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Inducible Costimulator (ICOS) and ICOS Ligand Signaling Has Pivotal Roles in Skin Wound Healing via Cytokine Production

2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2011.07.048

ISSN

1525-2191

Autores

Shintaro Maeda, Manabu Fujimoto, Takashi Matsushita, Yasuhito Hamaguchi, Kazuhiko Takehara, Minoru Hasegawa,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Skin wound healing is mediated by inflammatory cell infiltration of the wound site. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor–ligand pair. Although the ICOS-ICOSL pathway participates in adaptive immunity, its roles in skin wound healing, which is mediated by innate immune responses, remain unknown. To clarify these roles, repair of excisional wounds was examined in ICOS−/− mice, ICOSL−/− mice, and ICOS−/−ICOSL−/− mice. Each mutant strain showed similar, dramatic delays in wound healing, especially at early times. Knockout mice showed suppressed keratinocyte migration, angiogenesis, and granulation tissue formation, and diminished T-cell, macrophage, and neutrophil infiltration. The loss of ICOS and/or ICOSL resulted in marked suppression of cytokine expression in wounds, especially the Th2 cytokines interleukin (IL)-4, IL-6, and IL-10. T-cell transfer experiments and T-cell depletion therapy further clarified the important roles of ICOS expressed on T cells and its interaction with ICOSL. Application of IL-6, but not IL-4, to the wounds significantly increased the onset of early wound healing in mutant mice. Thus, our results indicate that ICOS-ICOSL costimulatory signaling has critical roles during wound healing, most likely by inducing IL-6 production. Skin wound healing is mediated by inflammatory cell infiltration of the wound site. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor–ligand pair. Although the ICOS-ICOSL pathway participates in adaptive immunity, its roles in skin wound healing, which is mediated by innate immune responses, remain unknown. To clarify these roles, repair of excisional wounds was examined in ICOS−/− mice, ICOSL−/− mice, and ICOS−/−ICOSL−/− mice. Each mutant strain showed similar, dramatic delays in wound healing, especially at early times. Knockout mice showed suppressed keratinocyte migration, angiogenesis, and granulation tissue formation, and diminished T-cell, macrophage, and neutrophil infiltration. The loss of ICOS and/or ICOSL resulted in marked suppression of cytokine expression in wounds, especially the Th2 cytokines interleukin (IL)-4, IL-6, and IL-10. T-cell transfer experiments and T-cell depletion therapy further clarified the important roles of ICOS expressed on T cells and its interaction with ICOSL. Application of IL-6, but not IL-4, to the wounds significantly increased the onset of early wound healing in mutant mice. Thus, our results indicate that ICOS-ICOSL costimulatory signaling has critical roles during wound healing, most likely by inducing IL-6 production. Skin wound healing starts immediately after an injury and consists of three general stages: i) an inflammatory stage that consists of platelet aggregation and recruitment of inflammatory cells to the wound site; ii) a proliferative phase that involves the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells, leading to re-epithelialization and granulation tissue formation; and iii) a long remodeling phase.1Singer A.J. Clark R.A.F. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4644) Google Scholar, 2Martin P. Wound healing-aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3729) Google Scholar Migration of inflammatory cells to the wound site is important during wound repair.1Singer A.J. Clark R.A.F. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4644) Google Scholar, 2Martin P. 