U.S. patent application number 13/283508 was filed with the patent office on 2012-06-07 for compositions for preventing, reducing or treating keratinocyte-mediated inflammation.
This patent application is currently assigned to UNIVERSITE D'ANGERS. Invention is credited to Francois-Xavier Bernard, Katia Boniface, Sylvie Chevalier, Caroline Diveu, Hughes Gascan, Jean-Claude Lecron, Franck Morel.
Application Number | 20120142755 13/283508 |
Document ID | / |
Family ID | 34931608 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142755 |
Kind Code |
A1 |
Lecron; Jean-Claude ; et
al. |
June 7, 2012 |
COMPOSITIONS FOR PREVENTING, REDUCING OR TREATING
KERATINOCYTE-MEDIATED INFLAMMATION
Abstract
The present invention relates to the field of epidermal repair.
More particularly, the invention concerns the use of a molecule
able to inhibit a heteromeric receptor comprising OSMR.beta. as a
subunit, for the preparation of a composition for inhibiting the
expression of inflammatory factors by the keratinocytes. In
particular, the invention concerns the use of antagonists and/or
expression inhibitors of OSM, IL-17, TNF.alpha., IL-31,
IFN-.gamma., and/or the OSMR.beta. subunit, for the preparation of
cosmetic or dermatologic compositions, especially for treating
inflammatory skin diseases. Methods for obtaining in vitro or
animal models of skin inflammation diseases are also disclosed.
These models can be used for screening molecules to find new drugs
to prevent or treat psoriasis and other dermatitis.
Inventors: |
Lecron; Jean-Claude;
(Bonnes, FR) ; Gascan; Hughes; (Angers, FR)
; Morel; Franck; (Savigney L'Evescault, FR) ;
Chevalier; Sylvie; (Angers, FR) ; Bernard;
Francois-Xavier; (Saint Maurice La Clouere, FR) ;
Boniface; Katia; (Mountain View, CA) ; Diveu;
Caroline; (Palo Alto, CA) |
Assignee: |
UNIVERSITE D'ANGERS
Angers
FR
BIOALTERNATIVES SAS
Gencay
FR
UNIVERSITE DE POITIERS
Poitiers cedex
FR
|
Family ID: |
34931608 |
Appl. No.: |
13/283508 |
Filed: |
October 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11721763 |
Feb 4, 2008 |
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PCT/EP2005/014198 |
Dec 15, 2005 |
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13283508 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61K 38/20 20130101;
A61P 37/08 20180101; A61P 17/06 20180101; C12N 5/0629 20130101;
A61K 38/204 20130101; A61P 17/00 20180101; A61P 17/12 20180101;
A61Q 19/00 20130101; A61P 17/08 20180101; A61P 17/02 20180101; A61P
29/00 20180101; A61K 8/64 20130101; A61P 17/04 20180101; C12N
2501/2306 20130101; A61K 38/20 20130101; A61K 38/1793 20130101;
C07K 16/28 20130101; A61K 38/217 20130101; A61P 17/10 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/217 20130101;
A61K 2300/00 20130101; C07K 2317/76 20130101; A61K 38/204 20130101;
A61K 38/1793 20130101; A61P 43/00 20180101; A61K 2300/00
20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61P 17/00 20060101 A61P017/00; A61P 29/00 20060101
A61P029/00; A61P 17/06 20060101 A61P017/06; A61P 17/12 20060101
A61P017/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 15, 2004 |
EP |
04293004.0 |
Claims
1. A method for improving epidermal repair in a patient in need
thereof, comprising a step of administering a composition to said
patient, wherein said composition comprises at least one molecule
selected from the group consisting of antagonists and expression
inhibitors of a cytokine selected amongst OSM, IL-17, TNF.alpha.,
IL-31 and IFN-.gamma., and antagonists and expression inhibitors of
the OSMR.beta. subunit.
2. The method according to claim 1, wherein said composition
prevents, reduces, or treats keratinocyte-mediated
inflammation.
3. The method according to claim 1, wherein said composition
comprises an OSM antagonist and/or an IL-17 antagonist and/or a
TNF.alpha. antagonist.
4. The method according to claim 1, wherein said composition
comprises an OSM expression inhibitor and/or an IL-17 expression
inhibitor and/or a TNF.alpha. expression inhibitor and/or an
OSMR.beta. expression inhibitor.
5. The method according to claim 4, wherein said expression
inhibitor is a siRNA.
6. The method according to claim 1 for preventing, alleviating or
treating a skin inflammation disease selected from the group
consisting of psoriasis, atopic dermatitis, bullous epidermolysis,
bullous phemphigoid, lichen, acne, eczema, professional dermatitis,
seborrheic dermatitis, keratosis, rosacea, erythema and
ichtyosis.
7. The method according to claim 1, wherein said composition
inhibits keratinocyte migration.
8. The method according to claim 1, wherein said composition
prevents or reduces epidermal thickening.
9. The method according to claim 1, wherein said composition slows
down epidermal healing.
10. The method according to claim 1, wherein said composition
prevents and/or attenuates chaps on hands, lips, face or body.
11. The method according to claim 1, wherein said composition
prevents and/or attenuates stretch marks.
12. The method according to claim 1, wherein said composition is
used for improving the aspect and comfort of scars.
13. The method according to claim 1, wherein said composition
improves the aspect and comfort of epidermal wounds during their
cicatrisation.
14. The method according to claim 1, wherein the composition is a
mixture containing at least two different molecules selected from
the group consisting of antagonists and expression inhibitors of a
cytokine selected amongst OSM, IL-17, TNF.alpha., IL-31 and
IFN-.gamma., and antagonists and expression inhibitors of the
OSMR.beta. subunit.
15. A method for preventing, reducing and/or treating
keratinocyte-mediated inflammation in a patient in need thereof,
comprising a step of administrating a composition to said patient,
wherein said composition comprises an antagonist of OSM.
16. The method according to claim 15, for preventing, reducing
and/or treating psoriasis.
17. The method according to claim 15, wherein said composition
comprises an antagonist of OSM and an antagonist of IL-17.
18. The method according to claim 17, for treating psoriasis.
Description
[0001] The present invention relates to the field of epidermal
repair. More particularly, the invention concerns the use of a
molecule able to inhibit a heteromeric receptor comprising
OSMR.beta. as a subunit, for the preparation of a composition for
inhibiting keratinocyte migration and/or the expression of
inflammatory factors by the keratinocytes. The invention also
pertains to the use of a molecule which activates heteromeric
receptor comprising OSMR.beta. as a subunit, for obtaining in vitro
and/or in vivo models of inflammatory skin diseases.
[0002] The skin is a large and complex tissue providing a
protective interface between an organism and its environment.
Epidermis forms its external surface, and is mainly constituted of
multiple layers of specialized epithelial cells named
keratinocytes. Skin can be injured by many different causes,
including micro-organisms, chemicals, behaviours, physical injury,
ageing, U.V. irradiation, cancer, autoimmune or inflammatory
diseases.
[0003] Epidermis homeostasis is regulated by a balance between
differentiation and proliferation of keratinocytes, differentiating
from the basal to the cornified layers of the skin. In response to
epidermal stress or in some skin diseases, this equilibrium is
broken. Keratinocytes become able to differentially respond to
soluble mediators such as Epidermal Growth Factor (EGF) family
members, and to additional growth factors and cytokines (FGFs,
IGF-1, PDGF, HGF, TGF.beta. family members, GM-CSF, TSLP, IL-1,
TNF-.alpha.). These modulators are produced by the keratinocytes
themselves, the skin fibroblasts, the Langerhans cells or by immune
infiltrating cells such as T lymphocytes. In response, the
keratinocytes release additional signaling molecules, modulate the
expression level of cell surface receptors, modify their
cytoskeleton morphology, and modulate their migration,
differentiation and proliferation capacities. These changes are
associated with an inflammatory response, leading to either wound
healing or to a chronic disease.
[0004] Psoriasis is the most common auto-immune disease of the
skin, affecting 2% of the population. The pathology results from a
hyperactivation of T lymphocytes that remain resident in the
epidermis of psoriatic lesions. Through the secretion of T helper
type 1-like cytokines, these T cells contribute to the epidermal
proliferation and thickness in psoriatic patients (Lew, Bowcock et
al. 2004).
[0005] Recently, it has been reported that epidermal keratinocytes
from transgenic mice that express constitutively active STAT3
showed up-regulation of several gene products that are linked to
the pathogenesis of psoriasis (Sano, Chan et al. 2005). However,
although required, STAT3 activation alone is not sufficient to
induce psoriatic lesions. The exact nature of the cytokines
secreted by activated intralesional T cells involved in
keratinocyte alterations, including cellular hyperproliferation,
abnormal differentiation and a specific gene expression profile,
characteristic of the psoriatic lesion, has not been
established.
[0006] The cytokines of the IL-6 family are multifunctional
proteins regulating cell growth and differentiation in a large
number of biological systems, such as immunity, hematopoiesis,
neural development, reproduction, bone modeling and inflammatory
processes. This cytokine family encompasses nine different members:
IL-6, IL-11, IL-27, leukemia inhibitory factor (LIF),
cardiotrophin-1, cardiotrophin-like factor, ciliary neurotrophic
factor, neuropoietin, and oncostatin M (OSM). The activities of
theses cytokines are mediated through ligand-induced
oligomerization of a dimeric or trimeric receptor complex. The IL-6
family of cytokines shares the gp130 receptor subunit in the
formation of their respective heteromeric receptors (Taga and
Kishimoto 1997). A recently described cytokine, named IL-31, has
been classified by Dillon et al as a novel member of the 130-IL6
family, because its receptor is a heterodimer comprising gp130-like
type I cytokine receptor (GPL) and an OSMR subunit (Dillon,
Sprecher et al. 2004).
