigate the
mechanisms involved in this pathologic activation, we studied
primary RF+ B cells from the spleens of mice genetically engineered
to express a pair of transgenes which would confer a particular RF
specific to most B cells. These B cells are prototypes of the
autoreactive B cells found in SLE patients.
[0080] Initially, we demonstrated that these RF+ B cells were
activated by serum samples from autoimmune mice, but not by serum
form non-autoimmune mice (Rifkin et al, 2000). We further proved
that the stimulatory factor in the serum was, in fact, IgG2a.
However, comparable amounts of IgG2a isolated from non-autoimmune
sera were not stimulatory, indicating that not all IgG2a was
sufficient to stimulate RF+ B cells. Therefore we concluded that
IgG2a in the autoimmune sera had unique properties. To further
identify the relevant IgG2a component, we used IgG2a monoclonal
antibodies (mAbs) and showed that mAbs specific for self-antigens,
such as DNA-histone complexes, were stimulatory while antibodies
specific for foreign or non-self antigens were not stimulatory.
[0081] We next considered the possibility that
autoantibody/autoantigen immune complexes were the relevant
component. When the assays with sera and mAbs were run in the
presence of DNAse, stimulation of the RF+ B cells was dramatically
reduced (FIG. 1). These data suggested that cleavage of the DNA
backbone results in the dissociation of the
autoantibody/autoantigen (DNA) immune complex. This strongly
indicates that immune complexes of autoantigens/autoantibodies have
unique properties which preferentially activate autoreactive B
cells. The inability of immune complexes containing non-self
antigen to activate autoreactive B cells is shown in FIG. 2.
[0082] We investigated the possibility that
autoantibody/autoantigen immune complexes are capable of engaging a
second receptor, which then facilitates the activation of the RF B
cells. Since recent studies have shown that a family of proteins
known as Toll-like receptors (TLR) can bind to bacterial DNA and
lead to macrophage, dendritic cell and B cell activation, we
considered the possibility that TLR could play a role in the
activation of these autoreactive RF B cells. TLR are currently a
very popular area of study, and have been shown to be responsible
to binding and recognition of many pathogen-associated molecular
patterns (PAMPS), so this link to innate immune responses would be
a very unique connection for autoimmunity. All known mammalian TLR
signal through the adaptor protein, MyD88. TLR-mediated activation
in mice in which the MyD88 gene has been eliminated by genetic
engineering is severely compromised and signals mediated through
TLR9 are completely abolished. We bred our transgenic RF+ mice onto
a MyD88-/- background. The resulting mice were positive for the RF
transgenes (FIG. 4A) and lacked MyD88 signaling molecules (FIG.
4B).
[0083] We found that RF MyD88-/- mice fail to respond to any of the
autoantibodies which otherwise stimulate proliferation of RF
MyD88+/+ B cells (see FIG. 4C). We tested multiple forms of
autoantibodies: serum, antinucleosome antibodies and anti-Sm
antibodies. This is consistent with the hypothesis that
autoantibody/autoantigen immune complexes contain repeating pattern
components such as DNA or RNA which engage Toll receptors either
inside the B cell (DNA/TLR9) or on the surface of the B cell
(RNA/TLR3). In the case of autoreactive RF+ B cells, the
autoantigen/autoantibody immune complexes likely sequentially
engage the BCR and the TLR, leading to increased activation and
proliferation. This is the first demonstration that co-engagement
of the BCR and TLR can result in the activation of autoreactive B
cells. This connection provides a very unique link between
autoimmunity and the body's innate immune response and may provide
an initiating mechanism for many of the pathogenic responses in
autoimmune diseases such as SLE and rheumatoid arthritis.
[0084] Subsequent studies demonstrated that drugs known to block B
cell activation by stimulatory CpG ODN via TLR9, such as
chloroquine and concanamycin A, completely blocked the ability of
the chromatin containing IC to stimulate RF+ B cells. The response
could also be blocked by inhibitory CpG ODN which specifically
inhibits activation through TLR9. These data demonstrate that TLR9
is a key receptor in the activation of autoreactive B cells. The
data demonstrate that the IgG component of the immune complex binds
to the BCR leading to the internalization of the immune complex and
delivery to an internal vesicular compartment. The subsequent
engagement of TLR9 by the chromatin component of the IC within this
internal compartment leads to B cell activation. Factors that
contribute to the enhanced availability and thus uptake of
chromatin per se (susceptibility factors for SLE) could also lead
to B cell activation through a similar mechanism. It is of note
that anti-chromatin antibodies are the first detectable
autoantibody present in the sera of SLE patients and animals models
of spontaneous SLE.
EXAMPLE 2
[0085] Patients afflicted with SLE and other systemic autoimmune
diseases produce a wide range of autoantibody specificities. Very
frequently these autoantibodies bind to chromatin or other
subcellular nucleic acid/protein particles (Tan, E. Adv. Immunol.
44, 93-151 (1989)). MRL/lpr mice, a well-studied murine model of
systemic lupus erythematosus and rheumatoid arthritis, also produce
exceedingly high titers of IgG anti-IgG rheumatoid factor (RF)
(Theofilopoulos, et al., J. Exp. Med. 162, 1-18 (1985); Wolfowicz,
et al., Clin. Immunol. Immunopath. 46, 382-395 (1988)). This RF
response has provided a highly relevant transgenic model for the
study of autoantibody regulation. B cells from the AM14 transgenic
mouse line express an antigen receptor specific for IgG2a.sup.a/j,
originally captured as a hybridoma product from the spleen of a
diseased MRL/lpr mouse (Shlomchik, et al., Int. Immunol. 5,
1329-1341 (1993)). The AM14 allotype specificity and relatively low
affinity for monomeric IgG2a are typical of the disease associated
RF repertoire (Jacobson, et al., J. Immunol. 152, 4489-99 (1994)).