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Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4644) Google Scholar, 2Martin P. Wound healing-aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3729) Google Scholar Inducible costimulator (ICOS) is the third member of the CD28 family of costimulatory molecules, and is induced on the cell surface following T-cell activation.5Hutloff A. Dittrich A.M. Beier K.C. Eljaschewitsch B. Kraft R. Anagnostopoulos I. Kroczek R.A. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.Nature. 1999; 397: 263-266Crossref PubMed Scopus (1203) Google Scholar, 6Yoshinaga S.K. Whoriskey J.S. Khare S.D. Sarmiento U. Guo J. Horan T. Shih G. Zhang M. Coccia M.A. Kohno T. Tafuri-Bladt A. Brankow D. Campbell P. Chang D. Chiu L. Dai T. Duncan G. Elliott G.S. Hui A. McCabe S.M. Scully S. Shahinian A. Shaklee C.L. Van G. Mak T.W. Senaldi G. T-cell co-stimulation through B7RP-1 and ICOS.Nature. 1999; 402: 827-832Crossref PubMed Scopus (683) Google Scholar, 7Greenwald R.J. Freeman G.J. Sharpe A.H. The B7 family revisited.Annu Rev Immunol. 2005; 23: 515-548Crossref PubMed Scopus (1934) Google Scholar ICOSL (also called B7-H2, B7RP-1, LICOS, and GL50), the ligand of ICOS, is weakly expressed on antigen-presenting cells in the steady state and is up-regulated after activation of these cells.6Yoshinaga S.K. Whoriskey J.S. Khare S.D. Sarmiento U. Guo J. Horan T. Shih G. Zhang M. Coccia M.A. Kohno T. Tafuri-Bladt A. Brankow D. Campbell P. Chang D. Chiu L. Dai T. Duncan G. Elliott G.S. Hui A. McCabe S.M. Scully S. Shahinian A. Shaklee C.L. Van G. Mak T.W. Senaldi G. T-cell co-stimulation through B7RP-1 and ICOS.Nature. 1999; 402: 827-832Crossref PubMed Scopus (683) Google Scholar, 7Greenwald R.J. Freeman G.J. Sharpe A.H. The B7 family revisited.Annu Rev Immunol. 2005; 23: 515-548Crossref PubMed Scopus (1934) Google Scholar, 8Swallow M.M. Wallin J.J. Sha W.C. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha.Immunity. 1999; 11: 423-432Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar The ICOS-ICOSL pathway promotes T-cell activation, differentiation, and effector responses, and T cell–dependent B-cell responses. ICOS-mediated costimulation of T cells leads predominantly to the production of effector cytokines such as IL-4 and IL-10 and, to a lesser extent, IL-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α,9McAdam A.J. Chang T.T. Lumelsky A.E. Greenfield E.A. Boussiotis V.A. Duke-Cohan J.S. Chernova T. Malenkovich N. Jabs C. Kuchroo V.K. Ling V. Collins M. Sharpe A.H. Freeman G.J. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.J Immunol. 2000; 165: 5035-5040PubMed Google Scholar thereby playing a more important role in Th2 responses than Th1 responses.5Hutloff A. Dittrich A.M. Beier K.C. Eljaschewitsch B. Kraft R. Anagnostopoulos I. Kroczek R.A. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.Nature. 1999; 397: 263-266Crossref PubMed Scopus (1203) Google Scholar, 10Nurieva R.I. Chung Y. Hwang D. Yang X.O. Kang H.S. Ma L. Wang Y.H. Watowich S.S. Jetten A.M. Tian Q. Dong C. Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages.Immunity. 2008; 29: 138-149Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 11Coyle A.J. Gutierrez-Ramos J.C. The role of ICOS and other costimulatory molecules in allergy and asthma.Springer Semin Immunopathol. 2004; 25: 349-359Crossref PubMed Scopus (52) Google Scholar, 12Lohning M. Hutloff A. Kallinich T. Mages H.W. Bonhagen K. Radbruch A. Hamelmann E. Kroczek R.A. Expression of ICOS in vivo defines CD4+ effector T cells with high inflammatory potential and a strong bias for secretion of interleukin 10.J Exp Med. 2003; 197: 181-193Crossref PubMed Scopus (204) Google Scholar However, recent studies demonstrated that ICOS influences the expansion of follicular helper T cells, Th17 cells, and regulatory T cells, indicating the complex roles of ICOS in several disease models.13Bauquet A.T. Jin H. Paterson A.M. Mitsdoerffer M. Ho I.C. Sharpe A.H. Kuchroo V.K. The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells.Nat Immunol. 