[0007] Different publications have reported that some members of
the IL-6 family may be implicated in certain skin diseases and
wound healing processes. IL-6, IL-11, LIF and OSM have been found
to be increased in psoriatic lesions (Bonifati, Mussi et al. 1998),
and IL-6 and LIF are produced by purified keratinocytes (Paglia,
Kondo et al. 1996; Sugawara, Gallucci et al. 2001). An impaired
wound healing process has been reported in IL-6 and STAT3 deficient
mice (Sano, Itami et al. 1999; Gallucci, Simeonova et al. 2000).
However, further studies on cultured keratinocytes isolated from
IL-6 deficient mice showed that the action of IL-6 on keratinocyte
migration is mediated by dermal fibroblasts. Indeed, IL-6 alone did
not significantly modulate the proliferation or migration of said
IL-6-deficient keratinocytes, whereas IL-6 significantly induced
their migration when co-cultured with dermal fibroblasts (Gallucci,
Sloan et al. 2004).
[0008] OSM is secreted from activated T cells, monocytes stimulated
by cytokines and from dendritic cells. OSM is a pro-inflammatory
mediator, which strongly triggers protein synthesis in hepatocytes
(Benigni, Fantuzzi et al. 1996). In humans, OSM and LIF display
overlapping biological functions in a number of tissues by
increasing growth regulation, differentiation, gene expression and
cell survival. OSM is also known to elicit some unique biological
functions, not shared with LIF, such as growth inhibition of some
tumor cell lines or stimulation of AIDS-associated Kaposi's sarcoma
cells. These shared and specific functions of OSM are explained by
the existence of two types of OSM receptor complexes. Beside the
common LIF/OSM receptor complex made of gp130/LIFR.beta.subunits,
OSM is also able to specifically recognize a type II receptor
associating gp130 with OSMR.beta. (also referred to as "OSMR" or
"OSM-R"), which is expressed by endothelial cells, hepatic cells,
lung cells, fibroblasts, hematopoietic cells and by some tumor cell
lines. The subsequent signaling cascade involves activation of the
Janus kinase (JAK 1, JAK 2, Tyk 2), followed by an activation of
the Signal Transducer and Activator of Transcription (STAT1, STAT3)
and of the Map kinase pathways.
[0009] In addition to its anti-neoplastic activity and its role in
the proinflammatory response (Wahl and Wallace 2001); Shoyab et al,
U.S. Pat. No. 5,451,506; Richards et al., U.S. Pat. No. 5,744,442),
OSM has been described as stimulating the growth of dermal
fibroblasts via a MAP kinase-dependent pathway, thereby promoting
dermal wound healing (Ihn and Tamaki 2000).
[0010] Other cytokines are also known to have an effect on dermis.
For example, Dillon et al (supra) suggest that overexpression of
IL-31 may be involved in promoting the dermatitis and epithelial
responses that characterize allergic and non-allergic diseases.
These authors do not suggest to use IL-31 for promoting skin
repair.
[0011] Presently, there exists a real need for novel dermatological
approaches for improving epidermal repair, especially in the
context of inflammation diseases. Inhibiting STAT3 and/or
pro-inflammatory cytokines would clearly ameliorate the status of
patients suffering from a variety of inflammatory diseases such as
psoriasis, atopic dermatitis, professional dermatitis, seborrheic
dermatitis, rosacea, erythema, eczema and certain kinds of acne.
Acting on the keratinocytes' differentiation and migration is also
necessary for treating other specific diseases such as eschars,
keratosis, squama, ulcers, ichtyosis, malum perforan pedis and
bullous epidermolysis. The phrase "bullous emolysis" designates a
number of dermatitis of different origins (such as bleds, burns,
autoimmune diseases, . . . ), leading to a detachment of the
epidermis and liquid accumulation between dermis and epidermis. A
particular example of bullous emolysis is bullous phemphigoid.
[0012] Improving epidermal repair is also important in the cosmetic
field, where no efficient compositions exist for improving the
aspect of scars, originating either from recent small wounds or
from old cuts, spots, stretch marks and the like.
[0013] In this context, the inventors found that several cytokines,
in particular OSM and IL-31, can enhance the expression of a number
of genes involved in inflammatory processes. As described above,
these two cytokines bind to different heteromeric receptors, that
both comprise OSM.beta. as a subunit. The inventors have shown that
normal human epidermal keratinocytes express gp130, GPL and
OSMR.beta..
[0014] As disclosed in the experimental examples below, OSM
recruits the STAT3 signaling pathways, as well as the MAP kinase
pathways in human epidermal keratinocytes. OSM up-regulates the
expression of pro-inflammatory genes in these cells, including
chemokines, defensin and the psoriasin. OSM also increases the
thickness of reconstituted human epidermis and down-regulates a set
of differentiation antigens. The inventors have obtained a
psoriasis-like phenotype in mice in which intradermal injections of
OSM were performed. Interestingly, other cytokines, especially
IL-17 and TNF.alpha., act synergistically with OSM and potentiate
its effects.
[0015] Experiments conducted by the inventors also revealed that
IL-31 can mediate keratinocyte migration. The inventors however
observed, in glioblastoma and melanoma tumor cells, that the action
of IL-31 depends on the type of GPL subunit involved with
OSMR.beta. in the formation of the heteromeric receptor. In
particular, they noticed that a short form of GPL receptor exerts a
profound inhibitory effect on the signaling of IL-31 and behaves as
a dominant negative receptor.
[0016] Taken together, these results indicate that OSM and IL-31
play important roles in skin inflammatory processes. These effects
require the signal transduction involving the OSMR.beta. subunit in
heteromeric receptors.
[0017] A first object of the present invention is hence the use of
at least one inhibitor of a heteromeric receptor having OSMR.beta.
as a subunit, for the preparation of a composition for improving
epidermal repair.
[0018] As used herein, the term "inhibitor" is defined as any
molecule able to inhibit the signal transduction involving the
OSMR.beta. subunit. Examples of such inhibitors are: [0019]
antagonists of the ligands of the heteromeric receptor having
OSMR.beta. as a subunit, in particular antagonists of OSM and
IL-31; [0020] antagonists of the potentiators of OSM and IL-31, in
particular antagonists of IL-17, TNF.alpha. and IFN-.gamma.; [0021]
antagonists of the OSMR.beta. subunit itself; [0022] expression
inhibitors of said ligands or potentiators, which means expression
inhibitors of OSM, IL-17, TNF.alpha., IL-31 and IFN-.gamma.; and
[0023] expression inhibitors of the OSMR.beta. subunit itself.
Examples of antagonists of OSM, IL-17, TNF.alpha., IL-31 and
IFN-.gamma. are antibodies, for example neutralizing monoclonal
antibodies (directed either against the cytokines, or against their
receptors, thereby preventing signal transduction), as well as
modified cytokines and soluble receptors. A number of neutralizing
antibodies against human OSM, IL-17 and TNF.alpha. have already
been described, some of which are commercialized by R&D,
Mineapolis, USA (www.RnDsystems.com): MAB295 (clone 17001) against
OSM, MAB317 (clone 41809) against IL-17, and MAB210 (clone 1825)
and MAB610 (clone 28401) against TNF.alpha.. Anti-TNF.alpha.
soluble receptors and antibodies are already available for
therapeutic use (against rheumatoid arthritis), among which
Enbrel.RTM. (etanercept), Remicade.RTM. (infliximab), and
Humira.RTM. (adalimumab). Neutralizing monoclonal antibodies can
also be used as antagonists of the OSMR.beta. subunit.
Alternatively, the inhibitor is a transmembrane receptor that acts
as a decoy receptor by "trapping" the ligands of the heteromeric
receptors having OSMR.beta. as a subunit. The short form of the GPL
subunit can be used according to this specific embodiment. Mutated
cytokines, native or mutated peptides different from a cytokine,
antibodies, and non-peptidic synthetic or natural molecules can
thus be used as inhibitors according to the invention.
[0024] Examples of expression inhibitors of OSM, IL-17, TNF.alpha.,
IL-31, IFN-.gamma. and the OSMR.beta. subunit are siRNAs,
ribozymes, antisens oligonucleotides and the like. An expression
inhibitor according to the invention can be, for example, an
anti-OSMR.beta. siRNA such as an isolated double-stranded RNA
molecule which is able to mediate interference of the human OSMR/3
mRNA, in particular to inactivate the gene encoding OSMR.beta., by
transcriptional silencing (of course, the same applies to OSM,
IL-17, TNF.alpha., IL-31 and IFN-.gamma.). Alternatively, it is
possible to use a DNA which is transcribed into anti-OSMR.beta.
siRNA in mammalian cells. For example, it can be a plasmid
comprising a palindromic sequence that will be transcribed in the
cell into a single-strand RNA molecule designed so that it forms a
short hairpin RNA (shRNA). In the cell, this shRNA will be
processed into siRNA (Yu, DeRuiter et al. 2002). A number of
protocols are now provided to help the skilled artisan design a
sequence for a shRNA. For example, mention can be made of the
instruction manuals for the pSilencer.TM. vectors (Ambion.RTM.
website: http://www.ambion.com). Of course, the obtained
polynucleotide sequence can be comprised in a vector for
introducing it into a mammalian cell. The skilled artisan will
choose, amongst the wide variety of vectors described in the
scientific literature, an appropriate vector, depending on the mode
of administration that is contemplated (intradermal, subcutaneous,
topic, etc.), the expected duration of expression of the
polynucleotide (depending on the disease), etc. Plasmids and
viruses, such as lentiviruses, can be cited as non-limitating
examples. All these siRNAs, shRNAs, either nude or included in a
vector, are herein considered as "expression inhibitors".