In wildtype mice, AM14 RF+ B cells develop normally and remain
functionally naive (Hannum, et al., J. Exp. Med. 184, 1269-1278
(1996)). However, on an autoimmune-prone lpr background of the
cognate allotype, they become activated, proliferate and secrete
autoantibody (Wang, and Shlomchik, J. Exp. Med. 190, 639-649
(1999)). A number of factors are likely to contribute to the
activation of AM14 B cells in lpr mice, not the least of which is
the status of the autoantigen, IgG2a.
RF+ B Cells are Activated by Autoantibody/Autoantigen Immune
Complexes
[0086] We have previously shown that AM14 RF+ B cells can be
activated in vitro by IgG2a.sup.a/j isolated from the sera of
autoimmune mice, but not by comparable levels of IgG2a present in
sera obtained from wildtype mice (Rifkin, et al., J. Immunol. 165,
1626-1633 (2000)). RF+ B cells were also found to proliferate
strongly in response to affinity purified IgG2a.sup.j mAbs specific
for nucleosomes, a self-antigen, whereas IgG2a.sup.a/j mAbs
specific for haptens or other foreign antigens produced little, if
any, response (Rifkin, et al., J. Immunol. 165, 1626-1633 (2000)).
These studies suggested that immune complexes (IC) formed between
IgG2a nucleosome-specific mAbs and chromatin fragments released
from co-cultured cells (Emlen, et al., J. Immunol. 148, 3042-3048
(1992)) could effectively activate RF+ B cells, while monomeric
anti-hapten IgG2a antibodies could not. To further test this
premise, DNase was added to the assay medium as a means of
disrupting the putative IC. The vigorous proliferative response
normally elicited by the purified anti-nucleosome mAb PL2-3
(Monestier and Novick, Mol. Immunol. 33, 89-99 (1996)) or by
autoimmune serum was dramatically reduced (FIG. 1). Nonspecific
toxicity of the DNase was not a factor as the responses to
F(ab').sub.2 anti-IgM and LPS were unaffected (FIG. 1). These data
confirm that RF+ B cells respond to IgG2a IC that contain DNA, not
to monomeric IgG2a antibodies alone.
[0087] If RF+ B cell activation by IgG2a IC simply resulted from
more effective crosslinking of the antigen receptor than would be
possible with monomeric IgG2a, then conventional
anti-hapten/hapten-protein complexes should also strongly stimulate
RF+ B cells. To address this issue, ICs were prepared by
pre-incubating IgG2a monoclonal anti-TNP antibodies with varying
concentrations of TNP-BSA; complex formation was confirmed by
demonstrating an increase in Clq binding activity (FIG. 2a). As
expected, IC of C4010, an IgG2a.sup.b anti-TNP mAb, failed to
stimulate RF+ B cells at all anti-TNP/TNP-BSA ratios. Surprisingly,
IC of Hy1.2, an IgG2a.sup.a anti-TNP mAb, only elicited a very
modest proliferative response relative to that elicited by
anti-nucleosome mAb-containing IC. This discrepancy indicated that
the extent of RF+ B cell proliferation elicited by an IC depends on
both the antibody and the nature of the (auto)antigen.
Activation by IC is not Dependent on Complement Receptor
Co-Engagement
[0088] A possible explanation for the dramatic difference in
stimulatory capacity of the anti-nucleosome and anti-TNP IC was
that only the former could synergistically engage both the B cell
receptor (BCR) and a second receptor on the B cell surface. One
potential candidate receptor was the complement receptor CD21, as
complement components have been shown to bind autoantigen IC and
could, in theory, serve to effectively co-engage CD19/CD21 and the
BCR (Carter et al., J. Immunol. 141, 457-463 (1988)). However, when
the AM14 heavy and light chain transgenics were bred onto mice
deficient for CR1/2Cr2 (Ahearn, et al. Immunity 4, 251-262 (1996)),
RF+ B cells from the CR1/2-deficient and CR1/2-sufficient
littermates responded comparably to IgG2a anti-nucleosome mAbs and
autoimmune sera (FIG. 3). B cells from control RF-- littermates
failed to respond to any form of IgG2a, confirming the specific
nature of the ligands used in this study. A mitogenic
hypomethylated CpG oligodeoxynucleotide was included as a control
stimulus. These data demonstrate that complement receptors are not
required for the RF+ B cell response to autoantibody/autoantigen
IC.
Role of a MyD88-Dependent Receptor
[0089] Potential candidate receptors included members of the
Toll-like receptor (TLR) family. These pattern recognition
receptors were first described in Drosophila where they were shown
to trigger the release of anti-fungal peptides (Lemaitre et al.,
Cell 86, 973-983 (1996)). Subsequently identified mammalian
homologues were found to recognize a series of conserved microbial
products (i.e. LPS, microbial lipoproteins, and hypomethylated CpG
DNA) referred to as pathogen associated molecular patterns (PAMPS)
(Medzhitov, et al., Nature 388, 323-324 (1997); Akira, et al., Nat.
Immunol. 2, 675-680 (2001)). Initial studies focused on the effects
of microbial products on cells of the innate immune system where
they were found to stimulate the release of a wide range of
inflammatory mediators (Akira, et al., Nat. Immunol. 2, 675-680
(2001)). However, it was later shown that in addition to exogenous
microbial ligands, TLRs could also recognize endogenous ligands
released from damaged or stressed mammalian cells (Li, et al., J.
Immunol. 166, 7128-7135 (2001)). All known mammalian TLRs signaling
pathways use the adapter protein MyD88 (Akira, et al., Nat.