2009; 10: 167-175Crossref PubMed Scopus (568) Google Scholar, 14Burmeister Y. Lischke T. Dahler A.C. Mages H.W. Lam K.P. Coyle A.J. Kroczek R.A. Hutloff A. ICOS controls the pool size of effector-memory and regulatory T cells.J Immunol. 2008; 180: 774-782PubMed Google Scholar, 15Akbari O. Freeman G.J. Meyer E.H. Greenfield E.A. Chang T.T. Sharpe A.H. Berry G. DeKruyff R.H. Umetsu D.T. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity.Nat Med. 2002; 8: 1024-1032Crossref PubMed Scopus (683) Google Scholar Because ICOS−/− mice and ICOSL−/− mice display similar defects in humoral immunity, ICOS and ICOSL have been considered a single receptor–ligand pair.16Mak T.W. Shahinian A. Yoshinaga S.K. Wakeham A. Boucher L.M. Pintilie M. Duncan G. Gajewska B.U. Gronski M. Eriksson U. Odermatt B. Ho A. Bouchard D. Whorisky J.S. Jordana M. Ohashi P.S. Pawson T. Bladt F. Tafuri A. Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses.Nat Immunol. 2003; 4: 765-772Crossref PubMed Scopus (175) Google Scholar, 17Nurieva R.I. Mai X.M. Forbush K. Bevan M.J. Dong C. B7h is required for T cell activation, differentiation, and effector function.Proc Natl Acad Sci U S A. 2003; 100: 14163-14168Crossref PubMed Scopus (99) Google Scholar, 18Dong C. Juedes A.E. Temann U.A. Shresta S. Allison J.P. Ruddle N.H. Flavell R.A. ICOS co-stimulatory receptor is essential for T-cell activation and function.Nature. 2001; 409: 97-101Crossref PubMed Scopus (759) Google Scholar It is generally accepted that ICOS-ICOSL signaling is critical for adaptive immunity, including autoimmunity, allergy, infectious diseases, and transplantation.18Dong C. Juedes A.E. Temann U.A. Shresta S. Allison J.P. Ruddle N.H. Flavell R.A. ICOS co-stimulatory receptor is essential for T-cell activation and function.Nature. 2001; 409: 97-101Crossref PubMed Scopus (759) Google Scholar, 19Rottman J.B. Smith T. Tonra J.R. Ganley K. Bloom T. Silva R. Pierce B. Gutierrez-Ramos J.C. Ozkaynak E. Coyle A.J. The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.Nat Immunol. 2001; 2: 605-611Crossref PubMed Scopus (244) Google Scholar, 20Kopf M. Coyle A.J. Schmitz N. Barner M. Oxenius A. Gallimore A. Gutierrez-Ramos J.C. Bachmann M.F. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection.J Exp Med. 2000; 192: 53-61Crossref PubMed Scopus (182) Google Scholar, 21Ozkaynak E. Gao W. Shemmeri N. Wang C. Gutierrez-Ramos J.C. Amaral J. Qin S. Rottman J.B. Coyle A.J. Hancock W.W. Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection.Nat Immunol. 2001; 2: 591-596Crossref PubMed Scopus (287) Google Scholar However, ICOSL is constitutively expressed, not only on B cells, but also on macrophages and dendritic cells that contribute to innate immunity.6Yoshinaga S.K. Whoriskey J.S. Khare S.D. Sarmiento U. Guo J. Horan T. Shih G. Zhang M. Coccia M.A. Kohno T. Tafuri-Bladt A. Brankow D. Campbell P. Chang D. Chiu L. Dai T. Duncan G. Elliott G.S. Hui A. McCabe S.M. Scully S. Shahinian A. Shaklee C.L. Van G. Mak T.W. Senaldi G. T-cell co-stimulation through B7RP-1 and ICOS.Nature. 1999; 402: 827-832Crossref PubMed Scopus (683) Google Scholar, 8Swallow M.M. Wallin J.J. Sha W.C. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha.Immunity. 