[0025] In a preferred embodiment of the invention, an OSM
antagonist and/or an IL-17 antagonist and/or a TNF.alpha.
antagonist is/are used for the preparation of a composition. In
another preferred embodiment, an OSM expression inhibitor and/or an
IL-17 expression inhibitor and/or a TNF.alpha. expression inhibitor
and/or an OSMR.beta. expression inhibitor is/are used for the
preparation of a composition.
[0026] The compositions obtained according to the invention can be
used for preventing, reducing and/or treating keratinocyte-mediated
inflammation.
[0027] In particular, the present invention pertains to the use of
at least one antagonist or expression inhibitor as described above,
for the preparation of a composition for preventing, alleviating or
treating a skin inflammation disease, such as, for example,
psoriasis, atopic dermatitis, bullous epidermolysis, bullous
phemphigoid, lichen, acne, eczema, professional dermatitis,
seborrheic dermatitis, rosacea, erythema, keratosis and
ichtyosis.
[0028] Wound healing often results in unsightly scars. Today, no
efficient treatment exists for attenuating hypertrophied scars and
cheloids. The only proposed solution is plastic surgery. However,
cheloids frequently reappear after excision. In such cases, as well
as in diseases comprising a thickening of the epidermis, it is most
desirable to control the keratinocyte migration. The present
invention hence provides novel applications of inhibitors of a
heteromeric receptor having OSMR.beta. as a subunit (like
antagonists and expression inhibitors as above-described), for
preparing compositions that inhibit keratinocyte migration, for
example for preventing or reducing epidermal thickening. Such
compositions can also be used for slowing down epidermal healing.
They can be used on established cheloids or scars, for attenuating
them.
[0029] According to specific embodiments of the present invention,
an inhibitor of a heteromeric receptor having OSMR.beta. as a
subunit, is used for the preparation of a composition for
preventing and/or attenuating chaps on hands, lips, face or body,
or for preventing and/or attenuating stretch marks. Other
applications of the compositions obtained according to the
invention are the improvement of the aspect and comfort of scars,
and/or the improvement of the aspect and comfort of epidermal
wounds during their healing.
[0030] The compositions prepared according to the invention are
preferably formulated for topic administration. They can for
example be in the form of a cream, lotion, ointment, or
dressing.
[0031] The invention also pertains to the use of at least two
different molecules selected in the group consisting of antagonists
and expression inhibitors of cytokines selected amongst OSM, IL-17,
TNF.alpha., IL-31 and IFN-.gamma., and antagonists and expression
inhibitors of the OSMR.beta. subunit, for the preparation of
dermatological and/or cosmetic compositions. These at least two
components can be either mixed in the same composition, or provided
in a kit of parts.
[0032] A cosmetic and/or dermatological composition comprising an
antagonist of OSM, for example a neutralizing anti-OSM monoclonal
antibody, is also part of the invention. In addition or
alternatively, a cosmetic and/or dermatological composition
according to the invention can comprise an antagonist of IL-17
and/or an antagonist of TNF.alpha..
[0033] According to another embodiment of the invention,
illustrated in the examples below, an activator of a heteromeric
receptor comprising an OSMR.beta. subunit is used for preparing a
composition that promotes keratinocyte-mediated inflammation. Such
a composition can be used, for example, for modifying the
secretion, differentiation and migration capacities of
keratinocytes, in order to mimic skin pathologies like psoriasis or
atopic dermatitis. This is useful for producing in vitro and animal
models of skin pathologies.
[0034] In particular, a method for preparing an in vitro model of
psoriasis, comprising a step of adding OSM and/or IL-17 and/or
TNF.alpha. to cultured keratinocytes, is part of the invention. For
example, this method can be performed by adding OSM and/or IL-17
and/or TNF.alpha. to the culture medium of said keratinocytes, at
concentrations preferably ranging 0.1 to 100 ng/ml, more preferably
from 1 to 10 ng/ml. Optionally, concentrations ranging 0.01 to 10
ng/ml, preferentially 0.1 to 2 ng/ml can be used for each cytokine,
especially when several of them are used.
[0035] The invention also concerns a method for obtaining an animal
model of skin diseases such as psoriasis, comprising a step of
administering at least one activator of a heteromeric receptor
comprising an OSMR.beta. subunit, in particular OSM and/or IL-17
and/or TNF.alpha., to said animal. In this method, the activator is
preferably administered subcutaneously and/or topically and/or via
intradermal injection. Animals that can used for performing this
method are preferably mammals, such as rodents (rats, mice, etc.),
dogs, pigs, primates, and the like. For example, 1 to 10 .mu.g of
murine OSM can be injected intradermally to mice, each day during 2
to 8 days, in order to obtain a murine model of psoriasis.
Concentrations corresponding to those indicated above for the in
vitro induction of a psoriasis-like phenotype can typically be used
also for obtaining an animal model of psoriasis.
[0036] The models of skin pathologies obtained according to the
invention can be used for the screening of molecules. In vitro
models will be referentially used for large (high throughput)
screening, and animal models for confirmation of results obtained
in vitro. These tools will be advantageously used for identifying
molecules exhibiting anti-inflammatory properties at the skin level
and, hopefully, new active principles for treating skin
inflammation diseases.
[0037] The invention is further illustrated by the following
figures and examples:
FIGURES LEGENDS
[0038] FIG. 1 shows expression of OSM receptor by NHEK.
[0039] (A) Total RNA was extracted from NHEK of 4 independent
donors. RT-PCR was performed with specific primers for OSMR, gp130,
LIFR and GAPDH genes. Serial dilutions of cDNA were amplified to
have a semi-quantitative analysis of transcripts expression level.
PCR products were analysed by agarose gel electrophoresis. (B)
Immunolabelling of cell surface NHEK and flux cytometry analysis.
Gp130 and OSMR are clearly detected on the cell, but not the LIFR.
(C) Twenty .mu.g of cell lysate from NHEK and the glioblastoma cell
line GO-G-UVM were separated by SDS-PAGE (10%) and transferred to
nitrocellulose membrane. Ponceau red staining was used to control
loading homogeneity. Detection of gp130, OSMR, LIFR and tubulin
bands were assessed by Western blot. The results are representative
of 3 independent experiments.
[0040] FIG. 2 shows induction of STAT3 and MAP kinase
phosphorylation by OSM in NHEK.
[0041] (A) NHEK were stimulated or not with LIF or OSM (50 ng/ml).
(B) NHEK were stimulated or not for 15 min with 50 ng/ml of IL-5
(negative control) or with 100, 50, 25, 12.5, 6.25 or 3.1 ng/ml of
OSM, and phospho-STAT3 (P-STAT3) and STAT3 protein levels were
assessed by Western blot. Before stimulation with the cytokines,
cells were incubated for 2 h in the presence of neutralizing
antibodies, an anti-gp130 (AN-HH1), or an anti-OSMR (XR-M70)
monoclonal antibody, or with an isotype control mAb MC192 (final
antibody concentration, 15 .mu.g/ml (C). Phospho-MAPK (P-MAPK) and
MAPK protein levels in response to OSM was assessed by Western blot
(D).
[0042] FIG. 3 shows effect of OSM on keratinocyte migration.
[0043] In vitro wounds were introduced in mitomycin-treated
confluent NHEK culture and the keratinocytes were cultured for 48 h
with or without 10 ng/ml of EGF or OSM. Cell migration to the cell
free area was assessed as described in Materials and Methods. Each
bar represents the mean.+-.SEM of migrating keratinocytes counted
in 4 non-overlapping fields. One experiment representative of
2.
[0044] * p<0.001 compared with respective control without
cytokine, based on Student's t test.
[0045] FIG. 4 shows expression profiles obtained from OSM
stimulated NHEK and OSM treated RHE.
[0046] NHEK (A) or RHE (B) were cultured with or without 10 ng/ml
of OSM for 24 h. Total RNA was isolated, treated with Dnase I, and
used to make .sup.33P-labelled cDNA probes, which were hybridized
to cDNA arrays. The computer images were obtained after 5 days
exposure to a Molecular Dynamics Storm storage screen and further
scanning. After local background substraction, an average signal
intensity from duplicate spots was normalized for differences in
probe labelling using the values obtained for housekeeping genes.
(C) The OSM-induced modulation was expressed as the percentage
ratio of the signal intensities for cells treated with each
cytokine over the signal intensity for unstimulated cells.
[0047] FIG. 5 shows effect of OSM on S100A7-9 synthesis by NHEK.
NHEK were cultured with or without 0.4, 0.8, 1.6, 3.1, 6.3, 12.5 or
25 ng/ml of OSM for 48 h (A) or with or without 10 ng/ml of OSM for
6, 12, 24, 48, 72, 96 h (B). Total RNA was extracted, reverse
transcribed, and S100A7 and HMBS mRNA relative expression was
quantified by real time PCR. HMBS was used as a housekeeping gene
to normalize gene expression as detailed in Materials and Methods.
Results, expressed as the relative expression of stimulated cells
over control cells, are representative of 2 independent
experiments. (C) NHEK were cultured with or without 10 ng/ml of OSM
for 48 h. Relative S100A8-calgranulin A, S100A9-calgranulin B,
(3-defensin 2 and filaggrin mRNA expression was quantified by
quantitative RT-PCR. Results are expressed as the relative
expression of stimulated cells over control cells. (D) NHEK were
cultured with or without 10 ng/ml of OSM for 48 and 96 h. Twenty
.mu.g of cell lysate were separated by SDS-PAGE (16%) and
transferred to nitrocellulose membrane. Ponceau red staining was
used to control loading homogeneity. S100A7, S100A8 and S100A9
protein levels was determined by Western blot. The results are
representative of 3 independent experiments.
