U.S. patent application number 10/513962 was filed with the patent office on 2005-06-30 for method of inhibiting the emigration of cells from the intravascular compartment into tissues.
Invention is credited to Elsner, Jorn, Escher, Sylvia, Forssmann, Ulf, Spodsberg, Nikolaj.
Application Number | 20050142101 10/513962 |
Document ID | / |
Family ID | 56290421 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050142101 |
Kind Code |
A1 |
Forssmann, Ulf ; et
al. |
June 30, 2005 |
Method of inhibiting the emigration of cells from the intravascular
compartment into tissues
Abstract
A method of inhibiting the emigration of cells from the
intravascular compartment into tissues (or through any membrane
limiting any body compartment from another) by confronting the
cells with an agonist specific for receptors involved with
migration of said cells via a receptor thereby making the cell
unresponsive to further activation.
Inventors: |
Forssmann, Ulf; (Hannover,
DE) ; Elsner, Jorn; (Hannover, DE) ; Escher,
Sylvia; (Hannover, DE) ; Spodsberg, Nikolaj;
(Copenhagen, DK) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
56290421 |
Appl. No.: |
10/513962 |
Filed: |
February 8, 2005 |
PCT Filed: |
May 9, 2003 |
PCT NO: |
PCT/EP03/04872 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60381802 |
May 21, 2002 |
|
|
|
Current U.S.
Class: |
424/85.1 ;
514/15.4; 514/15.7; 514/16.4; 514/17.5; 514/17.7; 514/19.5;
514/3.8; 514/6.9 |
Current CPC
Class: |
A61K 31/557 20130101;
A61K 38/195 20130101; Y10S 514/825 20130101; A61P 29/00 20180101;
Y10S 514/886 20130101; A61P 37/06 20180101; A61K 31/00 20130101;
Y10S 514/826 20130101 |
Class at
Publication: |
424/085.1 ;
514/012 |
International
Class: |
A61K 038/17; A61K
038/19 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2002 |
EP |
02010573.0 |
May 21, 2002 |
EP |
02011201.7 |
Claims
1. A method of inhibiting the emigration of cells from the
intravascular compartment into tissues (or through any membrane
limiting any body compartment from another) by confronting the
cells with an agonist specific for receptors involved with
migration of said cells via a receptor thereby making the cell
unresponsive to further activation.
2. A method according to claim 1, wherein the cells are blood
circulating cells and the intravascular compartiment is the blood
stream.
3. The method of claim 1 wherein the cells are leukocytes.
4. The method of claim 1 wherein the cell is unresponsive to
further activation for emigration to tissues after confrontation
with an agonist.
5. The method according to claim 1 wherein the agonist used to
inhibit the migration of the cells is a chemoattractant binding to
a corresponding receptor or molecule binding to such a
receptor.
6. The method of claim 5 wherein the chemo-attractant is selected
from the group consisting of chemokine, a defensine, a leukotriene,
a formyl-peptide or combinations thereof as well as mutants and/or
variants of the chemoattractant.
7. The method of claim 1 wherein the compound is selected from the
group consisting of R.sup.1-CCL14[10-74], R1-CXCL12[1-67],
R1-CXCL12V3I[1-67], R1-CXCL12[2-67], R1-CXCL12V3I[2-67],
R1-CXCL12[1-72], R1-CXCL12V3I[1-72], R1-CXCL12[2-72] and
R1-CXCL12V3I[2-72] wherein R.sup.1 is a lipophilic, hydrophobic or
polar aprotic residue.
8. The method of claim 7, wherein R.sup.1 is any organic residue
having up to 50 carbon atoms, which may be substituted by hetero
atoms, and which organic residue is branched, unbranched,
saturated, unsaturated or combinations thereof.
9. The method of claim 8, wherein R.sup.1 is an aromatic moiety,
polyethylenoxid, moiety with 2 to 18 units, comprising residue.
10. The method of claim 7, wherein R.sup.1 is any amino acid, or
CH.sub.3--(CH.sub.2).sub.n--X; in which (CH.sub.2).sub.n is
branched or unbranched X is --C(O)--NH--CH.sub.2--C(O)--,
--NHCH.sub.2--C(O)--, --ONH--CH.sub.2--C(O)--,
--OCH.sub.2--CH.sub.2-C(O)--, --CH.dbd.CH--C(O)--, --C(O)--, or a
covalent bond; and n is an integer of 1-17; or pharmaceutically
acceptable salt thereof.
11. A method of treating a disease state in mammals that is
alleviated by treatment with a compound of claim 7, which method
comprises administering to an mammal in need of such a treatment a
therapeutically effective amount of the compound.
12. The method of claim 5 wherein said method inhibits
inflammation.
13. The method of claim 12, wherein inflammation is selected from
the group consisting of allergic asthma, atopic dermatitis,
rheumatoid arthritis, and combinations thereof.
14. Use of an agonist specific for receptor involved with migration
of blood circulating cells from the blood stream for the
manufacturing of a medicament for the treatment of diseases
associated with migration of blood cells from the blood stream into
tissues.
15. Use according to claim 14 wherein the agonist is a
chemo-attractant.
16. Use according to claim 14 wherein the chemo-attractant is
selected from the group consisting of chemokine, defensin,
leukotriene, formyl-peptides as well as mutants and/or variants of
the chemo-attractants.
17. Use of a compound of the method of claim 7 for the
manufacturing of a medicament for the treatment of diseases
associated with migration of blood cells from the blood stream into
tissues.
18. A compound R.sup.1-CCL14[10-74], wherein R.sup.1 is a
lipophilic, hydrophobic or polar aprotic residue.
Description
BACKGROUND OF THE INVENTION
[0001] The role of inflammation in allergic diseases, especially in
asthma is widely recognized, and inflammation of the airways is one
of the three major characteristics of asthma.sup.1;2. The major
infiltrating effector cells in asthma contributing to the
inflammatory response are eosinophils, mast cells and Th2
lymphocytes.sup.3-5 all contributing to a complex pathologic
process that ultimately leads to reduced lung function.
[0002] The molecular mechanisms involved in the recruitment of
these cells from the circulation are complex.sup.6. Chemokines are
of fundamental importance in this multistep process. Being present
on the endothelium bound to glycosminoglycans, chemokines trigger
integrin activation on rolling leukocytes resulting in their firm
adhesion on the endothelial surface.sup.7. Transendothelial
migration of the leukocytes into the surrounding tissue also
strongly depends on chemokines and their receptors.sup.8. Key
molecules involved in asthma belonging to the chemokine system have
been recently elucidated. CCL11 (eotaxin) was the first specific
chemokine identified as an attractant for eosinophils in the
bronchoalveolar lavage fluid (BALF) obtained from an experimental
model of allergen exposure of sensitized guinea pigs.sup.9 and was
subsequently shown to be present in humans.sup.10. The functionally
related chemokines CCL24 (eotaxin-2) and CCL26 (eotaxin-3) were
described thereafter.sup.11;12. Besides the eotaxins, the MCPs,
CCL5 (RANTES) and a truncated chemokine derived from CCL14 (HCC-1)
are attracting the same type of inflammatory cells being involved
in asthma.sup.13;14. An important role of CCL11 for the attraction
of eosinophils into the lung was recently shown by several groups
in human asthmatic subjects. The influx of eosinophils correlates
strongly with increased peptide and mRNA expression of
CCL11.sup.15;16.