Immunol. 2, 675-680 (2001)), although in the case of TLR4, an
additional MyD88-independent pathway has been described (Horng, et
al., Nat. Immunol. 2, 835-841 (2001)).
[0090] To investigate the potential role of TLRs in mediating RF+ B
cell responses to autoantibody/autoantigen IC, we crossed the AM14
transgenes onto a MyD88-/- background (Adachi, et al., Immunity 9,
143-150 (1998)). B cells from RF-MyD88+/+ (wildtype), RF+ MyD88+/+,
and RF+ MyD88-/- offspring, identified by a combination of PCR and
FACS analysis (FIGS. 4A-4B), were assessed for their ability to
respond to anti-IgM, CpG ODN, and LPS, as well as to a panel of
anti-nucleosome mAbs and autoimmune sera. B cells from the
MyD88-deficient mice responded normally to anti-IgM, but were
unable to respond to stimulatory CpG DNA (Hacker, et al., J. Exp.
Med. 192, 595-600 (2000)). Their response to LPS was much lower
than in wildtype mice, but still detectable, consistent with
partial activation through MyD88, and responded only weakly to LPS,
which is known to stimulate partially through a MyD88-independent
mechanism (Horng, et al., Nat. Immunol. 2, 835-841 (2001)) (FIG.
4C). Most dramatically, B cells from the RF+ MyD88-deficient mice
were completely unresponsive to the anti-nucleosome mAbs PR1-3 or
PL2-3 or to any of the autoimmune sera that effectively stimulated
RF+ MyD88+/+ B cells (FIG. 4C). These results clearly demonstrate
that autoantibody/autoantigen IC are particularly stimulatory for
RF+ B cells because they synergistically engage both the BCR and a
MyD88-dependent receptor.
[0091] Hypomethylated CpG motifs are a common feature of bacterial
DNA, but they are also present in mammalian DNA promoter elements
(Singal and Ginder Blood 93, 4059-4070 (1999)). Since the B cell
response to CpG oligodeoxyribonucleotides (ODN) is mediated through
TLR9 (Hemmi, et al., Nature 408, 740-745 (2000)), we hypothesized
that chromatin-containing IC could stimulate RF B cells by
co-engaging TLR9. The TLR9-mediated response to CpG S-ODN is
distinguished from other known TLR signaling pathways by its
presumed requirement for endosome acidification and/or maturation
as determined by sensitivity to chloroquine and ammonium chloride
(Yi, et al., J. Immunol. 160, 4755-4761 (1998); Hacker, et al.,
EMBO J. 17, 6230-6240 (1998)). Concanamycin B and bafilomycin A are
specific inhibitors of the V-type ATPase responsible for
acidification of endosomes (Benaroch, et al., EMBO J. 14, 37-49
(1995)). To evaluate the role of TLR9 in the activation of RF+ B
cells, the effect of chloroquine, concanamycin B, bafilomycin A,
and ammonium chloride on the stimulatory capacity of mAb PL2-3 and
known TLR2 (lipopeptide, porin B) (Massari, et al., J. Immunol.
168:1533-1537, 2002), TLR4 (LPS), and TLR9 (CpG S-ODN 1826) ligands
was determined. As expected, chloroquine inhibited the CpG S-ODN
response and these inhibitors had little effect on the response to
the TLR2 and TLR4 ligands. Notably, all four agents that blocked
chloroquine also inhibited the RF+ B cell response to mAb PL2-3
(FIGS. 5A and 5B). The link to chloroquine is intriguing, as
chloroquine is an effective treatment for autoimmune diseases
including rheumatoid arthritis and SLE (Canadian Hydroxychloroquine
Study Group, N. Engl. J. Med. 324, 150-154 (1991); Furst, et al.,
Arth. Rheu. 42, 357-365 (1999)). Our data therefore suggest that
the therapeutic effects of chloroquine are due, at least in part,
to its ability to interfere with TLR-mediated signals that
contribute to autoantibody production or the production of
proinflammatory mediators.
[0092] The B cell response to the stimulatory CpG S-ODN 1826 can
also be blocked by a group of closely related inhibitory CpG S-ODN
such as S-ODN 2088 (Lenart, et al., Antisense Nucleic Acid Drug
Dev. 4, 247-256 (2001)). We found that S-ODN 2088 profoundly
inhibited the proliferative response to S-ODN 1826 but had no
effect on the response to anti-IgM or our TLR2 and TLR4 ligands
(FIGS. 5C and 5B). Most significantly, S-ODN 2088 also dramatically
blocked the RF+ B cell response to mAb PL2-3. Overall, these data
strongly implicate endosomal processing and/or engagement of an
endosome-associated TLR, most probably TLR9, in RF+ B cell
activation.
[0093] It has been clearly shown that autoreactive B cell clones
can undergo isotype switching and somatic mutation, similar to the
T-dependent features of conventional B cell responses to foreign
antigens (Shlomchik, et al., Nature 328, 805-811 (1987)). However,
more recent reports have suggested that autoreactive B cells may
segregate from conventional B cells in the peripheral lymphoid
tissues; autoreactive B cells tend to localize in the marginal zone
of the splenic white pulp (Zeng, et al., J. Immunol. 164, 5000-5004
(2000)), while conventional B cells home to the follicular regions.
In addition, in some instances, expansion and somatic mutation of
autoreactive B cells can take place outside of the germinal center.
These discrete localization patterns may reflect the preferential
accumulation of stimulatory autoantibody/autoantigen IC at these
sites. Alternatively, the relatively unique homing pattern may
result from distinct chemokine receptor profiles elicited in
response to the combined effects of BCR/TLR engagement. Future
studies will need to compare the functional properties of B cells
activated through BCR crosslinking alone from B cells activated
through BCR/TLR dual engagement.