[0048] FIG. 6 shows cytokines and chemokines production by NHEK.
IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF alpha, ENA-78, MIP 3
beta were measured by specific ELISA in 48 h NHEK culture
supernatants. Cells were cultured in the presence or not of OSM.
Dose-response (0.4 to 25 ng/ml) and kinetic (6 to 96 h) studies of
ENA78 production were also performed.
[0049] FIG. 7 shows histological and immunohistochemical analysis
of RHE stimulated or not with 10 ng/ml of OSM for 4 days. RHE were
fixed, embedded in paraffin. Four micron vertical sections were
stained with hematoxylin/eosin or reacted with anti-K10 keratin
mAb, anti-filaggrin mAb or anti-S100A7 mAb and then photographed
under a microscope (magnification.times.200).
[0050] FIG. 8 shows induction of STAT3 and MAP kinase
phosphorylation by IL-31 and OSM in NHEK.
[0051] NHEK were stimulated or not for 15 min with 10 ng/ml of OSM,
or with 100 ng/ml of IL-31 (A), or with different concentrations of
these cytokines (B), and phospho-STAT3 (P-STAT3) and STAT3 protein
levels were assessed by Western blot. Before stimulation with the
cytokines, cells were incubated for 2 h in the presence of
neutralizing antibodies, an anti-gp130 (AN-HHI), or an anti-OSMR
(XR-M70) monoclonal antibody, or with an isotype control mAb MC192
(final antibody concentration, 15 .mu.g/ml) (C). Phospho-MAPK
(P-MAPK) and MAPK protein levels in response to IL-31 and OSM was
assessed by Western blot (D).
[0052] FIG. 9 shows effect of IL-31 and OSM on S100A7 mRNA
expression.
[0053] NHEK were cultured with or without 3.1, 6.3, 12.5, 25, 50 or
100 ng/ml of IL-31 (A) or with or without 0.4, 0.8, 1.6, 3.1, 6.3,
12.5 or 25 ng/ml of OSM (B) for 48 h. Kinetic study of S100A7 mRNA
expression in the absence or presence of 100 ng/ml of IL-31 (C) or
10 ng/ml of OSM (D). Total RNA was extracted, reverse transcribed,
and S100A7 and HMBS mRNA relative expression was quantified by real
time PCR. HMBS was used as a housekeeping gene to normalize gene
expression as detailed in Material and Methods. Results, expressed
as the relative expression of stimulated cells over control cells,
are representative of 2 independent experiments.
[0054] FIG. 10 shows effect of IL-31 and OSM on keratinocyte
migration.
[0055] In vitro wounds were introduced in mitomycin-treated
confluent NHEK culture and the keratinocytes were cultured for 48 h
with or without 100 ng/ml of IL-31, or 10 ng/ml of EGF or OSM. Cell
migration to the cell free area was assessed as described in
Materials and Methods. Each bar represents the mean.+-.SEM of
migrating keratinocytes counted in 4 non-overlapping fields. One
experiment representative of 2. *p<0.001 compared with
respective control without cytokine, based on Student's t test.
[0056] FIG. 11 shows the effect of several cocktails of cytokines
on S100A7, hBD4/2, and KRT10 mRNA expression.
[0057] Confluent normal human keratinocytes (NHEK) were treated for
24 hrs with the indicated mixed cytokine (each cytokine at 1 ng/ml
final concentration). Total RNA was extracted, reverse-transcribed
and the expression of the selected genes was analyzed by real-time
PCR.
[0058] FIG. 12 shows OSM and type H OSM receptor expression in
psoriasis
[0059] (A). Ten .mu.m cryostat sections of skin biopsy from healthy
donors or psoriatic patients were fixed and subjected to
immunohistochemistry for OSM, gp130, OSMR.beta., LIFR.beta., S100A7
and filaggrin. One experiment representative of 4
(magnification.times.100).
[0060] (B). OSM transcripts were PCR-quantified from total RNA
extracted from healthy or psoriatic lesional skins.
[0061] (C). Lesion infiltrating T cells or peripheral blood
circulating T cells were in vitro expanded by one stimulation using
anti-CD3, anti-CD28 mAbs and IL-2. After a 12-days culture period,
cells were stimulated again under the same conditions and 24 h
culture supernatants harvested for OSM ELISA determination.
p-values were calculated using Student's t test.
[0062] (D). The co-localization of OSM and CD3 expression in
lesional skin infiltrating T cells was analyzed by double
immunofluorescent staining. Dotted lines indicate the border
between epidermis (top) and dermis (bottom).
[0063] FIG. 13 shows the result of intradermal injection of OSM in
129S6/SvEv mice
[0064] Four mice were treated with 5 .mu.g of OSM or vehicle alone
for 4 days.
[0065] (A). Ten .mu.m cryostat sections of skin biopsies were
counterstained with hematoxylin and eosin and photographed under a
microscope (magnification.times.400). One mouse representative of
4.
[0066] (B). Quantitative RT-PCR analysis of S100A8, S100A9,
MIP-113, MDC and TARC mRNA expression in skin sample from control
or OSM-treated mice (mean.+-.SEM of 4 independent mice).
EXAMPLE 1
Material and Methods
[0067] Cell Culture
[0068] NHEK were obtained from surgical samples of healthy breast
skin. The use of these samples for research studies was approved by
the Ethical Committee of the Poitiers Hospital. Skin samples were
incubated overnight at 4.degree. C. in a dispase solution (25 U/ml;
Invitrogen Life Technologies, Cergy Pontoise, France). Epidermal
sheets were removed from dermis and NHEK were dissociated by
trypsin digestion (trypsin-EDTA, Invitrogen) for 15 min at
37.degree. C. Cells were cultured in Serum-Free Keratinocyte Medium
(Keratinocyte SFM) (Invitrogen, Cergy Pontoise, France)
supplemented with bovine pituitary extract (25 .mu.g/ml) and
recombinant epidermal growth factor (0.25 ng/ml; all purchased from
Invitrogen). NHEK were starved for 48 h in Keratinocyte SFM without
addition of growth factors before stimulation.
[0069] RHE were purchased from SkinEthic Laboratories (Nice,
France). They consist of a multi-layered epidermis grown for 12
days at the air-medium interface and which exhibits morphological
and growth characteristics similar to human skin (Rosdy et al.,
1997). Skin-infiltrating or peripheral blood T cells were expanded
using magnetic beads coated with anti-CD3 (SPV-T3b) and anti-CD28
(L293) mAbs (Expander Beads.RTM.: Dynal, Oslo, Norway) as described
previously (Yssel, Lecart et al. 2001; Trickett and Kwan 2003).
[0070] Skin-infiltrating T cells were generated from 3 mm punch
biopsies from active psoriatic lesions that were extensively washed
and transferred to a well of a 24-well tissue culture plate (Nunc,
Roskilde, Denmark), in the presence of 5 .mu.l of Expander
Beads.RTM. in RPMI medium, supplemented with 10% Fetal Calf Serum
and 10 ng/ml IL-2 in a final volume of 1 ml and incubated at
37.degree. C. at 5% CO.sub.2. Peripheral blood mononuclear cells
were isolated via Ficoll Hypaque density centrifugation and
10.sup.5 lymphocytes were cultured in the presence of Expander
Beads.RTM., as described above. After 3 days of culture, fresh
culture medium, containing 10 ng/ml IL-2 was added to the cultures
and growing T cells were collected after 10-14 days of culture for
use in subsequent experiments. In order to preserve the functional
and phenotypic properties of the skin-infiltrating and peripheral
blood T lymphocytes, cells were stimulated with Expander Beads.RTM.
only once and not restimulated for further propagation. Results
from immunofluorescence and flow cytometric analysis showed that T
cells expanded from each of psoriatic skin lesions were 60%-80%
CD8.sup.+ (data not shown). This typical CD4/CD8 ratio was not due
to the preferential outgrowth of CD8.sup.+ T cells under these
experimental conditions. For analysis of cytokine production,
10.10.sup.6 T cells were activated with 10 .mu.g/ml of immobilized
anti-CD3 mAb and 2 .mu.g/ml of anti-CD28 mAb in the presence of 10
ng/ml of IL-2 for 24 h and cytokine production was analyzed by
ELISA.
[0071] Cytokines and Reagents
[0072] Cytokines were purchased from R&D Systems (Oxon, UK) and
IL-31 was produced in the laboratory as described previously
(Diveu, Lak-Hal et al. 2004). The IgG1 isotype control (MC192),
anti-gp130 (AN-HH1, AN-HH2, AN-G30), anti-OSMR antibody (AN-A2),
anti-OSMR.beta. (AN-N2, AN-R2, AN-V2), anti-GPL (AN-GL1, AN-GL3)
and anti-LIFR.beta. (AN-E1) mAbs were produced in the laboratory,
the neutralizing anti-OSMR.beta. Ab (XR-M70) was obtained from
Immunex (Seattle, Wash.) and the anti-OSM mAb (17001) from R&D
Systems. Polyclonal anti-gp130, anti-LIFR.beta., anti-STAT3,
anti-S100A8-calgranulin A and anti-S100A9-calgranulin B Abs were
from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Anti-S
100A7-psoriasin and anti-.beta.-tubulin Abs were purchased from
Imgenex (San Diego, Calif., USA) and Sigma (St. Louis, Mo.)
respectively. Antibodies raised against phospho-STAT3, phospho-MAPK
and MAPK were from Upstate Biotechnology (Lake Placid, N.Y.).
Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 and
phycoerythrin (PE)-conjugated anti-CD4 and anti-CD8 mAbs were
purchased from BD Biosciences. Antibodies against CK-10 and
filaggrin were from Lab Vision Corporation (Fremont, Calif., USA).