[0003] The common feature of the eotaxins, the MCPs and CCL5 are
their ability to mediate chemotaxis via the chemokine receptor
CCR3, which has been shown to be expressed on eosinophils.sup.17,
mast cells.sup.18, basophils.sup.19, and Th2-cells.sup.20;21. The
involvement of other chemokines was clearly demonstrated in vivo
showing that different chemokines, in the majority activators of
CCR3, contribute at different levels to the complex pathophysiology
of asthma.sup.22 Only this year a major breakthrough for the
extraordinary role of CCR3 in asthma was achieved. Targeted
disruption of CCR3 was successfully performed showing that
eosinophils and other inflammatory cells were arrested in the
subendothelial space of pulmonary vessels after bronchial allergen
challenge in OVA-sensitized mice.sup.23, implicating that the local
inflammatory response can be abolished targeting CCR3 already in
the circulation. Furthermore airway hyperresponsiveness (AHR) is
completely abrogated in CCR3-deficient mice in which the animals
are sensitized by the epicutaneous route.sup.24. Therefore the
common receptor CCR3 is exceptionally attractive as a drug target,
and its blockade is already propagated as a therapy for
asthma.sup.25.
[0004] CCL14 was recently isolated from human hemofiltrate based on
its high concentrations in human blood plasma.sup.26. The
originally isolated molecular form of CCL14 containing 74 amino
acid residues was shown to be a very weak activator of monocytes.
Later on, chemically synthesized N-terminal truncated forms of
CCL14 were shown to be more potent activators of monocytes acting
via CCR1.sup.27. Further screening of human hemofiltrate fractions
for novel natural ligands of chemokine receptors led to the
identification of the variant CCL-14[9-74], being a potent agonist
for CCR1, CCR3, and CCR5.sup.28.
[0005] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in the art
from the following description. It should be understood, however,
that the following description and the specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1: Alignment of N-terminal sequences of CCL14
derivatives and CCL11.
[0007] FIG. 2: CRIC3 induces the release of reactive oxygen species
(ROS) from human eosinophils with more potency than CCL11.
[0008] FIG. 3: CRIC3 induces an internalization of CCR3 from human
eosinophils in the same range than CCL11.
[0009] FIG. 4: CRIC3 induces chemotaxis of eosinophils but also
inhibits CCL11.
[0010] FIG. 5: CRIC3 induces a functional desensitization of
CCR3.
[0011] FIG. 6: CRIC3 is not processed by CD26/DPP IV.
[0012] FIG. 7: CRIC3 prevented slightly the influx of eosinophils
into the lung tissue of OVA-sensitized mice.
[0013] FIG. 8: CRIC3 prevented the influx of eosinophils into the
BALF in OVA-sensitized mice.
SUMMARY OF THE INVENTION
[0014] The chemokine receptor CCR3 is expressed on different
inflammatory cells, such as eosinophils, basophils, and Th.sub.2
cells, and is responsible for the invasion of these cells to the
site of inflammation, particularly in allergic disease. Whereas
most anti-inflammatory strategies act on their target cells after
migration to the site of inflammation, a method is disclosed on how
to prevent the cellular recruitment by inactivation of the
chemokine receptor by means of a potent agonist, before blood
circulating cells, such as leukocytes, leave the blood vessels. An
object of the present invention is therefor to provide a method for
preventing the migration of blood circulating cells out of the
blood.
[0015] Surprisingly, by confronting the cells with an agonist
specific for receptors involved with migration of said cells via a
receptor, the cell is rendered unresponsive to further activation
and is not leaving the blood stream. For this purpose, in
particular different truncated and chemically modified peptides
derived from the chemokine CCL14 (HCC-1) can be used. Replacement
of the ultimate N-terminal amino acid of CCL14[9-74] by nonanoic
acid (NNY) converts it into a potent agonist, termed CD26-resistant
inactivator of CCR3 (CRIC3) exhibiting an activity profile
identical to CCL11 (eotaxin), the most potent chemokine agonist of
CCR3 known with respect to the release of reactive oxygen species,
chemotaxis, and CCR3-internalization. This modification results in
a resistance to degradation by dipeptidyl peptidase IV (CD26/DPP
IV) due to the substitution of the Gly-Pro motif by the NNY-Pro
motif. In contrast to CRIC3, CCL11 is processed completely by
CD26/DPP IV into CCL11[3-74] thereby reducing its biological
activity. Interestingly, intravenous administration of CRIC3 in
ovalbumin-sensitized mice, prior to allergen challenge, resulted in
a significant reduction of eosinophils in the BALF and lung tissue.
Therefore, CRIC3 can be used to induce internalization of CCR3 on
blood leukocytes in the circulation. As a consequence of the
inactivation of CCR3, the recruitment of eosinophils, basophils,
and Th.sub.2 cells into tissues could be prevented in CCR3-driven
pathologies.
[0016] The object addressed by the present invention is solved by a
method of inhibiting the emigration of blood circulating cells from
the blood stream by confronting the cells with an agonist specific
for receptors involved with migration of said cells via a receptor
thereby making the cell unresponsive to further activation. In
particular, the cells are leukocytes.
[0017] According to the invention the cell is unresponsive to
further activation for emigration to tissues after confrontation
with an agonist.
[0018] In one embodiment of the invention the agonist used to
inhibit the migration of the cells is a chemoattractant binding to
a corresponding receptor or molecule binding to such a receptor.
The chemoattractant is in particular selected from the group
consisting of a chemokine, a defensine, a leukotriene, a
formyl-peptide or combinations thereof. The proteins and peptides
may exist and can be used also in form of mutants and variants. In
particular this may be the case for chemokines. Mutants or variants
of peptides differ to some extent in their amino acid sequences
from the respective consensus sequence. The alterations may be due
to exchanges of single or multiple amino acids or deletions of
single or multiple amino acids. For example, so called conservative
amino acid exchanges do not significantly change structure and
function of the protein or polypeptide. If a hydrophobic amino acid
such as Valin is exchanged for example against another hydrophobic
amino acid, such as Alanin, this is understood as an example for
conservative exchange. Such changes in most instances will not
change structure and function of the protein dramatically. Another
example for a conservative exchange is for example the exchange of
Lysine against another charged amino acid.
[0019] A protein is constructed by domains for example a series of
hydrophobic or hydrophilic amino acids. If one amino acid is
deleted it may also not alter the function and structure
dramatically. Mutants and variants can occur naturally, but may
also be generated by well known genetic engineering methods such as
site directed mutagenesis. The alterations may be such that they do
not lead to loss of function of the polypeptide in particular
receptor internalisation and desensitisation.