[0094] Autoantibodies that recognize DNA/nucleosomes are the
defining and most prevalent specificity in SLE patients and murine
lupus models of SLE, while RFs are characteristic of a subset of
SLE patients, autoimmune-prone lpr mice, and RA patients.
Remarkably, these same autoantigens are the targets in a number of
new murine autoimmunity models generated by mutations that disrupt
lymphocyte homeostasis or autoantigen metabolism (Botto, et al.,
Nature Genetics 19, 56-59 (1998); Bickerstaff, et al., Nature
Medicine 5, 694-697 (1999); Napirei, et al., Nat. Gen. 25, 177-180
(2000); Scott, et al., Nature 411, 207-211 (2001)). The reason for
the predominance of these particular specificities has been
long-sought. Our data provide an explanation, namely the potent
synergistic interaction of BCR/TLR signaling events mediated by
chromatin containing immune complexes. In our experimental system,
dependent on endosome/lysosome-localized TLR9 (Hacker, et al., EMBO
J. 17, 6230-6240 (1998)), it is reasonable to conclude that
autoantibody/autoantigen IC engagement of the BCR triggers the
endocytosis of IC-associated antigen that then results in the
highly efficient delivery of chromatin fragments to
endosome-associated TLR9. In contrast to published studies (Bell,
et al., J. Clin. Invest. 85, 1487-1496 (1990); Bell, et al., Clin.
Immunol. Immunopath. 60, 1326 (1991)), we have been unable to
activate B cells with either culture supernatants or chromatin
fragments alone. Whether other strains of mice will prove more
responsive to our fragments remains to be determined.
[0095] Beyond the current experimental model, the principle of
BCR/TLR dual-engagement has wide ranging implications for
autoimmunity in general. Model ligands such as haptenated-LPS and
haptenated LPSLPS-coupled SRBC can also co-signal BCRs and TLRs and
are remarkably potent and specific for the B cells that have the
relevant receptors (Pike, Methods Enzymol. 150:265-75, 1987).
Activation in this mode is therefore likely to be a fundamental
event in the loss of peripheral B cell tolerance in a wide variety
of settings; other autoantigens may signal through TLRs other than
TLR9. Overall, the data establish a critical role for endogenous
TLR ligands in the aberrant activation of the adaptive immune
system in autoimmunity and explain why the autoantibody repertoire
is often skewed toward the recognition of subcellular nucleic
acid/protein particles (Tan, E. Adv. Immunol. 44, 93-151
(1989)).
Methods
[0096] Mice. The MRL+/+AM14 BCR Tg mice described previously
(Hannum, et al., J. Exp. Med. 184, 1269-1278 (1996); Rifkin, et
al., J. Immunol. 165, 1626-1633 (2000)) were crossed to
Cr2-deficient mice, kindly provided by Dr. Michael Carroll (Harvard
Medical School, Boston), and the F1 offspring were intercrossed to
generate AM14 Cr2 deficient and Cr2 sufficient control mice.
MyD88-/- mice, originally produced by Dr. Shizuo Akira (Osaka
University, Osaka, Japan) (Adachi, et al., Immunity 9, 143-150
(1998)) and kindly provided through Dr. Douglas Golenbock
(University of Massachusetts Medical School, Worcester, Mass.) were
crossed to AM14 MRL+/+ mice to generate AM14 MyD88-/- and control
littermate offspring. The RF+ offspring were initially identified
by PCR, and their identity was confirmed by flow cytometric
analysis of peripheral blood lymphocytes or spleen cells, using the
4-G7 monoclonal anti-idiotype as described (Shlomchik, et al., Int.
Immunol. 5, 1329-1341 (1993)). Complement receptor genotype was
determined by flow cytometry using the FITC-conjugated 7G6 antibody
(Pharmingen, San Diego, Calif.). MyD88 genotype was determined by
PCR using the primers: MyD88F (5'-TGG CAT GCC TCC ATC ATA GTT AAC
C-3') (SEQ ID NO: 1), MyD88R (5'-GTC AGA AAC AAC CAC CAC CAT GC-3')
(SEQ ID NO: 2), and neoR (5'-ATC GCC TTC TAT CGC CTT CTT GAC G-3')
(SEQ ID NO: 3) (MWG Biotech, High Point, N.C.) to yield wild type
and knockout products of approximately 550 bp and 750 bp
respectively.
[0097] Cell culture and reagents. Spleen cell preparations were
T-depleted and cultured with the appropriate ligands for 40-48 hrs.
In some experiments, B cells were preincubated with CD40L as
described (Rifkin, et al., J. Immunol. 165, 1626-1633 (2000)).
Proliferation was assessed by .sup.3[H]-thymidine incorporation
during the final 16 hours of culture. Data are presented as the
mean percentage of the anti-IgM response from triplicate cultures.
The percentage of the anti-IgM response was calculated according to
the formula: [(cpm experimental condition-cpm CD40L alone)/(cpm
anti-IgM-cpm CD40L alone).times.100].
[0098] Ligands included: goat anti-mouse IgM F(ab').sub.2 (15
.mu.g/ml, Jackson ImmunoResearch Laboratories, West Grove, Pa.);
the nucleosome specific mAbs PR1-3 (IgG2a.sup.j), PL2-3
(IgG2a.sup.j), and PL2-8 (IgG2b) (all at 50 .mu.g/ml), kindly
provided by Dr. Marc Monestier (Temple University School of
Medicine, Philadelphia, Pa.) (Monestier and Novick Mol. Immunol.