Goat anti-mouse and anti-rabbit peroxidase-labeled immunoglobulins
were from Clinisciences (Montrouge, France), rabbit anti-goat
peroxidase-conjugated Ab was from Sigma, and streptavidin-Alexa
Fluor 568 conjugated from Invitrogen. IL-1.beta., IL-6, IL-8,
IL-10, IL-12p70 and TNF.alpha. were quantified using BD.TM.
Cytometric Bead Array (CBA) Human Inflammation kit (BD Biosciences,
Mountain View, Calif., USA). Detection of OSM, ENA-78, MIP-3.beta.,
TARC and VEGF was carried out using ELISA kits purchased from
R&D Systems.
[0073] RT-PCR and RT-Real Time PCR Analysis
[0074] Total cellular RNA was isolated using Trizol reagent
(Invitrogen) and treated with DNase I (0.05 U/.mu.l; Clontech, Palo
Alto, Calif., USA). cDNAs were synthesised from 2 .mu.g of total
RNA by random hexamer primers using MMLV reverse transcriptase
(Promega, Madison, Wis.). Reverse transcription products were
subsequently amplified by 25 cycles of PCR using primers for OSMR
(forward 5'-CCTGCCTACCTGAAAACCAG-3' (SEQ ID No: 1) and reverse
5'-ACATTGGTGCCTTCTTCCAC-3' (SEQ ID No: 2)), gp130 (forward 5%
GGGCAATATGACTCTTTGAAGG-3' (SEQ ID No: 3) and reverse
5'-TTCCTGTTGATGTTCAGAATGG-3' (SEQ ID No: 4)), LIFR (forward
5'-CAGTACAAGAGCAGCGGAAT-3' (SEQ ID No: 5) and reverse
5'-CCAGTCCATAAGGCATGGTT-3' (SEQ ID No: 6)) and GAPDH (forward
5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID No: 7) and reverse
TCCACCACCCTGTTGCTGTA (SEQ ID No: 8)). Amplified products were
analysed by 2% agarose gel electrophoresis.
[0075] Quantitative real time PCR was carried out using the
LightCycler-FastStart DNA Master.sup.PLUS SYBR Green I kit (Roche,
Mannheim, Germany). The reaction components were 1.times. FastStart
DNA Master SYBR Green 1 and 0.5 .mu.M of forward and reverse
primers for S100A7 (forward 5'-GCATGATCGACATGTTTCACAAATACAC-3' (SEQ
ID No: 9) and reverse 5'-TGGTAGTCTGTGGCTATGTCTCCC-3' (SEQ ID No:
10)), S100A8 (Pattyn, Speleman et al. 2003), S100A9 (forward
GCTCCTCGGCTTTGACAGAGTGCAAG-3' (SEQ ID No: 11) and reverse
5'-GCATTTGTGTCCAGGTCCTCCATGATGTGT-3' (SEQ ID No: 12)), hBD2/4
(forward 5'-GCCATCAGCCATGAGGGTCTTG-3' (SEQ ID No: 13) and reverse
5'-AATCCGCATCAGCCACAGCAG-3' (SEQ ID No: 14)), KRT10 (forward
5'-GCCCGACGGTAGAGTTCTTT-3' (SEQ ID No: 15) and reverse
5'-CAGAAACCACAAAACACCTTG-3' (SEQ ID No: 16)), OSM (forward
5'-TCAGTCTGGTCCTTGCACTC-3' (SEQ ID No: 17) and reverse
5'-CTGCAGTGCTCTCTCAGTTT-3' (SEQ ID No: 18)), and GAPDH (forward
5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID No: 19) and reverse
5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID No: 20)) and
hydroxymethyl-bilane synthase (HMBS) (Vandesompele, De Preter et
al. 2002) as housekeeping genes. After cDNA fluorescent
quantification using propidium iodide, 250 ng, 25 ng and 2.5 ng of
cDNA were added as PCR template in the LightCycler glass
capillaries. The cycling conditions comprised a 10 min polymerase
activation at 95.degree. C. and 50 cycles at 95.degree. C. for 10
s, 64.degree. C. for 5 s and 72.degree. C. for 18 s with a single
fluorescence measurement. Melting curve analysis, obtained by
increasing temperature from 60.degree. C. to 95.degree. C. with a
heating rate of 0.1.degree. C. per second and a continuous
fluorescence measurement, revealed a single narrow peak of
suspected fusion temperature. A mathematical model was used to
determine the relative quantification of target genes compared to
HMBS reference gene (Pfaffl 2001).
[0076] For in vivo studies, RNA from control or skin site injected
with OSM were extracted, reverse-transcribed and used to perform
quantitative RT-PCR with specific primers for murine S100A8
(forward 5'-TCCAATATACAAGGAAATCACC-3' (SEQ ID No: 21) and reverse
5'-TTTATCACCATCGCAAGG-3' (SEQ ID No: 22)), murine S100A9 (forward
5'-GAAGGAATTCAGACAAATGG-3' (SEQ ID No: 23) and reverse
5'-ATCAACTTTGCCATCAGC-3' (SEQ ID No: 24)), murine MIP-1.beta.
(forward 5'-CCTCTCTCTCCTCTTGCTC-3' (SEQ ID No: 25) and reverse
5'-AGATCTGTCTGCCTCTTTTG-3' (SEQ ID No: 26)), murine MDC (forward
5'-TGCTGCCAGGACTACATC-3' (SEQ ID No: 27) and reverse
5'-TAGCTTCTTCACCCAGACC-3' (SEQ ID No: 28)), murine TARC (forward
5'-CATTCCTATCAGGAAGTTGG-3' (SEQ ID No: 29) and reverse
5'-CTTGGGTTTTTCACCAATC-3' (SEQ ID No: 30)) and murine GAPDH
(forward 5'-ATCAAGAAGGTGGTGAAGC-3' (SEQ ID No: 31) and reverse
5'-GCCGTATTCATTGTCATACC-3' (SEQ ID No: 32)) as housekeeping
gene.
[0077] Gene Expression Profiling Using cDNA Macroarrays
[0078] Total RNA was isolated as described for PCR studies. DNase
treatment, polyA.sup.+ RNA enrichment, .sup.33P-labelled cDNA probe
synthesis, purification and hybridization to custom Atlas array
membranes (Bernard, Pedretti et al. 2002) were performed according
to Clontech's recommendations (Clontech, Palo Alto, USA). Membranes
were exposed for 5 days to a Molecular Dynamics Storm storage
screen and scanned using a phosphorimager scanner (Molecular
Dynamics Storm analyser, Amersham Biosciences, Uppsala, Sweden).
After local background substraction, average signal intensity from
duplicate spots was normalized for differences in probe labelling
using the values obtained for housekeeping genes (Bernard et al.,
2002). For each gene, the OSM-induced modulation was expressed as
the relative expression value for stimulated versus control sample.
Arbitrarily, only modulation above 2 was considered significant for
confirmation using RT-real time PCR assay.
[0079] Western Blotting Analysis
[0080] For STAT3 and MAPK phosphorylation, NHEK were stimulated for
15 min in the presence of the indicated cytokine. Cells were lysed
in SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10%
glycerol, 50 mM DTT, 0.1% bromophenol blue), sonicated and then
submitted to SDS-PAGE and transferred onto an Immobilon membrane.
The membranes were subsequently incubated overnight with the
primary antibody, before being incubated with the appropriate
peroxidase-labelled secondary antibody for 60 min. The reaction was
visualized by chemiluminescence according to the manufacturer's
instructions. Membranes were stripped in 0.1 M glycine pH 2.8 for 2
h and neutralized in 1 M Tris-HCl pH 7.6 before reblotting. For
neutralizing experiments, NHEK were incubated with the appropriate
antibodies for 2 h before stimulation.
[0081] To determine the expression of gp130, LIFR and OSMR, the
cells were lysed in 10 mM Tris HCl pH 7.6, 5 mM EDTA, 50 mM NaCl,
30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium
orthovanadate proteinase inhibitor and 1% Brij 96. After lysis and
centrifugation to remove cellular debris, the supernatants were
then treated as described above.
[0082] For S100 proteins expression, NHEK were stimulated for 2
days in the presence of OSM (10 ng/ml). Cell lysis was performed
with 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton, 1%
sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM sodium
orthovanadate, 1% proteases inhibitors. S100A7, S100A8 and S100A9
were detected by immunochemistry as described above. Ponceau red
staining was used to control loading homogeneity.
[0083] In Vitro Keratinocyte Migration Assay
[0084] Keratinocytes were cultured in wells pre-coated with type I
collagen (200 .mu.g/ml, Institut Jacques Boy, Reims, France) until
they reached 80% confluency. Cells were starved for 48 h in
Keratinocyte SFM and then treated with 10 .mu.g/ml of mitomycin C
(Sigma) for 2 h to prevent cell proliferation. A cell-free area was
created by scraping the keratinocyte monolayer with a plastic
pipette tip. Keratinocytes migration to the cell-free area was
evaluated after 48 h of culture in the absence or presence of EGF
or OSM. Using an inverted phase contrast microscope. The number of
migrating keratinocytes was counted in 4 non-overlapping fields.
Values represent the mean.+-.SEM of cells per mm.sup.2 beyond the
frontiers of the in vitro injury. Student's t test was used for
statistical analysis.
[0085] Reconstituted Human Epidermis Model
[0086] For histological and immunohistochemical studies, RHE, grown
for 12 days at the air-medium interface, were purchased from
SkinEthic Laboratories (Nice, France). They consist of a
multi-layered epidermis which exhibit morphological and growth
characteristics similar to human skin (Rosdy, Bertino et al. 1997).