[0020] According to the present invention the following compounds
can be used:
[0021] R.sup.1-CCL14[10-74], R1-CXCL12[1-67], R1-CXCL12V3I[1-67],
R1-CXCL12[2-67], R1-CXCL12V3I[2-67], R1-CXCL12[1-72],
R1-CXCL12V3I[1-72], R1-CXCL12[2-72] and R1-CXCL12V3I[2-72]
[0022] wherein R.sup.1 is a lypophilic, hydrophobic or polar
aprotic residue. A polar aprotic residue is a residue, which shows
some polarity, but does not have active hydrogen or can be
associate in H.sup.+ and an ion. Typical polar aprotics compounds
are for example organic ethers. In particular R.sup.1 is an organic
residue having up to 50 carbon atoms, which may be substituted with
heteroatoms and which organic residue is branched, unbranched,
saturated, unsaturated or combinations thereof. In a specific
embodiment R.sup.1 is a residue containing aromatic moieties,
polyethylenoxide moieties with 2 to 18 units.
[0023] Furthermore, R.sup.1 may be any amino acid, or
CH.sub.3--(CH.sub.2).sub.n--X; in which
[0024] (CH.sub.2).sub.n is branched or unbranched
[0025] X is --C(O)--NH--CH.sub.2--C(O)--, --NHCH.sub.2--C(O)--,
--ONH--CH.sub.2--C(O)--, --OCH.sub.2--CH.sub.2-C(O)--,
--CH.dbd.CH--C(O)--, --C(O)--, or a covalent bond; and n is an
integer of 1-17;
[0026] or pharmaceutically acceptable salt thereof.
[0027] It will be appreciated, as is well known and as noted above,
that polypeptides are not always entirely linear. For instance,
polypeptides may be branched as a result of ubiquitination, and
they may be circular, with or without branching, generally as a
result of posttranslation events, including natural processing
event and events brought about by human manipulation which do not
occur naturally. Circular, branched and branched circular
polypeptides may be synthesized by non-translation natural
processes and by entirely synthetic methods, as well. Modifications
can occur anywhere in a polypeptide, including the peptide
backbone, the amino acid side-chains and the amino or carboxyl
termini. In fact, blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally occurring and synthetic polypeptides and such
modifications may be present in polypeptides of the present
invention, as well. For instance, the amino terminal residue of
polypeptides made in E. coli, prior to processing, almost
invariably will be N-formylmethionine.
[0028] It is also an object of the invention to provide a method
for the treatment of a patient having need of the agonist
comprising administering to the patient a therapeutically effective
amount (normally in the range of 1-1,000 nmol/kg body weight) of
the ligand, wherein said patient is suffering from a disease or a
disorder, including, but not limited to, inflammatory diseases,
particularly allergic asthma, atopic dermatitis, and rheumatoid
arthritis, infections such as bacterial, fungal, protozoan and
viral infections, particularly infections caused by HIV-1 or HIV-2;
pain; cancers; diabetes; Parkinson's disease; both acute and
congestive heart failure; hypotension; hypertension; urinary
retention; osteoporosis; angina pectoris; myocardial infarction;
ulcers; allergies; benign prostatic hypertrophy; chronic renal
failure; renal disease; impaired glucose tolerance; sexual
dysfunction and psychotic and neurological disorders, among
others.
[0029] The agonists are produced by standardized chemical methods
as described later. Alternatively they are produced by recombinant
methods, such as phage display or expression cloning in E. coli.
The agonists are purified and galenically prepared for application
using established methods.
[0030] The medicaments of the invention are prepared in suitable
galenic formulation.
[0031] Preferred formulations are lyophilized with mannitol or
similar carbohydrates in a sterile container. These can be used for
repeated injection or continuous infusion upon uptake in a
physiologic medium. Preferred amounts are 1 to 1,000 nmol/kg body
weight of the patient, calculated on the pure agonist.
[0032] Preferably the medicament is a galenic formulation
comprising biocompatible microspheres. The route of administration
is preferably selected from aerosols, intravenous or subcutaneous
application for systemic or local administration. Subject matter of
the present invention is also a method of treating a disease state
in mammals that is alleviated by treatment with a compound of the
invention, which method comprises administering to an mammal in
need of such a treatment a therapeutically effective amount of the
compound. In particular the method of the invention is able to
inhibit inflammation.
[0033] The invention is also concerned with the use of an agonist
specific for receptor involved with migration of blood circulating
cells from the blood stream for the manufacturing of a medicament
for the treatment of diseases associated with migration of blood
cells from the blood stream into tissues. According to the
invention the agonist is e.g. a chemo-attractant, in particular
selected from the group consisting of chemokines, defensines,
leukotrienes, formyl-peptides.
[0034] The use of a compound selected from the group consisting
of
[0035] R.sup.1-CCL14[10-74], R1-CXCL12[1-67], R1-CXCL12V3I[1-67],
R1-CXCL12[2-67], R1-CXCL12V3I[2-67], R1-CXCL12[1-72],
R1-CXCL12V3I[1-72], R1-CXCL12[2-72] and R1-CXCL12V3I[2-72]
[0036] wherein R1 is an aromatic or non-aromatic, branched or
unbranched compound comprising 1 to 50 atoms selected from C, H, O,
N, S, P, F, Cl, Br and I,
[0037] preferably
[0038] R.sup.1 is any amino acid, or CH.sub.3--(CH.sub.2).sub.n--X;
in which
[0039] (CH.sub.2).sub.n is branched or unbranched
[0040] X is --C(O)--NH--CH.sub.2--C(O)--, --NHCH.sub.2--C(O)--,
--ONH--CH.sub.2--C(O)--, --OCH.sub.2--CH.sub.2-C(O)--,
--CH.dbd.CH--C(O)--, --C(O)--, or a covalent bond; and n is an
integer of 1-17;
[0041] or pharmaceutically acceptable salt thereof
[0042] for the manufacturing of a medicament for the treatment of
inflammation and tumors, especially their metastatic spread, and to
modulate the homing of any cell, such as lymphocytes or stem cells
is also subject of the present invention.
[0043] The invention discloses and utilizes the effects of the
N-terminal modification of CCL14.sup.26;28 on its biological
activity mediated via the CCR3. Chemical modification of the most
active form was performed with the intention to generate a
CCR3-ligand leading to the inactivation of CCR3. Replacement of the
ultimate N-terminal amino acid of CCL14[9-74] by nonanoic acid led
to the identification of a potent agonistic inactivator of CCR3,
being as active as eotaxin, but fully resistant to cleavage by
dipeptidyl peptidase IV (CD26/DPP IV, EC 3.4.14.5) and therefore
termed CD26-resistant inactivator of CCR3 (CRIC3). CD26/DPP IV is
an abundant peptidase found in serum, tissue and on the cell
surface of different cell types. It is responsible for the
inactivation of chemokines, preferentially due to hydrolysis of
peptides with N-terminal Xaa-Pro and Xaa-Ala motifs.sup.29. Whereas
most anti-inflammatory strategies act on the target cells after
they migrated to the site of inflammation.sup.30, the present
invention discloses a method in which the cellular recruitment is
prevented by inactivation of the CCR3 before cells leave the blood
vessels. CRIC3 was also able to impair airway influx of CCR3.sup.+
eosinophils in OVA-sensitized mice. The application of this
compound represents a new concept for the therapy of CCR3-driven
pathologies.
[0044] The invention is further described by means of the following
non-limiting examples.