33, 89-99 (1996)); 10 .mu.g/ml LPS (Sigma, St. Louis, Mo.); 0.3-2
.mu.g/ml stimulatory CpG S-ODN 1826 (Yi, et al., J. Immunol. 160,
4755-4761 (1998)) (Oligo's Etc, Wilsonville, Ohio), 10 .mu.g/ml
Neisseria meningitidis porin B (kindly provided by Dr. Lee Wetzler,
Boston University School of Medicine, Boston, Mass.); 10 .mu.g/ml
synthetic lipopeptide Pam.sub.3Cys-Sk.sub.4 (Dr. G. Jung, Univ. of
Tuebingen, Germany, kindly provided by Dr. Doug Golenbock); and the
anti-TNP mAbs Hy1.2 (IgG2a.sup.a) and C1040 (IgG2a.sup.b) (Hannum,
et al., J. Exp. Med. 184, 1269-1278 (1996)) (both at 50 .mu.g/ml)
complexed to varying concentrations of TNP-BSA. In some
experiments, DNase I (type IV) (Sigma, St. Louis, Mo.), chloroquine
(Sigma), concanamycin B, bafilomycin A (Sigma), or 12 .mu.g/ml of
the inhibitory CpG S-ODN 2008 (Lenart, et al., Antisense Nucleic
Acid Drug Dev. 4, 247-256 (2001)) (Oligo's Etc.) were added to the
cultures 15-30 min-2 hr before the addition of the ligands. All mAb
and ODN preparations were shown to be endotoxin free by Limulus
Amebocyte Lysate ELISA (Bio-Whittaker, Walkersville, Md.).
EXAMPLE 3
[0099] We have further shown that dendritic cells are activated by
chromatin-containing immune complexes through sequential engagement
of the Fc-receptor and TLR 9. This is a similar mechanism to that
we have described for B cells except that in the case of the B
cell, the relevant cell surface receptor is the B cell antigen
receptor, whereas in dendritic cells the relevant cell surface
receptor is an Fc gamma receptor (Fc.gamma.R). Both cell surface
receptors serve to deliver chromatin to TLR9 located within the
endosome/lysosome compartment.
[0100] In FIGS. 8A-8C we show that dendritic cells can be
specifically activated through TLR9 and this activation can be
blocked by TLR9 inhibition with ODN 2088. These experiments
demonstrate that the dendritic cells that we use in the subsequent
experiments (shown in Tables 1 and 2 below) express functional
TLR9, in that the specific TLR9 activating ligand ODN 1826 induces
TNF-.alpha. and IL-12 production by the dendritic cells (8A, 8B)
and furthermore induces upregulation of co-stimulatory molecules
(8C). ODN 2088 is a specific inhibitor of the TLR9 mediated
activation because (as shown in 8A, 8B, 8C), ODN 2088 blocks ODN
1826 mediated activation but has no effect on LPS mediated
activation.
[0101] Dendritic cells were generated from the bone marrow of
C57BL/6 mice by in vitro culture with GM-CSF and IL-4 for 6 days.
On day 6, the CD11c positive dendritic cells were isolated using
magnetic beads (Miltenyi Biotec) and either pre-incubated, or not
pre-incubated, for 30 minutes with ODN 2088, an inhibitory
oligodeoxynucleotide that specifically blocks signaling through
TLR9. The stimulatory ODN 1826 (CpG, 3 .mu.g/ml), that specifically
activates through TLR9, or lipopolysaccharide (LPS, 10 .mu.g/ml),
that activates through TLR4, or culture medium only (media), was
then added to the cultures. After 48 hours, levels of TNF-.alpha.
(8A) and IL-12 (8B) in the culture supernatants were measured by
ELISA (results shown as OD units determined by absorption at 405
nm). After removal of the supernatants, cells were collected and
analyzed by flow cytometry for expression of the co-stimulatory
molecule CD86 (8c). Unstimulated refers to the level of expression
of CD86 in the presence of media alone, whereas stimulated refers
to the level of expression of CD86 in the presence of the stimulus
(ODN 1826 or LPS).
[0102] Table 1 below shows that chromatin-containing immune
complexes (containing autoantibody in a complex with the chromatin
autoantigen) activate dendritic cells to produce TNF-.alpha.
whereas immune complexes containing a foreign antigen (TNP-BSA) do
not induce this dendritic cell activation. Additionally, the
activation induced by the chromatin-containing immune complexes is
completely blocked by the TLR9 specific inhibitor ODN 2088,
indicating that engagement of TLR9 (presumably by the chromatin
within the chromatin-containing immune complex) is an essential
component of the activation process.
TABLE-US-00001 TABLE 1 Stimulus None 2088 Media (no stimulus)
<50 <50 ODN 1826 2347 <50 LPS 2514 2393 PL2-3 1653 <50
TNP/.alpha.TNP-BSA IC 1:1 <50 <50 TNP/.alpha.TNP-BSA IC 4:1
<50 <50 TNP/.alpha.TNP-BSA IC 16:1 <50 <50
[0103] Table 1 shows that dendritic cell TNF-.alpha. production
induced by chromatin-containing immune complexes is blocked by the
TLR9 specific inhibitor ODN 2088. Dendritic cells were generated
from the bone marrow of C57BL/6 mice by in vitro culture with
GM-CSF and IL-4 for 6 days. On day 6, the CD11c positive dendritic
cells were isolated using magnetic beads (Miltenyi Biotec). The
dendritic cells were then either pre-incubated, or not
pre-incubated, for 30 minutes with ODN 2088 at 12 .mu.g/ml. The
stimulatory ODN 1826 (3 .mu.g/ml), that specifically activates
through TLR9, lipopolysaccharide (LPS, 10 .mu.g/ml), that activates
through TLR4, the anti-chromatin antibody PL2-3 (50 .mu.g/ml),
three different antibody/protein ratios of .alpha.-TNP/TNP-BSA
immune complexes (50 .mu.g/ml), or the culture medium only (media),
was then added to the cultures. After 48 hours, levels of
TNF-.alpha. in the culture supernatants was measured by ELISA
(results shown as pg/ml; lower level of sensitivity of assay is 50
pg/ml). A representative experiment of 3 similar experiments is
shown.