As recommended, RHE were grown for 1 day in SkinEthic growth medium
prior to stimulation in the absence or presence of OSM for 4 days.
They were then fixed in a balanced 10% formalin solution and
embedded in paraffin. Four micron vertical sections were stained
with heamatoxylin/eosin or with specific Ab and
peroxidase-conjugated Antibodies, and counterstained with
haematoxylin according to standard protocols (Rosdy, Bertino et al.
1997). Anti-K10 keratin and anti-filaggrin monoclonal Antibodies
were from Lab Vision Corporation (Fremont, Calif., USA).
[0087] For gene expression profiling using cDNA macroarrays, 17
days old RHE were grown for 1 day in SkinEthic maintenance medium
prior to stimulation in the absence or presence of OSM for 24 h.
Total RNA was isolated and cDNA arrays were performed as described
above.
[0088] Immunofluorescence and Immunohistochemistry Analyses
[0089] Cells were incubated for 30 min at 4.degree. C. with the
appropriate primary Ab (AN-G30, AN-N2, AN-E1) or with an isotype
control Ab followed by a PE-conjugated anti-mouse Ab incubation.
Fluorescence was analysed on a FACSCalibur.RTM. flow cytometer
equipped with Cellquest software (Becton-Dickinson). For
immunohistochemistry, 10 .mu.m cryostat sections were fixed,
permeabilized and immunostained with the relevant primary Ab
(AN-HH2, AN-R2, AN-GL1, AN-E1, 17001) and the avidin peroxidase
method (Vector Laboratories, Burlingame, Calif., USA). Sections
were counterstained with Mayer's hematoxylin (Sigma) before
mounting. For immunofluorescent staining, a biotin-labeled anti-OSM
mAb was used and detected with streptavidin-Alexa Fluor 568
conjugated together with a FITC-conjugated anti-CD3 mAb. Nuclei
were counterstained with To-Pro 3 (Invitrogen). Immunostainings
were analyzed with the Olympus FV1000 confocal microscope.
Twelve-day grown RHE were cultured for 4 additional days in the
presence or absence of OSM or IL-31. Cultures were then fixed with
a formalin solution and embedded in paraffin. Four .mu.m vertical
sections were stained according to standard protocols.
[0090] Murine Model
[0091] All animal experiments were approved by the Institutional
Animal Care Committee. Hair was removed from the back of 129S6/SvEv
9-week old mice. Three days later, mice were injected intradermally
with 5 .mu.g of murine OSM or vehicle control in two locations on
either side of the back. Injections were carried out daily during 4
days until sacrifice. Mice were euthanized using carbon dioxide.
Skin samples were removed from the prepared area and were embedded
and frozen in Tissue Tek OCT compound for histological analysis or
directly frozen in liquid nitrogen for mRNA extraction. Ten .mu.m
sections were fixed and counterstained with Mayer's hematoxylin and
eosin (Sigma).
EXAMPLE 2
Human Keratinocytes Express the Type OSM Receptor on their
Surface
[0092] To show the potential functions of OSM in normal human
keratinocytes the inventors first undertook an analysis of its
receptor chain expression. To determine the nature of expressed
type I or type II receptors, RT-PCR for gp130, LIFR.beta. and
OSMR.beta. were carried out starting from primary cultures of
keratinocytes. CO-G-UVM and glioblastoma cells were used as
controls for LIFR. Obtained results show that NHEK predominantly
expressed transcripts for OSMR and the gp130, whereas only low
levels of the LIFR chain could be evidenced (FIG. 1A). The RNA
analysis was further reinforced by measuring the expression levels
of corresponding proteins by flow cytometry. Fluorescence analyses
revealed a clear expression of gp130 and OSMR.beta. on the NHEK
cell surface (FIG. 1B). In contrast no detection of membrane
LIFR.beta. expression could be evidenced, whereas the
anti-LIFR.beta. antibody gave the expected result when incubated
with a cell line used as a positive control. This was further
supported by western blot analyses showing the detection of gp130
and OSMR.beta. chains, and an absence of LIFR.beta. expression in
NHEK (FIG. 1C). Similar experiments carried out on samples coming
from four different donors led to the same results, ruling out the
possibility for variations in type I or type II OSM receptor
expression from donor to donor. These first results indicate that
human keratinocytes preferentially expressed the specific type II
OSM receptor.
EXAMPLE 3
STAT-3 and MAP Kinase Pathways are Recruited by OSM in Human
Keratinocytes
[0093] The inventors show OSM-induced signal transduction in NHEK.
Since STAT3 is usually recruited by the OSM type II receptor
pathway, they analyzed the tyrosine phosphorylation of the
signaling molecule in response to increasing concentrations of the
cytokine. A strong induction of tyrosine phosphorylation was
observed for STAT3, with plateau level values still present down to
3 ng/ml OSM (FIG. 2B). The involvment of gp130 and OSMR.beta.
subunits was further demonstrated by blocking of the STAT3
phosphorylation when adding receptor neutralizing mAbs to the NHEK
culture before OSM contact (FIG. 2C). Importantly, the complete
neutralization of STAT3 phosphorylation observed in the presence of
the anti-OSMR.beta. mAb further demonstrated the absence of
recruitment by OSM of the share LIF/OSM type I receptor in NHEK. In
agreement with this observation, after a LIF contact no evidence
for a STAT3 activation could be observed in NHEK.
[0094] In addition, type II OSM receptor complex is also known to
be a more potent activator for the recruitment of the Map kinase
pathway compared to the shared LIF/OSM receptor. A cooperative
effect between ERK1/ERK2 and the Shc adaptor, and mediated through
OSMR.beta., but not through the LIFRb, explains this strong
activation the MAP kinase pathway in response to OSM (Boulton,
Stahl et al. 1994). The ERK1/2 signaling in response to the
cytokine in NHEK was therefore analyzed by determining their
tyrosine phosphorylation level. As expected, NHEK stimulation with
OSM quickly increased the MAP kinase phosphorylation (FIG. 2D).
Taken together, these results demonstrated that gp130/OSMR.beta.
receptor complex expressed in human keratinocytes is fully
functional, and that the entire observed signals are mediated
through the type II receptor.
EXAMPLE 4
OSM is a Potent Inducer of Keratinocyte Migration
[0095] To underline the functional responses of NEHK to OSM, The
inventors analyzed the potential effect of OSM on an in vitro model
mimicking the wound healing and based on the keratinocyte migration
(Kira, Sano et al. 2002). Forty eight hours after initiation of the
culture, cells present in the middle of the well were removed by
scratching, and the remaining keratinocytes were stimulated with
either EGF, known to trigger the keratinocyte migration, or with
OSM. After an additional 36 h of culture, the cytokine potential
for inducing cell migration was visually determined or by cell
counting (FIG. 3). Obtained results show that OSM led to an
important migration of NHEK, similar to that observed in the
presence of EGF.
EXAMPLE 5
Identification of OSM-Induced Gene Expression in Human
Keratinocytes
[0096] To have a better view of the NHEK functional response the
inventors analyzed the modification of keratinocyte gene expression
profile induced by OSM using cDNA arrays. Used arrays were
specially designed for the study of keratinocytes, and consisting
of 586 different cDNAs spotted in duplicate. They consisted in
genes involved in keratinocyte cell structure, metabolism,
extracellular matrix, adhesion, differentiation, signaling, signal
transduction, apoptosis and stress (Bernard et al., 2002). RNA
extracted from control or OSM-stimulated NHEK were used to generate
labeled cDNA probes by reverse transcription. Probing the Atlas
cDNA array membranes with these cDNA probes revealed that OSM
increased the expression of 36 genes and decreased the expression
of 38 genes. OSM down regulates a large set of genes associated
with keratinocyte differentiation, such as cytokeratin (CK)1, CK10,
fillagrin and loricrin genes. Among the up-regulated genes, the
inventors found a marked increase for the calcium binding proteins,
psoriasin (S100A7), calgranulins (S100A8, S100A9) and the S100
neutrophil protein (FIG. 4). Interestingly, the expression of these
proteins is known to be up-regulated in inflammatory tissues
(Madsen, Rasmussen et al. 1991; Nagase and Woessner 1999; Roth,
Vogl et al. 2003). OSM also induced the G-protein-coupled receptor
HM74, the super oxyde dismutase 2 and the beta-defensin genes,
involved in tissue protection. Genes involved in tissue remodelling
such as matrix metalloproteinase 1 and tenascin were also induced
by OSM. In addition, OSM increased the expression of the chemokines
CXCL1 (MIP-2a), CXCL5 (epithelial-derived neutrophil-activating
peptide (ENA 78) and CXCL8 (IL-8), and the platelet-derived growth
factor A (PDGF-A) genes.
[0097] Obtained results indicate that, in human keratinocytes, OSM
was able to recruit a number of genes involved in inflammatory
processes and in innate immune response.
EXAMPLE 6
OSM Induced Keratinocytes to Produce Psoriasin, Calgranulin, .beta.
Defensin, and Chemokines
[0098] To further reinforce the results obtained using
designed-arrays, quantitative analyses at mRNA and protein levels
were carried out for a selected number of identified genes.