EXAMPLES
[0045] Chemokines.
[0046] CCL11 and CXCL12 (SDF-1.alpha.) were obtained from PeproTech
(London, U.K.). C5a was obtained from Sigma (Deisenhofen, Germany).
CCL14[1-74] was prepared as previously described.sup.26.
[0047] Synthesis of CCL14 Derivatives.
[0048] CCL14[6/7/8/9/10/11/12-74] and N-terminally modified
derivatives were prepared by Fmoc based solid-phase peptide
synthesis as described.sup.31. The synthesis of CCL14 peptides were
carried out on a 433A peptide synthesizer (Applied Biosystems) at a
scale of 0.1 mmol with a ten fold excess Fmoc amino acid using
HBTU/HOBt activation. After peptide chain assembly, nonanoic acid
was coupled as symmetrical anhydride (Sigma-Aldrich, Taufkirchen,
Germany) in N-methylpyrrolidinone (65 eq.) to the obtained
polypeptide. The resulting peptides were cleaved and deprotected in
the presence of TFA: H2O: EDT: phenol (86:6:6:2, v:v:v:w, 15 ml/g),
precipitated in cold TBME and purified chromatographically. The
resulting chromatographically homogeneous peptides were analyzed by
capillary zone electrophoresis and electrospray mass spectrometry.
The purified derivatives were used for biological testing according
to the net peptide content as determined by amino acid
analysis.
[0049] Antibodies.
[0050] The rat mAb against CCR3 (clone 61828.111; IgG2a) and the
murine mAb against CCR1 (IgG2b) were obtained from R&D Systems
(Wiesbaden Germany). The rat IgG2a and mouse IgG2b isotype control
Ab were obtained from Immunotech (Hamburg, Germany).
[0051] Eosinophil Isolation.
[0052] Eosinophils were purified from the venous blood of normal
nonatopic healthy or atopic donors using Ficoll (Pharmacia,
Erlangen, Germany) density gradient centrifugation and a negative
selection based on CD16 Microbeads (Miltenyi Biotec, Auburn,
Calif.) as described previously.sup.32. The resulting eosinophil
purity was >990/% as determined by microscopic examination with
Kimura staining.
[0053] Lucigenin-Dependent Chemiluminescence.
[0054] Lucigenin-dependent chemiluminescence representing a
sensitive method to measure the generation of reactive oxygen
species (ROS).sup.33 was performed using a single-photon imaging
system with a two-dimensional photon counting system allowing the
simultaneous measurement and analysis of 96 wells as previously
described in detail.sup.33. Data are expressed as the ratio between
stimulus-induced intensity integral counts and medium-induced
intensity integral counts.
[0055] Internalization of CCR3.
[0056] These experiments were performed as described in detail
previously.sup.32;33. For flow cytometry analysis 10.sup.5
eosinophils were incubated at 4.degree. C. for 30 min with
anti-chemokine receptor mAb or isotype control at the
concentrations recommended by the supplier. In a second step the
cells were stained by FITC-conjugated goat anti-rat (Immunotech) or
goat-anti mouse Ab (Immunotech) and thereafter analyzed by flow
cytometry (FACScan, Becton Dickinson, Heidelberg, Germany).
[0057] For internalization of CCR3, the cells were preincubated for
30 min at 37.degree. C. with the indicated chemokines in a total
volume of 100 .mu.l RPMI 1640 prior to staining. The inhibition of
CCR3 internalization was achieved by initial treatment of the cells
for 5 min with 8 .mu.M phenylarsine oxide (PAO) at 37.degree. C.
and alternatively by treatment of cells with the indicated
chemokines at 4.degree. C. Both strategies are suitable to inhibit
internalization of 7-transmembraneous receptors from the cell
surfaces as described in detail earlier.sup.32.
[0058] In Vitro Chemotaxis.
[0059] Chemotaxis was assessed in 48-well chambers (NeuroProbe,
Cabin John, MD) using polyvinylpyrrolidone-free polycarbonate
membranes with 5-.mu.m pores (Nucleopore, NeuroProbe, Cabin John,
MD) for 5.times.10.sup.4 eosinophils as previously
described.sup.11. For inhibition of CCL11 induced chemotaxis,
eosinophils were preincubated for 15 min at room temperature with
CRIC3 and thereafter directly placed in the upper compartment of
the chemotaxis chamber.
[0060] Measurement of Intracellular Calcium Concentration
[Ca.sup.2+].sub.i
[0061] Eosinophils were loaded with 2 .mu.M fura-2-AM (Molecular
Probes, Eugene, Oreg.) and processed as described
previously.sup.13. Receptor desensitization was tested by
monitoring [Ca.sup.2+].sub.i changes in response to sequential
stimulation with chemokines as described.
[0062] Kinetics of CD26/DPP IV Processing.
[0063] To analyze the processing of the naturally occurring
chemokine CCL14[9-74] and the modified derivative CRIC3, an in
vitro kinetic study was made by incubating 10 .mu.M chemokine with
6,6.times.10.sup.-4 units of porcine kidney DPP IV/CD26 [lot:
100K38002, Sigma, Deisenhofen, Germany] in Tris-HCl, pH 7.5 at
37.degree. C. At specific time intervals, the reactions were
stopped with 0.1% trifluoroacetic acid and placed on ice. For
comparison, the DPP IV/CD26 processing of the previously analyzed
chemokines CCL11 and CXCL12.sup.34 were examined in parallel. The
composition of the reactions was determined offline with a MALDI
mass spectrometer (Voyager DE-Pro, Applied Biosystems, Weiterstadt,
Germany) in linear mode accumulating eight spectra of 100 shots
each. The instrument uses a high-potential acceleration source (20
kV), and other parameters were optimized for measurement of
chemokines.
[0064] Animals
[0065] Syngenic female Balb/c mice, obtained from Charles River
(Sulzfeld, Germany) with an age of 8 weeks and an average weight of
19 g were used in this experiment as described previously.sup.35.
Mice were maintained on laboratory food and tap water ad libitum
under pathogen free conditions in a regular 12 h dark/light cycle
with a temperature of 22.degree. C. and were allowed to become
acclimated to their environment for a period of 7 days prior to
experiment.
[0066] Protocol of Allergic Sensitization and CRIC3 Treatment
[0067] Animals were divided into two groups of n=4. Sensitization
of the animals was carried out via the intraperitoneal route on day
0, 14 and 21 each with 10 .mu.g OVA (chicken ovalbumin grade VI,
Sigma) together with 1 mg Al(OH).sub.3 (Alum Inject, Pierce,
Rockford, Ill.) as adjuvant dissolved in sterile saline.sup.35. To
provoke an allergic airway response, aerosol challenge was
performed using a Pari Master nebulisation system (MMAD 2,5 .mu.m)
and 1% OVA solution. Animals were exposed to allergen on day 28 for
10 min, yielding to a calculated airway allergen deposition of
approximately 10 .mu.g OVA. To examine the inhibitory effect of
CRIC3, four mice were treated with 10 nmol/kg CRIC3 diluted in
sterile saline (applicated via the tail vein) 30 min prior to
allergen exposure and 3 h and 8 h after the challenge. The other
group was injected with sterile saline at identical time
points.