[0104] Table 2 shows that chromatin-containing immune complexes
induce TNF-.alpha. production by dendritic cells from wildtype mice
(as was also seen in the studies shown in table 1 in the absence of
ODN 2088) but completely fail to induce TNF-.alpha. production by
dendritic cells from Fc receptor .gamma. chain deficient mice. This
demonstrates that an activating Fc receptor is absolutely required
for dendritic cell activation by chromatin-containing immune
complexes.
TABLE-US-00002 TABLE 2 Fc receptor .gamma. Stimulus wildtype chain
deficient Media (no stimulus) <50 <50 ODN 1826 7899 2373 LPS
7056 3411 PL2-3 4554 <50 PL2-8 3964 <50 TNP/.alpha.TNP-BSA IC
1:1 <50 <50 TNP/.alpha.TNP-BSA IC 4:1 <50 <50
[0105] Table 2 demonstrates that chromatin-containing immune
complexes induce TNF-.alpha. production by dendritic cells from
wildtype mice but do not induce TNF-.alpha. production by dendritic
cells from Fc receptor .gamma. chain deficient mice. Dendritic
cells were generated from the bone marrow of C57BL/6 wildtype mice
and from C57BL/6 mice lacking the Fc receptor .gamma. chain in by
in vitro culture of the bone marrow cells with GM-CSF and IL-4 for
6 days. On day 6, the CD11c positive dendritic cells were isolated
using magnetic beads. The stimulatory ODN 1826 (3 .mu.g/ml), that
specifically activates through TLR9, lipopolysaccharide (LPS, 10
.mu.g/ml), the anti-chromatin antibodies PL2-3 (50 .mu.g/ml; IgG2a)
and PL2-8 (50 .mu.g/ml; IgG2b), two different antibody/protein
ratios of .alpha.-TNP/TNP-BSA immune complexes (50 .mu.g/ml), or
the culture medium only (media), was then added to the dendritic
cell cultures. After 48 hours, levels of TNF-.alpha. in the culture
supernatants was measured by ELISA (results shown as pg/ml; lower
level of sensitivity of assay is 50 pg/ml). A representative
experiment of 2 similar experiments is shown.
[0106] Taken together, the data shown in Table 1 and Table 2 above
demonstrate that dendritic cell activation induced by
chromatin-containing immune complexes requires a signal through an
activating Fc.gamma. receptor as well as a signal mediated through
TLR9.
[0107] Additional experiments to evaluate mechanisms and
consequences of this Fc.gamma.R/TLR dual engagement pathway are
outlined below.
Characterization of the IFN-.alpha. Producing Dendritic Cell
Subsets.
[0108] Mouse strains. Differences in dendritic cell and macrophage
function between autoimmune lupus-like and non-autoimmune mouse
strains have been described. These include differences in immune
complex handling (Jones et al., Clin. Immunol. Immunopathol. 36:
30-39, 1985; Magilavy et al., J. Immunol. 131: 2784-2788, 1983),
phagocytosis (Russell and Steinberg Clin. Immunol. Immunopathol.
27: 387-402, 1983), Fc receptor expression and function (Pritchard
et al., Current Biology. 10: 227-230, 2000; Jiang et al.,
Immunogenetics. 51: 429-435., 2000) and cytokine production (Alleva
et al., J. Immunol. 159: 5610-5619., 1997; Koh et al., J. Immunol.
165: 4190-4201., 2000). It is therefore important to directly
compare dendritic cells derived from both autoimmune and
non-autoimmune strains. MRL+/+ and (NZBxNZW) F1 are the two
autoimmune lupus-like strains and C57BL/6 and BALB/c are the two
non-autoimmune strains that are used.
[0109] Purification of dendritic cell subsets. There are 2
dendritic cell subsets which are particularly important in the
pathogenesis of SLE: CD11c+ CD8.alpha.+ and CD11c+ B220+ Gr-1+.
These cells are either obtained directly from the spleen (Hochrein
et al., J. Immunol. 166: 5448-5455, 2000; Nakano et al., J. Exp.
Med. 194: 1171-1178, 2001) or generated by in vitro culture of bone
marrow cells in the presence of Flt3 ligand (Brasel et al., Blood.
96: 3029-3039, 2000; Gilliet et al., J. Exp. Med. 195: 953-958,
2002). Splenic dendritic cells are isolated as described (Vremec et
al., J. Immunol. 164: 2978-2986, 2000) and, after appropriate
fluorescent antibody staining, the specific dendritic cell subsets
are fractionated and collected using a MoFlo cell sorter
(Cytomation, Fort Collins, Colo.). The CD11c+ CD8.alpha.- dendritic
cell subset, which is also found in the spleen, are isolated at the
same time as the target CD11c+ CD8.alpha.+ dendritic cell subset
and used as an experimental control.
[0110] To generate bone marrow derived dendritic cells, bone marrow
cells are harvested and cultured in vitro for 6-10 days in the
presence of recombinant murine Flt3 ligand using established
protocols (Gilliet et al., J. Exp. Med. 195: 953-958, 2002; Labeur
et al., J. Immunol. 162: 168-175, 1999). Bone marrow cells grown in
Flt3 ligand are enriched in the target populations, namely CD11c+
CD8+ and CD11c+ B220+ Gr-1+ dendritic cells. These populations are
fractionated by cell sorting as described above for the
spleen-derived dendritic cells.