Quantitative analysis of psoriasin/S100A7 mRNA expression in
reponse to OSM was performed by RT-real-time PCR along kinetic and
dose-response studies. The inventors show that psoriasin/S100A7
mRNA was up-regulated in a dose-dependent manner in response to OSM
ranging from 1.6 to 6.3 ng/ml after a 48-h treatment, and the
plateau was reached for 6.3 ng/ml OSM with a fifty fold increase of
the signal above control (FIG. 5A). Kinetic study revealed an
increase in psoriasin/S100A7 mRNA expression starting at 12 h
following stimulation with 10 ng/ml of OSM (FIG. 5B). It still
increased up to 96 h, with a strong induction of about 290 folds
above the control value. This was confirmed at the protein level by
western blot analyses of the psoriasin/S100A7, as well as of two
related calcium binding proteins, the S100A8 and S100A 9
calgranulins (FIG. 5D). Results show that NHEK exposure to 10 ng/ml
of OSM resulted in an increased expression of studied proteins that
was strongest at day 4 than at day 2 (FIG. 5D). FIG. 5C depicts the
results obtained by analyzing the RNA quantitative expression of
filagrin and .beta.defensin-2, two important markers of skin
activation.
[0099] The production of the chemokines CXCL5 and CXCL8 in 48 h
NHEK is also clearly enhanced under OSM stimulation (FIG. 6B).
EXAMPLE 7
OSM Triggers Hyperplasia of Reconstituted Human Epidermis and
Modulates the Expression of Differentiation Related Antigens
[0100] To further approach the dynamic of epidermal
differentiation, the inventors tested the biological effect of OSM
on in vitro RHE in order to assess the basal cell layer
proliferation and the graduated epidermal differentiation
processes. Histological analysis of control RHE showed a
keratinised multi-stratified epithelium resembling epidermis in
vivo, containing intact basal, spinous, granulous and cornified
cell-layers, and numerous keratohyalin granules in the upper
granular layer (FIG. 7). OSM triggered the hyperplasia of the
keratinocytes layers, leading to an increase in the overall
thickness of the RHE. In addition, they observed a loss of
keratohyalin granules in the granular layer and the presence of
picnotic nuclei. cDNA array profile analysis of RHE confirmed that
OSM strongly up-regulated S100A7, S100A8, S100A9 and S100
neutrophil protein genes, as previously described on NHEK (FIG. 4).
By immunohistochemistry, we confirmed the S100A7 protein
up-regulation in OSM-treated RHE (FIG. 7). In agreement with the
data on NHEK, OSM treatment on RHE also up-regulated CXCL5, CXCL8
chemokine genes, and the PDGF-A and the cadherin 3 gene
transcription. Specific to RHE but not detected in NHEK, OSM
up-regulated the CK6A, 6B, 6D, 7, 13, 14, 16, the skin-derived
antileukoproteinase and the TGF.beta.-inducible early protein.
[0101] On the other hand, OSM down-regulates genes associated with
keratinocyte differentiation, such as involucrin, filaggrin, and
calmodulin-like skin protein (Mehul, Bernard et al. 2000; Rogers,
Kobayashi et al. 2001; Jonak, Klosner et al. 2002; Wagener, van
Beurden et al. 2003). Immunohistochemical analysis performed on RHE
sections confirmed the inhibition of keratinocyte differentiation,
as indicated by the decrease of filaggrin and keratin 10 expression
in OSM treated RHE (FIG. 7).
EXAMPLE 8
Discussion
[0102] The use of a cDNA array approach, specially designed for the
analysis of gene expression in human skin, enabled the
identification of OSM target genes in human keratinocytes and the
demonstation of the involvement of OSM in a variety of processes,
including migration and differentiation. In particular, the strong,
dose dependent, OSM-mediated induction of the expression of S100A7,
S100A8 and S100A9 proteins in NHEK and RHE demonstrates the
pro-inflammatory and chemotactic effects of the cytokine. The
opposing effects of IL-10 and OSM in cutaneous inflammation are
underscored by the IL-10-induced down-regulation of S100A8 and
S100A9 release by monocytes (Lugering, Kucharzik et al. 1997).
S100A7, S100A8 and S100A9 belong to the pleiotropic 5100 family of
calcium-binding proteins (Roth, Vogl et al. 2003). Although their
main functions are as yet unclear, they appear to play prominent
inflammatory functions (Watson, Leygue et al. 1998; Donato 1999;
Roth, Vogl et al. 2003) and to be involved in the tight regulation
of a large number of intra- and extracellular activities such as
the dynamic of motility of cytoskeletal components or chemotaxis
(Ryckman, Vandal et al. 2003). Interestingly, whereas all three
S100A7, S100A8 and S100A9 proteins have been reported to be
expressed at low or undetectable levels in normal skin epidermis
and non-differentiated cultured keratinocytes, they are highly
expressed in abnormally differentiated psoriatic keratinocytes
(Broome, Ryan et al. 2003), during wound repair (Thorey, Roth et
al. 2001) and in epithelial skin tumors (Watson, Leygue et al.
1998; Gebhardt, Breitenbach et al. 2002; Alowami, Qing et al.
2003). Because of the chemotactic effects of S100A7 on inflammatory
cells, in particular neutrophils and CD4.sup.+ T lymphocytes, it
has been suggested that S100A7 may be involved in the genesis of
psoriatic lesions (Watson, Leygue et al. 1998). Since S100A7 acts
upstream of these mechanisms, the inventors demonstrate that OSM is
a key molecule for the induction of S100A7 under pathological
conditions, and is involved in the pathological state. The
modulation of additional genes by OSM is also in favour of the
pro-inflammatory and chemotactic roles of OSM. Indeed, the
induction of neutrophil attractant chemokine CXCL5/ENA-78 together
with the down-regulation of heme oxygenase 1, which antagonizes
inflammation by attenuating adhesive interaction and cellular
infiltration in skin, could contribute to the neutrophil influx in
skin (Koch, Kunkel et al. 1994; Wagener, van Beurden et al.
2003).
[0103] OSM-induced MMP-3 expression is also of interest in the
context of inflammatory cutaneous diseases and wound repair.
Whereas MMP-3 cannot be detected in normal skin, it is expressed by
proliferative keratinocytes of the basal layer after injury
(Pilcher, Wang et al. 1999). During progression of many diseases,
MMP-3 is involved in epidermis remodeling by removal of
extracellular matrix during tissue resorption (Nagase and Woessner
1999; Pilcher, Wang et al. 1999), and mice that lack the MMP-3 gene
are deficient in wound repair of the epidermis (Bullard, Lund et
al. 1999). Using an in vitro wound assay, the inventors
demonstrated that keratinocyte migration is strongly increased by
OSM stimulation. These data are in agreement with the demonstration
that STAT3 deficiency in keratinocytes leads to an impaired
migration (Sano, Itami et al. 1999). Under inflammatory conditions,
OSM appears to be one essential mediator enhancing keratinocyte
migration and wound healing, via MMP-3 or S100A8-S100A9 dependent
mechanisms. Additional evidence to establish the involvement of OSM
in wound healing is the strong induction of PDGF in RHE, a major
proliferative and migratory stimulus for connective tissue during
the initiation of skin repair processes (Rollman, Jensen et al.
2003).
[0104] The inventors also showed that OSM increases the overall
thickness of the keratinocyte layer of RHE. This process seems not
to be related to basal cell hyperproliferation since Ki67
expression is not induced in response to OSM, but more likely
results from an inhibition of terminal keratinocyte
differentiation, as shown by the decreased production of filaggrin,
loricrin or involucrin. OSM down-regulates the expression of the
calmodulin-like skin protein (CLSP) and calmodulin-related protein
NB-1, two members of the calmodulin family, directly related to
keratinocyte differentiation (Mehul, Bernard et al. 2001; Rogers,
Kobayashi et al. 2001). CLSP binds transglutaminase-3, a key enzyme
implicated in the formation and assembly of proteins, such as
loricrin or involucrin, to form the cornified cell envelop of the
epidermis (Mehul, Bernard et al. 2000). The modulating effects of
OSM on the keratin expression profile, i.e., keratin 6
over-expression and keratin 10 inhibition, also supports the notion
of an inhibition of epidermal differentiation. Keratin 6 is known
to be induced under hyperproliferative and/or inflammatory
situations, including wound healing, psoriasis, carcinogenesis, or
by agents such as retinoic acid that provoke epidermal hyperplasia
(Navarro, Casatorres et al. 1995; Komine, Rao et al. 2000). In
contrast, keratin 10, normally expressed in terminally
differentiating epidermal keratinocytes, is reduced during wound
healing (Paramio, Casanova et al. 1999).
EXAMPLE 9
Similar Results Obtained with IL-31
[0105] Studies presented in examples 3, 4 and 5 have been realized
with IL-31 instead of OSM and similar results were obtained: IL-31
recruits STAT3 signaling pathways (FIG. 8) in NHEK and induces
expression of psoriasin (S100A) and calgranulin A and B (S100A8-9)
(FIG. 9). IL-31 is also able to induce keratinocyte migration (FIG.
10).
EXAMPLE 10
Gamma Interferon Potentiates the Action of IL-31 on the Signal
Transduction
[0106] When NHEK were preincubated 24 h in the presence of 50 g/ml
of gamma Interferon (INF.gamma.) before IL-31 stimulation (50
ng/ml), P-STAT3 level were increased 3 to 4 folds when compared to
NHEK preincubated in medium alone (same studies as those realized
for the FIG. 8). This demonstrates that INF.gamma. is a modulator
of the action of IL-31 on signal transduction.
EXAMPLE 11
IL-17 and TNF.alpha. Potentiate the Action of OSM on the Expression
of Several Inflammation Markers
[0107] The combined effect of several cytokines on keratinocytes
was then tested by measuring the expression of the keratinocyte
inflammation markers psoriasin (S100A7) and defensin beta-2/beta-4
(hBD2/4) mRNAs, in the presence of various cytokines cocktails. The
effect of these cocktails was also tested on keratin 10 (KRT10)
mRNA, since KRT10 is a differentiation marker associated with
tissue healing.