[0068] BALF and Histological Evaluation
[0069] Animals were sacrificed 24h after OVA-challenge by injecting
an overdose of pentobarbital-Na (Narcoren.RTM.)
intraperitoneally.sup.35. The trachea was cannulated, airways were
lavaged with 0,8 ml cold 0.90/% NaCl, and lung dissection was
performed. Total cell numbers in BALF were counted and cytospins
were evaluated. The left lungs were dissected and fixed in formalin
for further histological examinations using hematoxylin-eosin
staining.sup.35.
[0070] Statistical Analysis
[0071] The number of experiments is stated in the legends of the
Fig.s as n and represents different donors. Unless otherwise
stated, the data in the text and Fig.s were expressed as
mean.+-.standard error of the mean (SEM) as determined by
SigmaStat.TM. (SPSS Inc.) analysis. Values of p<0.05 were
accepted as significant using Student's t test.
[0072] Results
[0073] Derivatives of CCL14.
[0074] To characterize the functional importance of the N-terminal
domain of CCL14, the biological activity of ten different CCL14
analogues was investigated. These included: CRIC3
(NNY-CCL14[10-74]), Bis-NNY-CCL14[10-74], full size CCL14[1-74] and
seven N-terminally truncated variants that have been described
previously.sup.26;28;36. The N-terminal sequences of these
derivatives and of CCL11 are shown in FIG. 1.
[0075] Human blood eosinophils as a natural system to study the
effects of CCL14 derivatives on CCR3.
[0076] To study the effects of the CCL14 derivatives on CCR3,
freshly isolated human blood eosinophils were used being a natural
cell population expressing high surface levels of CCR3. Eighty
donors were screened for CCR1 expression and found only two
individuals with significant CCR1 surface expression (data not
shown), which were excluded from the following experiments.
[0077] CCL14 analogs are potent activators of the respiratory burst
mediated by CCR3.
[0078] First, the effects of all CCL14 derivatives on the release
of reactive oxygen species (ROS) were compared using
lucigenin-dependent chemiluminescence, which is established as a
sensitive method to study effector functions mediated by chemokine
receptors on human eosinophils. Among all derivatives studied only
CCL14[8-74], [9-74], [10-74] and CRIC3 induced a significant
release of ROS at concentrations up to 10.sup.-7 M (FIG. 2). These
derivatives were compared at different doses with CCL11, which has
been described as the most potent activator of the respiratory
burst in eosinophils.sup.37 and CCL14[1-74] as the naturally
abundantly occurring form. As shown in FIG. 2, CCL14[9-74] and
CRIC3 are as potent as CCL11 in inducing the release of an
identical amount of ROS at a concentration of 10.sup.-7 M, while
CCL14[8-74] and [10-74] are less potent, and full-length
CCL14[1-74] is virtually inactive. CRIC3 is the most potent
stimulus tested being already significantly active at a
concentration of 10.sup.-9 M (FIG. 2) and almost reaching its
maximal effect at 10.sup.-8 M. The inactive analogs were further
analyzed for antagonistic effects. Pretreatment of human
eosinophils with CCL14[1-74], [6-74], [7-74], [11-74], [12-74] or
Bis-NNY-CCL14[10-74] 10.sup.-7 M did not result in significant
inhibition of ROS release following stimulation with CCL11 at
identical concentrations (data not shown).
[0079] CRIC3 induces internalization of CCR3 as efficient as
CCL11.
[0080] In the next set of experiments, human eosinophils were
incubated for 30 min with all CCL14 derivatives or CCL11 as
positive control at a concentration of 10.sup.-7 M at 37.degree. C.
The cells were then stained with anti-CCR3 mAb and receptor
expression was measured by flow cytometry. Preincubation of human
eosinophils with CCL14[9-74], CCL14[10-74], and CRIC3 led to a
significant down-regulation of CCR3 (FIGS. 3a and b). The other
derivatives including CCL14 [8-74], which was a weak inducer of
ROS, did not affect CCR3 expression (FIG. 3a). At optimal doses
CRIC3 and CCL11 both removed 800% of cell surface CCR3 and were
significantly more effective than CCL14[9-74] (50%) and
CCL14[10-74] (30%). At lower concentrations, CCL11 was superior to
all CCL14 derivatives (FIGS. 3a and b).
[0081] As reduced staining intensity in flow cytometry could also
depend on altered receptor accessibility of the antibody after
preincubation with ligands, the same experiments were performed
with the active ligands at 4.degree. C., a temperature at which
receptor internalization is prevented.sup.38. In the same set of
experiments, the influence of PAO on the expression of CCR3 were
also studied. PAO inhibits the protein tyrosine phosphatase and has
been widely used as a general inhibitor of receptor
internalization.sup.32. Both treatments clearly prevented the
disappearance of CCR3, which was between 30% and 80% at 37.degree.
C. for the active CCL14 derivatives in the same experimental
setting (FIG. 3c). These data clearly show that the induced
decrease of cell surface CCR3 on human eosinophils was due to
receptor internalization and that CRIC3 induced internalization of
CCR3 is as efficient as by CCL11.
[0082] CRIC3 is the most potent eosinophil chemoattractant among
CCL14 derivatives and inhibits CCL11-induced chemotaxis.
[0083] To evaluate whether the most active CCL14 derivatives induce
chemotaxis of CCR3-positve cells, migration assays were carried out
in 48-well micro chemotaxis chambers. As shown in FIG. 4a CCL14
derivatives are effective chemoattractants for human eosinophils.
The activity of the derivatives tested was similar in terms of the
efficacy, as indicated by the number of migrated cells. Maximal
responses to CCL14[9-74] and CCL14[10-74] were reached at 300 and
1,000 nM, respectively. CRIC3 is more potent than the other
derivatives, as its maximal effect observed was at 10 nM, which is
in the range of eotaxin as previously shown.sup.11. When
eosinophils are treated with 100 nM CRIC3 prior to application to
the migration chamber, it dramatically decreases the migratory
response towards CCL11 close to medium response (FIG. 4b).
[0084] CRIC3 Induces a Functional Desensitization of CCR3
[0085] CCL11 induced [Ca.sup.2+].sub.i changes in human eosinophils
are exclusively mediated via CCR3.sup.39. In order to study the
potential of the active CCL14 derivatives to desensitize CCR3,
heterologous desensitization experiments were performed with the
most active CCL14 derivatives and CCL11. Stimulation of eosinophils
with CCL14[9-74] or CRIC3 at 10.sup.-7 M completely desensitized
the cells to CCL11 at the same dose (FIGS. 5a and c). The less
active form CCL14[10-74] did virtually not desensitize eotaxin at
equal doses (FIG. 5e), which is in agreement with the results
obtained for the release of ROS and the moderate CCR3
internalization after preincubation with this ligand. Stimulation
of eosinophils with CCL11 completely desensitized the active CCL14
derivatives in all experiments performed (FIGS. 5b, d, and f),
indicating that all donors used did not express functional CCR1.
These results demonstrate that CRIC3 induces a functional
desensitization of CCR3 and makes it resistant to its activation by
CCL11.