[0111] Determining the expression of TLR9 in the specific dendritic
cell subsets. TLR9 messenger RNA is measured by quantitative
reverse transcription polymerase chain reaction (RT-PCR). In
addition, the response of the dendritic cell subsets to a
TLR9-specific stimulatory ligand, S-ODN 1826 (Ballas et al., J.
Immunol. 167: 4878-4886, 2001), are compared by measuring
expression of co-stimulatory molecules and cytokine production.
Specific antibodies against murine TLR9 can be produced using
techniques known to one skilled in the art.
Preparation of IC and Assessment of Ic Binding and Uptake by
Dendritic Cells.
[0112] Immune complex preparation. Anti-nucleosome/chromatin IC is
prepared by incubating the anti-nucleosome monoclonal antibodies
PL2-3 (IgG2a), PL2-8 (IgG2b) and PL9-7 (IgG3) with supernatant
obtained from 24 hour in vitro cultures of spleen cells. Chromatin
is spontaneously released from spleen cells in culture (Bell et
al., J. Clin. Invest. 85: 1487-1496, 1990) and binding of the
anti-nucleosome antibodies to this chromatin results in the
formation of IC which can be measured using a Clq immunoassay
(Rifkin et al., Journal of Immunology. 165: 1626-1633, 2000). We
have further shown that biotinylated PL2-3 preincubated with
culture fluid binds specifically to rheumatoid factor B cells as
detected by flow cytometry; monomeric biotinylated PL2-3 does not
bind. Non-self antigen control IC is made using the anti-TNP
monoclonal antibodies Hy1.2 (IgG2a) or C4010 (IgG2b) and incubating
them with TNP-BSA, with confirmation of IC formation also by Clq
binding as demonstrated (Leadbetter et al., Nature. 416: 603-607,
2002). Additional non-self antigen control IC is made using the
anti-ovalbumin monoclonal antibodies Ov1 (IgG2a) and Ov2 (IgG2b)
prepared in this laboratory and incubating them with ovalbumin. As
well as demonstrating IC formation in a Clq binding assay, FPLC is
also used as an additional confirmatory assay.
[0113] Immune complex binding and uptake by dendritic cells. Before
setting up the actual experimental cultures containing the IC and
dendritic cells, it is necessary to determine the extent to which
the purified dendritic cell subsets from the different strains are
able to bind and take up IC. This is particularly important given
the reported differences between autoimmune and non-autoimmune
mouse strains as regards IC handling (Jones et al., Clin. Immunol.
Immunopathol. 36: 30-39, 1985; Magilavy et al., J. Immunol. 131:
2784-2788, 1983) and Fc receptor expression (Pritchard et al.,
Current Biology. 10: 227-230, 2000; Jiang et al., Immunogenetics.
51: 429-435, 2000). The experimental approach is to track the
antibody component of the immune complex using a combination of
flow cytometry and confocal microscopy. The anti-nucleosome
antibodies PL2-3 (IgG2a), PL2-8 (IgG2b) and PL9-7 (IgG3) are
biotin-conjugated, using standard procedures, and
biotin-anti-nucleosome/chromatin IC are prepared as above. To
confirm that the biotin-anti-nucleosome/chromatin IC retains
functionality it is assessed whether the biotin-PL2-3/chromatin IC
is able to induce proliferation in rheumatoid factor B cells to the
same extent as the non-biotin-PL2-3/chromatin IC (Rifkin et al.,
Journal of Immunology. 165: 1626-1633, 2000). The
biotin-PL2-8/chromatin IC and the biotin-PL9-7/chromatin IC cannot
be tested in this way because IgG2b and IgG3 are not recognized by
the rheumatoid factor B cell receptor, but it is assumed that the
functional effect of biotin-conjugation will be similar
irrespective of isotype. Isotype-matched biotin-anti-TNP/TNP-BSA IC
and biotin-anti-ovalbumin/ovalbumin IC are prepared similarly.
Biotin-conjugated antibodies alone not made into complexes are used
as controls. The biotin-conjugated IC and biotin-conjugated
antibodies alone are added to the different dendritic cell subsets,
and flow cytometry is used to detect cell surface binding. The
biotin-labeled compounds are visualized with an anti-biotin
monoclonal antibody conjugated to a specific fluorochrome
(Molecular Probes: anti-biotin mouse monoclonal 2F5 Alexa Fluor 488
conjugate). Subsequently, confocal microscopy is used to assess
dendritic cell internalization of the anti-nucleosome antibody
(biotin-anti-nucleosome/chromatin IC), the anti-TNP antibody
(biotin-anti-TNP/TNP-BSA IC) or the anti-ovalbumin antibody
(biotin-anti-ovalbumin/ovalbumin IC) following incubation periods
of 1-12 hours and fixation with 3% paraformaldehyde.
[0114] In addition, co-localization studies are performed by
co-staining with fluorescent-labeled monoclonal antibodies specific
for endosomal/lysosomal compartments, or by the use of fluorescent
pH indicators that partition into acidic organelles (Molecular
Probes).
Measurement of Dendritic Cell Activation by Chromatin-Containing IC
and Establishment of the Role of Toll-Like Receptors and Fc Gamma
Receptors.
[0115] Measuring dendritic cell activation by IC. The
anti-nucleosome/chromatin IC, anti-TNP/TNP-BSA IC and
anti-ovalbumin/ovalbumin IC outlined above are incubated with the
dendritic cell subsets and dendritic cell activation determined by
measuring upregulation of co-stimulatory molecules and cytokine
production. Controls include the monomeric non-complexed antibodies
alone, antigens alone, stimulatory CpG S-ODN 1826 as a positive
control for TLR9 signaling, LPS as a positive control for TLR4
signaling and porin B as a positive control for TLR2 signaling.