[0108] To that aim, confluent normal human keratinocytes (NHEK)
were treated for 24 hrs with the indicated mix of cytokines (each
cytokine at 1 ng/ml final concentration). Total RNA was extracted,
reverse-transcribed and the expression of the selected genes was
analyzed by real-time PCR as described.
[0109] The results are shown in Table 1 below and in FIG. 11.
TABLE-US-00001 TABLE 1 cytokines 1 ng/ml RT-Q-PCR X/GAPDH # mix
IL-22 OSM IL-17 TFN.alpha. IL-1.alpha. IFN.gamma. S100A7 hBD2/4
KRT10 M3 + + + + + + 6 350 1 441 000 36 M1 + + + + - + 5 450 1 380
000 38 M4 + + + + + - 7 270 1 185 000 30 M8 - + + + + - 7 880 1 165
000 36 M7 + - + + + - 2 550 475 000 51 M6 + + - + + - 1 610 32 585
45 M5 + + + - + - 2 450 211 000 49 M2 + + + + - - 6 570 1 331 000
40 - - - - - - 100 100 100
[0110] The mix of the 6 selected cytokines (M3) exhibited a strong
synergic effect on the expression of the keratinocyte inflammation
markers psoriasin (S100A7) and defensin beta-2/beta-4 (hBD2/4). The
depletion of the mixes in either IFN.gamma., IL-22, or IL-1.alpha.,
did not significantly decrease the activity of the complete
cocktail of cytokines; these cytokines are hence probably not
directly involved in the observed synergy. To the contrary, [0111]
the omission of OSM from the 5 cytokines reference mix (IFN.gamma.
has been omitted because it is inactive) led to a decrease of the
activity of the mix by 3-fold for S100A7 and by 2.5-fold for
hBD2/4; [0112] the omission of TNF.alpha. led to a decrease of the
activity of the mix by 3-fold for S100A7 and by 5.6-fold for
hBD2/4; and [0113] the omission of IL-17 led to a decrease of the
activity of the mix by 4.5-fold for S100A7 and by 36-fold for
hBD2/4.
[0114] These results indicate a strong synergy between OSM,
TNF.alpha. and IL-17 for a maximal response in keratinocytes.
[0115] Hence, it appears that OSM is able to synergize with IL-17
and/or with TNF.alpha. in pathologic situations such as psoriasis
and that the blockage of the signalling of these two cytokines may
lead to a dramatic inhibition of the physiopathologic response of
the target keratinocytes, allowing to a reversion of the
pathology.
EXAMPLE 12
Involvement of the Type II OSM Receptor and OSM in the Pathogenesis
of Psoriasis
[0116] Since OSM is a potent inducer of keratinocyte motility and
triggers hyperplasia of RHE, the expression of the OSMR.beta.,
LIFR.beta. and gp130 receptor subunits in active cutaneous lesions
from psoriatic patients was analyzed by immunohistochemistry. This
type of lesions is characterized by a thickened epidermis, a
decreased stratum granulosum, parakeratosis and a strong
up-regulation of S100A7-psoriasin expression, together with a
concomitant decrease in the expression of filaggrin (FIG. 12A).
Both OSMR.beta. and gp130 were highly expressed in psoriatic, as
compared to normal skin (FIG. 12A). The expression of LIFR.beta.
was undetectable (FIG. 12A), in agreement with the above results
obtained on NHEK. Taken together, these results indicate that under
conditions of cutaneous inflammation, the expression of the type II
OSMR. is up-regulated on human keratinocytes and furthermore
confirm the absence of the type I OSMR in both healthy and
psoriatic skin.
[0117] Concomitantly, OSM mRNA and protein were strongly expressed
in psoriatic lesions as compared to normal skin where no signal
could be detected (FIGS. 12A and 12B), in line with the previously
reported release of OSM by short term culture of psoriatic skin
(Bonifati et al., 1998).
EXAMPLE 13
OSM Production by Lesion-Infiltrating T Cells and, to a Lower
Extend, by Keratinocytes
[0118] In order to extend the observation of example 12, the
inventors tested the possibility whether skin-infiltrating T cells
could be a source of OSM in these lesions. Indeed, as shown in FIG.
12D, a subset of CD3.sup.+ T lymphocytes were OSM producers. This
observation was confirmed in anti-CD3/CD28 mAb activated T cells
isolated and expanded from the psoriatic lesion that were found to
produce high levels of OSM, as compared to those obtained with
peripheral blood T cells from the same patients or from healthy
donors (FIG. 12C). Nonetheless, residual anti-OSM reactivity was
detected in lesional keratinocyte themselves both by
immunoenzymatic and immunofluorescent stainings (FIGS. 12A and
12D). To precise this point, OSM mRNA was measured by quantitative
RT-PCR in NHEK, cultured in the presence or absence of OSM or
culture supernatants from activated psoriatic skin-infiltrating T
cells. These results showed that control or stimulated NHEK express
20 to 100 fold less OSM mRNA than in activated T cells derived from
psoriatic skin. Finally, the inventors failed to detect any OSM
production by ELISA in NHEK supernatants, indicating that
keratinocytes themselves synthesized very limited amounts of the
cytokine. Furthermore, no circulating OSM could be detected in the
sera of these patients. Taken together, these results demonstrate
that T cells infiltrating the psoriatic lesions are a major source
of OSM and are likely to contribute, via the induction of
keratinocyte inflammation, to the pathogenesis of psoriasis.
EXAMPLE 14
OSM Induces a Psoriasis-Like Phenotype in Mice
[0119] To analyze the direct contribution of OSM to the
pathogenesis of psoriasis, intradermal injections of OSM were
performed in mice. After 4 days of treatment, the site of injection
showed a 2 to 3 fold epidermal thickening in OSM-treated mice, as
compared to animals treated with vehicle alone (FIG. 13A). Further
characterization of these skin samples by quantitative RT-PCR
analysis showed that OSM enhances the expression of S100A8, S100A9,
MIP-1.beta. and macrophage-derived chemokine (MDC) genes (FIG.
13B). These data support the notion that OSM is an important
mediator directly involved in epidermal chemotaxis, thickening and
inflammation of the skin.
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Sequence CWU 1
1
32120DNAArtificialforward primer for reverse transcription of OSMR
1cctgcctacc tgaaaaccag 20220DNAArtificialreverse primer for reverse
transcription of OSMR 2acattggtgc cttcttccac
20322DNAArtificialforward primer for reverse transcription of gp130
3gggcaatatg actctttgaa gg 22422DNAArtificialreverse primer for
reverse transcription of gp130 4ttcctgttga tgttcagaat gg
22520DNAArtificialforward primer for reverse transcription of LIFR
5cagtacaaga gcagcggaat 20620DNAArtificialreverse primer for reverse
transcription of LIFR 6ccagtccata aggcatggtt
20720DNAArtificialforward primer for reverse transcription of GAPDH
7accacagtcc atgccatcac 20820DNAArtificialreverse primer for reverse
transcription of GAPDH 8tccaccaccc tgttgctgta
20928DNAArtificialforward primer for Q-RT-PCR of S100A7 9gcatgatcga
catgtttcac aaatacac 281023DNAArtificialreverse primer for Q-RT-PCR
of S100A7 10tggtagtctg tggctatgtc tcc 231126DNAArtificialforward
primer for Q-RT-PCR of S100A9 11gctcctcggc tttgacagag tgcaag
261230DNAArtificialreverse primer for Q-RT-PCR of S100A9
12gcatttgtgt ccaggtcctc catgatgtgt 301322DNAArtificialforward
primer for Q-RT-PCR of hBD2/4 13gccatcagcc atgagggtct tg
221421DNAArtificialreverse primer for Q-RT-PCR of hBD2/4
14aatccgcatc agccacagca g 211520DNAArtificialforward primer for
Q-RT-PCR of KRT10 15gcccgacggt agagttcttt
201621DNAArtificialreverse primer for Q-RT-PCR of KRT10
16cagaaaccac aaaacacctt g 211720DNAArtificialforward primer for
Q-RT-PCR of OSM 17tcagtctggt ccttgcactc 201820DNAArtificialreverse
primer for Q-RT-PCR of OSM 18ctgcagtgct ctctcagttt
201919DNAArtificialforward primer for Q-RT-PCR of GAPDH
19gaaggtgaag gtcggagtc 192020DNAArtificialreverse primer for
Q-RT-PCR of GAPDH 20gaagatggtg atgggatttc
202122DNAArtificialforward primer for Q-RT-PCR of murine S100A8
21tccaatatac aaggaaatca cc 222218DNAArtificialreverse primer for
Q-RT-PCR of murine S100A8 22tttatcacca tcgcaagg
182320DNAArtificialforward primer for Q-RT-PCR of murine S100A9
23gaaggaattc agacaaatgg 202418DNAArtificialreverse primer for
Q-RT-PCR of murine S100A9 24atcaactttg ccatcagc
182519DNAArtificialforward primer for Q-RT-PCR of murine MIP-1 beta
25cctctctctc ctcttgctc 192620DNAArtificialreverse primer for
Q-RT-PCR of murine MIP-1 beta 26agatctgtct gcctcttttg
202718DNAArtificialforward primer for Q-RT-PCR of murine MDC
27tgctgccagg actacatc 182819DNAArtificialreverse primer for
Q-RT-PCR of murine MDC 28tagcttcttc acccagacc
192920DNAArtificialforward primer for Q-RT-PCR of murine TARC
29cattcctatc aggaagttgg 203019DNAArtificialreverse primer for
Q-RT-PCR of murine TARC 30cttgggtttt tcaccaatc
193119DNAArtificialforward primer for Q-RT-PCR of murine GAPDH
31atcaagaagg tggtgaagc 193220DNAArtificialreverse primer for
Q-RT-PCR of murine GAPDH 32gccgtattca ttgtcatacc 20
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References