[0086] CRIC3 is Resistant to CD26/DPP IV Processing.
[0087] CD26/DPP IV processing of CCL11, CCL14[9-74] and CRIC3 was
analyzed in vitro, essentially as described by Lambeir et
al..sup.34. First the amount of enzyme applied was optimized to
give a kinetic profile for CCL11 and CXCL12 comparable to that
previously reported (FIG. 6a). CXCL12 was completely processed
within 10 min and CCL11[1-74] was fully converted into CCL11[3-74]
after 1 h at 37.degree. C. Using these conditions, the processing
of CCL14[9-74] was followed over time in a similar fashion.
Although significantly slower than for CCL11 and CXCL12, a
virtually complete conversion of CCL14[9-74] into CCL14[11-74] was
achieved within 12 h. In contrast to these chemokines CRIC3
remained completely resistant to CD26/DPP IV treatment after 24 h
(FIGS. 6b and c) and even after 90 h of incubation (not shown).
[0088] CRIC3 is an Effective Inhibitor of Eosinophil Infiltration
in a Murine Model of Allergic Lung Inflammation
[0089] To test the hypothesis that CRIC3 may influence the influx
of eosinophils in vivo a murine model of allergic asthma was used.
The intravenous application of 10 nM/kg CRIC3 30 min before and 3 h
and 8 h after allergen aerosol provocation significantly reduced
the infiltration of eosinophils into the airways in comparison to
the saline treated control group. Eosinophil infiltration into the
lung tissue was demonstrated by standard staining procedures and
was clearly reduced in the CRIC3 treated animals (FIGS. 7a and b).
For quantification of the effect on inflammation, differential cell
counts were performed on cytospins of BALF. Application of CRIC3
significantly reduced the influx of eosinophils into the airways
when compared to the saline treated group (0.2 vs.
1.62.times.10.sup.4 cells per ml BALF, p<0.005) (FIG. 8).
[0090] Discussion
[0091] Recent discoveries on the immunological mechanisms of asthma
have markedly altered our understanding of this common respiratory
disorder. These insights have been gained during a persistent
period of rising disease incidence and severity and are now being
applied to develop improved therapies.sup.40. Whereas steroids are
one of the most potent and broad-spectrum drug for the therapy of
the inflammatory phase in asthma.sup.41, many new concepts focus to
block a specific cytokine, chemokine or cell type in the expectance
to reduce side effects and to be more successful. In this context
humanized antibodies, e.g. against IL-5.sup.42, and chemokine
receptor antagonist have been developed to hinder the invasion of
leukocytes to the site of inflammation.sup.43. Whereas most of
these anti-inflammatory strategies act on the target cells after
they migrated to the site of inflammation, a concept is proposed in
which the cellular recruitment is prevented by inactivation of the
chemokine receptor before cells leave the blood vessels. Herein it
is demonstrated that the CD26-resistant inactivator of CCR3, CRIC3,
which derives from the recently identified chemokine CCL14[9-74] is
a potent ligand of the human CC chemokine receptor CCR3 and able to
prevent eosinophil invasion into the lung in a murine model of
allergic asthma.
[0092] Recently, the importance of the CC chemokine receptor CCR3
in allergic asthma has been highlighted. This receptor is expressed
constitutively or upon activation of cytokines on eosinophils, Th2
cells, basophils and mast cells.sup.17-20. All these cells
contribute to the inflammatory infiltrate in allergic asthma.sup.1.
The deletion of the CCR3 locus in the germ line of mice gave new
insight in the role of this receptor for the trafficking of
different immune cells into the lung.sup.23;24. These studies
showed in a model of allergen-induced airway inflammation, that
allergen challenge results in subendothelial trapping of
eosinophils in CCR3 deficient mice, while wild-type controls had an
impressive infiltration of the lung accompanied by lymphocytes,
which were both not found in CCR3-/- mice. Moreover, the
CCR3-deficient mice are completely protected from allergen-induced
AHR, if the epicutaneous route is used for sensitization instead of
the intraperitoneal, underlining the relevance of CCR3 in several
phases of asthma.sup.23;24.
[0093] In the past, the naturally occurring mature form of CCL14
were isolated, circulating in nanomolar levels in the plasma,
exhibiting minimal biological effects.sup.26. Further
investigations led to the identification of the N-terminal
truncated form CCL14[9-74], which can be generated in
nature.sup.28;44. This variant was shown to be a good agonist of
CCR3. In this study, it was our aim to design a ligand of CCR3
leading to its inactivation. Therefore, several forms of CCL14 with
different N-terminal length were synthesized. However, screening of
the variants for their potential to inhibit the respiratory burst
induced by CCL11, did not result in the identification of an
antagonist. As several studies have demonstrated, that modification
of the N-terminal amino acid reveal dramatic changes in the
activity of chemokines, the ultimate amino acid of the most active
variant were replaced. For this replacement, nonanoic acid was used
as NNY-RANTES was recently shown to be a more potent inhibitor of
HIV infection than AOP-RANTES.sup.45.
[0094] Here it is shown that the modified variant of CCL14, CRIC3
acts as a high-affinity agonist of CCR3, as only human eosinophils
were used expressing CCR3 but not CCR1 (see results). Screening of
different variants of CCL14 using an assay for the respiratory
burst revealed that CRIC3 is a highly potent agonist of CCR3
identical to CCL11. In addition, this modified peptide induces
surface internalization of CCR3 as effective as CCL11. Performing
chemotaxis assays with eosinophils revealed an activity profile
being very similar to CCL11.sup.11. Furthermore, in desensitization
experiments the usage of CCR3 by CRIC3 was confirmed which makes it
resistant to activation by CCL11.
[0095] As expected from the amino acid sequence it was demonstrated
that CRIC3 was not degraded by DPP IV/CD26. This is a relevant
difference in comparision to CCL11. The latter was processed
rapidly in its inactivated form, CCL11[3-74]. This processing
reduces the interaction of CCL11 with CCR3.sup.46 CD26/DPP IV
occurs in highly active levels in human plasma and is also well
known as a widely distributed cell-surface glycoprotein.sup.47;48.
The plasma stability of other peptides such as GLP-1, a potent
insulinotropic hormone expressed by intestinal L-cells, have been
shown to depend on degradation by CD26/DPP IV.sup.49. The
anti-diabetogenic effect of GLP-1 was clearly demonstrated in
patients with type-II diabetes.sup.50 and therefore it is not
surprising, that several antagonists of CD26/DPP IV are already
launched for clinical trials.sup.51. However, as a suppressor of
CCR3 induced inflammation, the CD26-resistant inactivator of CCR3,
CRIC3, is most likely superior to a combination of CCL11 and an
inhibitor of the broad spectrum enzyme DPP IV/CD26. The most potent
CCR3 peptide antagonist yet described, I-Tac/E.sub.0H1 contains the
eight N-terminal amino acids of CXCL11 (1-TAC).sup.53. As CXCL11 is
efficiently cleaved by CD26/DPP IV.sup.52, I-Tac/E.sub.0H1 is
likely to share a similar fate, resulting in a short plasma
half-life.