Cytokines are measured by ELISA and include IL-12, TNF-.alpha.,
IL-10 and IFN-.alpha.. IL-12 and TNF-.alpha. are the two cytokines
most commonly used as markers of dendritic cell activation
(Banchereau et al., Annu. Rev. Immunol. 18: 767-811, 2000) and
IFN-.alpha. is the cytokine most strongly associated with the
activation of the CD11c+ CD8.alpha.+ and the CD11c+ B220+ Gr-1+
dendritic cell subsets. It is necessary to measure IL-10 as it has
been shown that Fc receptor engagement in macrophages can lead to
IL-10 production induced by a subsequent stimulus which, in the
absence of Fc receptor engagement, leads to IL-12 and not IL-10
production (Gerber and Mosser J. Immunol. 166: 6861-6868., 2001).
The increased expression of MHC class II and the co-stimulatory
molecules CD80, CD86, and CD40 is determined by flow cytometry.
[0116] Role of Toll-like receptors and Fc gamma receptors in
IC-mediated dendritic cell activation. S-ODN 2088 is an inhibitory
CpG S-ODN which specifically blocks signaling through TLR9 (Lenert
et al., Antisense and Nucleic Acid Drug Development. 11: 247-256,
2001). We have shown that S-ODN 2088 strongly inhibits the
chromatin-containing IC-induced proliferation of B cells from
rheumatoid factor B cell receptor transgenic mice (Leadbetter et
al., Nature. 416: 603-607, 2002). Also, we have shown that S-ODN
2088 completely prevents dendritic cell activation by stimulatory
CpG S-ODN (such as S-ODN 1826) acting through TLR9. The experiments
outlined above are done in the presence or absence of S-ODN 2088 to
assess the role of TLR9 in IC-mediated dendritic cell activation.
They are also done in the presence and absence of inhibitors of
endosomal acidification, including chloroquine, concanamycin B and
bafilomycin A, which prevent signaling through TLR9. In addition,
dendritic cells from C57BL/6 MyD88+/+ mice are compared to
dendritic cells from C57BL/6 MyD88-/- mice to establish the
requirement for TLR signaling.
[0117] In order to assess the requirement for Fc gamma receptor
(Fc.gamma.R) signaling, experiments are also done in the presence
and absence of 2.4.G2, a monoclonal antibody that specifically
blocks murine Fc.gamma.RII and Fc.gamma.RIII (Araujo-Jorge et al.,
Infect Immun. 61: 4925-4928, 1993). However, the third type of
murine Fc gamma receptor, Fc.gamma.R I, is not blocked by 2.4G2 and
can mediate IC-induced inflammatory responses (Ioan-Facsinay et
al., Immunity. 16: 391-402., 2002; Barnes et al., Immunity. 16:
379-389, 2002). Dendritic cells express all three classes of
Fc.gamma.R (Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII) (Ravetch
and Bolland, Ann. Rev. Immunol. 19: 275-290, 2001). Therefore,
additional studies can be performed using Fc.gamma.R knockout mice.
The mice are available from Jackson Laboratories, and include i)
mice rendered genetically deficient in Fc.gamma.RI and
Fc.gamma.RIII by knockout of the common stimulatory
signal-transducing gamma chain shared by these two receptors (Takai
et al., Cell. 76: 519-529., 1994) ii) Fc.gamma.RII knockout mice
(Takai et al., Nature. 379: 346-349., 1996) and iii) Fc.gamma.RIII
knockout mice (Hazenbos et al., Immunity. 5: 181-188., 1996).
[0118] Role of IC and TLR9 in antigen processing and presentation
by dendritic cells. CD4+ autoreactive T cells are central to the
pathogenesis of SLE, both in humans and in murine models of the
disease (Craft et al., Immunol. Res. 19, 1999; Hoffman, Front.
Biosci. 6: D1369-78, 2001; Shlomchik et al., Nature Rev. Immunol.
1: 147-153., 2001). Dendritic cells are the key antigen presenting
cells involved in initiating T cell responses (Banchereau et al.,
Annu. Rev. Immunol. 18: 767-811, 2000) and TLR9 engagement has been
shown to strongly promote development of Th1-type immune responses
(Jakob et al., J. Immunol. 161: 3042-3049, 1998; Lipford et al., J.
Immunol. 165: 1228-1235, 2000). To show whether TLR9 engagement
alters the T cell response to antigen contained within immune
complexes, ovalbumin is conjugated to the stimulatory CpG S-ODN
1826 as described (Shirota et al., J. Immunol. 164: 5575-5582.,
2000). IC is consequently made by incubating unconjugated ovalbumin
or the ovalbumin-CpG conjugate with the anti-ovalbumin monoclonal
antibodies Ov1 (IgG2a) or Ov2 (IgG2b). Dendritic cell subsets from
BALB/c mice (H-2.sup.d) are pulsed with these IC. CD4+ T cells,
which recognize ovalbumin in the context of H-2A.sup.d, are
purified from the lymph nodes of ovalbumin-specific T cell receptor
transgenic mice (DO11.10 mice) (Murphy et al., Science. 250:
1720-1723, 1990). These CD4+ T cells are cultured with the
IC-pulsed dendritic cells and T cell proliferation and cytokine
production (IFN-gamma, IL-4 and IL-2) is measured during primary
and secondary T cell responses. S-ODN 2088 and endosomal
acidification inhibitors is used to assess the role of TLR9.
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[0164] The references cited herein and throughout the specification
are herein incorporated by reference in their entirety.
Sequence CWU 1
1
3125DNAArtificial sequenceSynthetic oligonucleotide 1tggcatgcct
ccatcatagt taacc 25223DNAArtificial sequenceSynthetic
oligonucleotide 2gtcagaaaca accaccacca tgc 23325DNAArtificial
sequenceSynthetic oligonucleotide 3atcgccttct atcgccttct tgacg
25
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