[0096] The therapeutic use of chemokines or their derivatives with
antagonistic or agonistic properties such as Met-RANTES.sup.54,
AOP-RANTES.sup.55, or NNY-RANTES.sup.56 has been intensively
discussed in the literature especially for the inhibition of HIV
infection.sup.57 The treatment of asthma is one rationale for the
development of CCR3 antagonists like Met-RANTES or
ITAC/EoH1.sup.53. Other modifications like the replacement of the
first amino-terminal residue by aminooxypentane, which was done to
create an antagonist.sup.55 revealed at least a partial
agonist.sup.33. Besides peptides, small molecular weight compounds
are also considered as potential drugs for the blockade of
chemokine receptors.
[0097] Since the latter compounds derived from piperazine and
piperidine may exhibit unexpected side effects (particularly heart
and central nervous system), peptide ligands inactivating chemokine
receptors represent a respectable alternative due to a better
tolerance.
[0098] Based oh our observations, it is not devious to propose the
use of full agonists for the treatment of disease. Many antagonists
used in other medical areas have an intrinsic activity, which is
well tolerated. There are also agonists on the market, which are
applied to induce inhibitory effects, such as LHRH analogs, which
are used to down-regulate hormone release.sup.58. Therefore, the
present invention discloses a method of an agonistic receptor
inactivator mediating its effects through desensitization and
internalization. CRIC3 might be applied into a defined body
compartment to activate cells at sites were they are not harmful
and thereby rendering them insensitive to further activation
through the same receptor. This inactivation is the result of
desensitization depending most likely on the uncoupling of the
receptor from heterotrimeric G proteins in response to receptor
phosphorylation and the internalization of receptors to
intracellular membranous compartments.sup.59.
[0099] To prove whether CRIC3 is able to block the infiltration of
CCR3+ cells into the lung tissue we used a murine model of allergic
asthma. Intravenous administration of CRIC3 prior and after
allergen challenge was well tolerated without any side effects.
Remarkably, the number of eosinophils in the BALF as well as in the
lung tissue dramatically decreased in the CRIC3-treated group.
Therefore, a blockade of the migration towards the inflammatory
focus is the result of the unresponsiveness. The blood represents
the ideal compartment for application of such a CD26-resistant
inactivator of CCR3, as generated toxic substances like ROS
accumulate much slower than directly in the inflamed tissues. Other
forms of application than systemic administration might cause
adverse events. Therefore, peptides would be interesting candidates
as they are mainly administered parenteral and are usually highly
specific and have therefore relatively low systemic
toxicity.sup.60. It should also be mentioned that agonists such as
CRIC3 have to be given at much lower doses and less frequently to
desensitize the effector cells than antagonists like Met-RANTES to
block cellular responsiveness.sup.61, as down-regulation of surface
receptors might prolong the effects of agonists in comparison to
antagonists. The reason that we were not able to prevent the influx
of all eosinophils into the lung (quantified by BALF analysis) may
be due to the application and provocation protocol in our study.
Further studies are necessary for pharmacokinetic evaluation and to
show the effect on AHR. Nevertheless, we could clearly demonstrate
that CRIC3 principally acts in vivo.
[0100] Up to date, peptides with agonistic activities have not been
proposed for the treatment of CCR3 driven pathologies. As shown by
our study, CRIC3 represents such a candidate. Therefore, CRIC3
could be useful in diseases which are dominated by effects
depending on CCR3, but also in diseases with a broader spectrum of
chemokine receptors involved, which might be true for asthma. In
accordance with this, the broad-spectrum chemokine receptor
antagonist vMIPII has been shown to be more effective in a model of
spinal cord injury with posttraumatic inflammation than the more
specific antagonist MCP-1(9-76).sup.62;63. The results of our study
implicate the new concept to generate an inactivating agonist which
is resistant to degradation by serum and tissue peptidases. As a
consequence of the inactivation of receptors by an agonist, the
recruitment of eosinophils, basophiles, and Th.sub.2 cells into
tissues could be prevented in CCR3-driven pathologies as suggested
by our study.
[0101] Abbreviations
[0102] CCL, CC chemokine ligand; CRIC3, n-nonanoyl-CCL14[10-74];
bis-NNY-CCL14[10-74], Bis-n-nonanoyl-CCL14[10-74]; RANTES,
regulation upon activation and T cell secreted; BALF,
bronchoalveolar lavage fluid; AHR, airway hyper responsiveness;
OVA, ovalbumin
[0103] FIG. 1:
[0104] Alignment of N-terminal sequences of CCL14 derivatives and
CCL11. The cleavage motif for CD26/DPP IV of CCL14[9-74] and CCL11
(eotaxin) is marked in gray.
[0105] FIG. 2:
[0106] CRIC3 induces the release of reactive oxygen species (ROS)
from human eosinophils with more potency than CCL11.
[0107] The release of ROS was measured using lucigenin-dependent
chemiluminescence. Human eosinophils were stimulated with different
concentrations of the indicated chemokine. Data (n=7) are expressed
as relative ROS release that is expressed as the ratio of
stimulus-stimulated and medium-stimulated cells.
[0108] FIG. 3:
[0109] CRIC3 induces an internalization of CCR3 from human
eosinophils in the same range than CCL11.
[0110] Human eosinophils were treated with the indicated CCL14
derivatives (10.sup.-7 M) and CCL11(10.sup.-7 M), respectively, for
30 min at 37.degree. C. Thereafter cells were stained with
anti-CCR3 mAb and analyzed by flow cytometry. A: Data (n=4) are
expressed as the mean.+-.SEM of relative fluorescence intensity as
described in Materials and Methods. B: Histogram analysis of one
representative experiment shown in Fig. A. Bold line, anti-CCR3
staining before chemokine treatment; dotted line, isotype control;
broken line, anti-CCR3 staining after chemokine treatment. C: Cells
were incubated with the indicated indicated chemokine (10.sup.-7
M), at 37.degree. C., 4.degree. C. or pretreated with PAO. Data
(n=4) are expressed as the mean.+-.SEM of relative fluorescence
intensity.
[0111] FIG. 4:
[0112] CRIC3 induces chemotaxis of eosinophils but also inhibits
CCL11.
[0113] A) CCL14 derivatives induce in vitro chemotaxis of human
eosinophils. Numbers of migrating cells per five high power
(.times.1000) fields are given. One out of three similar
experiments performed with cells from different donors is shown. B)
Pretreatment of eosinophils with 100 nM CRIC3 15 min before loading
to the chemotaxis chamber dramatically inhibits CCL11 induced
migration.
[0114] FIG. 5:
[0115] CRIC3 induces a functional desensitization of CCR3.
[0116] Human eosinophils were loaded with Futa-2 and [Ca.sup.2+]
was measured using spectrofluometry. Cells were stimulated with the
indicated chemokine (10.sup.-7 M) and the anaphylatoxin C5a
(10.sup.-8 M). Data are expressed as original plot of one
representative experiment out of five.
[0117] FIG. 6:
[0118] CRIC3 is not processed by CD26/DPP IV. A and B)
CCL11/eotaxin[1-74], CXCL12/SDF-1.alpha.[22-89], and
CCL14/HCC-1[9-74] (10 .mu.M each) were incubated for the indicated
times with porcine kidney CD26/DPP IV as described in Materials and
Methods and analyzed using mass spectrometry. Processing was
calculated as amount of full-length chemokine related to total
amount of the full-length and processed forms as defined by the
peak heights. C) Partial MALDI mass spectrometry spectra of
chemokines after incubation for different times. The relative
molecular masses of unprocessed chemokines are indicated on the
right side of the peaks, and of processed (minus 2 N-terminal amino
acids) on the left.
[0119] FIG. 7.
[0120] CRIC3 prevents the diapedesis of eosinophils into the lung
tissue of OVA-sensitized mice. The photographs represent the
reduced diapedesis of eosinophils being arrested on the venule
eridothelium in CRIC3-treated mice (A), in comparison to the
saline-treated group having a marked peribronchial infiltration
primarily consisting of eosinophils (B). Original magnification
630.times..
[0121] FIG. 8.
[0122] CRIC3 prevents the migration of eosinophils into the BALF of
OVA-challenged mice. OVA-challenged mice were treated with CRIC3
(3.times.10 nmol/kg) or saline, respectively. Cell composition in
BALF 24 h after allergen challenge was analyzed differentiating 500
cells per cytospin and expressed as total cell numbers.
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Sequence CWU 1
1
20 1 17 PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CCL14 derivative CCL14[1-74] 1 Thr Lys Thr
Glu Ser Ser Ser Arg Gly Pro Tyr His Pro Ser Glu Cys 1 5 10 15 Cys 2
12 PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CCL14 derivative CCL14[6-74] 2 Ser Ser Arg
Gly Pro Tyr His Pro Ser Glu Cys Cys 1 5 10 3 11 PRT Artificial
Sequence Description of Artificial Sequence N-terminal sequence of
CCL14 derivative CCL14[7-74] 3 Ser Arg Gly Pro Tyr His Pro Ser Glu
Cys Cys 1 5 10 4 10 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CCL14 derivative
CCL14[8-74] 4 Arg Gly Pro Tyr His Pro Ser Glu Cys Cys 1 5 10 5 9
PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CCL14 derivative CCL14[9-74] 5 Gly Pro Tyr
His Pro Ser Glu Cys Cys 1 5 6 9 PRT Artificial Sequence Description
of Artificial Sequence N-terminal sequence of CCL11 (eotaxin) 6 Gly
Pro Ala Ser Val Pro Thr Cys Cys 1 5 7 8 PRT Artificial Sequence
Description of Artificial Sequence N-terminal sequence of CCL14
derivative CCL14[10-74] 7 Pro Tyr His Pro Ser Glu Cys Cys 1 5 8 7
PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CCL14 derivative CCL14[11-74] 8 Tyr His Pro
Ser Glu Cys Cys 1 5 9 6 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CCL14 derivative
CCL14[12-74] 9 His Pro Ser Glu Cys Cys 1 5 10 8 PRT Artificial
Sequence Description of Artificial Sequence N-terminal sequence of
CCL14 derivative CRIC3 10 Pro Tyr His Pro Ser Glu Cys Cys 1 5 11 8
PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CCL14 derivative Bis-NNY-CCL14 11 Pro Tyr
His Pro Ser Glu Cys Cys 1 5 12 65 PRT Artificial Sequence
Description of Artificial Sequence N-terminal sequence of CCL14
derivative CCL14[10-74] 12 Pro Tyr His Pro Ser Glu Cys Cys Phe Thr
Tyr Thr Thr Tyr Lys Ile 1 5 10 15 Pro Arg Gln Arg Ile Met Asp Tyr
Tyr Glu Thr Asn Ser Gln Cys Ser 20 25 30 Lys Pro Gly Ile Val Phe
Ile Thr Lys Arg Gly His Ser Val Cys Thr 35 40 45 Asn Pro Ser Asp
Lys Trp Val Gln Asp Tyr Ile Lys Asp Met Lys Glu 50 55 60 Asn 65 13
67 PRT Artificial Sequence Description of Artificial Sequence
N-terminal sequence of CXCL12 derivative CXCL12[1-67] 13 Lys Pro
Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20
25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg
Gln 35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr
Leu Glu Lys 50 55 60 Ala Leu Asn 65 14 67 PRT Artificial Sequence
Description of Artificial Sequence N-terminal sequence of CXCL12
derivative CXCL12V3I[1-67] 14 Lys Pro Ile Ser Leu Ser Tyr Arg Cys
Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val
Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Leu Gln
Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile
Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Ala
Leu Asn 65 15 66 PRT Artificial Sequence Description of Artificial
Sequence N-terminal sequence of CXCL12 derivative CXCL12[2-67] 15
Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser His 1 5
10 15 Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro
Asn 20 25 30 Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn
Arg Gln Val 35 40 45 Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu
Tyr Leu Glu Lys Ala 50 55 60 Leu Asn 65 16 66 PRT Artificial
Sequence Description of Artificial Sequence N-terminal sequence of
CXCL12 derivative CXCL12V3I[2-67] 16 Pro Ile Ser Leu Ser Tyr Arg
Cys Pro Cys Arg Phe Phe Glu Ser His 1 5 10 15 Val Ala Arg Ala Asn
Val Lys His Leu Lys Ile Leu Asn Thr Pro Asn 20 25 30 Cys Ala Leu
Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln Val 35 40 45 Cys
Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys Ala 50 55
60 Leu Asn 65 17 72 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CXCL12 derivative
CXCL12[1-72] 17 Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe
Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys
Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg
Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro Lys Leu
Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Ala Leu Asn Lys Arg
Phe Lys Met 65 70 18 72 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CXCL12 derivative
CXCL12V3I[1-72] 18 Lys Pro Ile Ser Leu Ser Tyr Arg Cys Pro Cys Arg
Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu
Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Leu Gln Ile Val Ala
Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro Lys
Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Ala Leu Asn Lys
Arg Phe Lys Met 65 70 19 71 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CXCL12 derivative
CXCL12[2-72] 19 Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe
Glu Ser His 1 5 10 15 Val Ala Arg Ala Asn Val Lys His Leu Lys Ile
Leu Asn Thr Pro Asn 20 25 30 Cys Ala Leu Gln Ile Val Ala Arg Leu
Lys Asn Asn Asn Arg Gln Val 35 40 45 Cys Ile Asp Pro Lys Leu Lys
Trp Ile Gln Glu Tyr Leu Glu Lys Ala 50 55 60 Leu Asn Lys Arg Phe
Lys Met 65 70 20 71 PRT Artificial Sequence Description of
Artificial Sequence N-terminal sequence of CXCL12 derivative
CXCL12V3I[2-72] 20 Pro Ile Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe
Phe Glu Ser His 1 5 10 15 Val Ala Arg Ala Asn Val Lys His Leu Lys
Ile Leu Asn Thr Pro Asn 20 25 30 Cys Ala Leu Gln Ile Val Ala Arg
Leu Lys Asn Asn Asn Arg Gln Val 35 40 45 Cys Ile Asp Pro Lys Leu
Lys Trp Ile Gln Glu Tyr Leu Glu Lys Ala 50 55 60 Leu Asn Lys Arg
Phe Lys Met 65 70
* * * * *