1999; 11: 423-432Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Therefore, it is possible that ICOS-ICOSL signaling may be contributing to innate immunity as well as adaptive immunity. In fact, recent studies suggest involvement of ICOS and ICOSL in innate immune disease models.22Tanaka C. Fujimoto M. Hamaguchi Y. Sato S. Takehara K. Hasegawa M. Inducible costimulator ligand regulates bleomycin-induced lung and skin fibrosis in a mouse model independently of the inducible costimulator/inducible costimulator ligand pathway.Arthritis Rheum. 2010; 62: 1723-1732Crossref PubMed Scopus (38) Google Scholar, 23Dianzani C. Minelli R. Mesturini R. Chiocchetti A. Barrera G. Boscolo S. Sarasso C. Gigliotti C.L. Sblattero D. Yagi J. Rojo J.M. Fantozzi R. Dianzani U. B7h triggering inhibits umbilical vascular endothelial cell adhesiveness to tumor cell lines and polymorphonuclear cells.J Immunol. 2010; 185: 3970-3979Crossref PubMed Scopus (28) Google Scholar As skin wound healing involves a strong innate immune component, we applied this model to mice deficient for ICOS and ICOSL to investigate the roles of these molecules in modulating innate immunity. ICOS−/− mice and ICOSL−/− mice were purchased from Jackson Laboratory (Bar Harbor, ME). ICOS−/− mice and ICOSL−/− mice were backcrossed 10 and 8 generations onto the C57BL/6 genetic background, respectively. Mating these ICOS−/− mice with ICOSL−/− mice generated ICOS+/− ICOSL+/− mice, which were crossed to generate ICOS−/− ICOSL−/−. To verify the ICOS or ICOSL genotype, PCR amplification of each gene was conducted using genomic DNA from each mouse. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. Eight- to 10-week-old male mice were used. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science approved all studies and procedures, including the use of diethyl ether for anesthesia. Mice were anesthetized and their backs shaved and cleaned with 70% alcohol. Four full-thickness excisional wounds per mouse were made using a disposable, sterile 6-mm biopsy punch (Kai, Tokyo, Japan), as described elsewhere.24Mori R. Kondo T. Nishie T. Ohshima T. Asano M. Impairment of skin wound healing in beta-1,4-galactosyltransferase-deficient mice with reduced leukocyte recruitment.Am J Pathol. 2004; 164: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar After surgery, mice were caged individually. Photos of the wounds were taken each day, and the open wound area was calculated and compared to that of the previous day using the free-hand tool of Photoshop Elements (Adobe Systems, Tokyo, Japan). No signs suggestive of local infection were detected in the wounded skin. After the mice were euthanized, wounds were harvested with a 2-mm rim of unwounded skin tissue. The wounds were cut into halves laterally, fixed in 3.5% paraformaldehyde, and were then paraffin embedded. Six-micrometer sections were stained with hematoxylin and eosin (H&E), or were immunostained. For immunohistochemistry, deparafinized sections were treated with endogenous peroxidase blocking reagent (DAKO Cytomation A/S, Copenhagen, Denmark) and proteinase K (DAKO Cytomation A/S) for 6 minutes at room temperature. Sections were then incubated with rat monoclonal antibodies specific for myeloperoxidase (Neomarkers, Fremont, CA), macrophages (clone F4/80; Abcam, Cambridge, UK), CD3 (Serotec, Kidlington, UK), CD31 (BD Pharmingen, Franklin Lakes, NJ), and α-smooth muscle actin (α-SMA; Sigma-Aldrich, St. Louis, MO). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 minutes, 37°C) with a biotinylated rabbit anti-rat IgG secondary antibody (Ab) (Vectastain ABC method, Vector Laboratories, Burlingame, CA), then horseradish peroxidase–conjugated avidin-biotin complexes. Sections were washed three times with PBS between incubations. Sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and then counterstained with methyl green. Numbers of myeloperoxidase-positive neutrophils, F4/80-positive macrophages, and CD3-positive T cells were determined by counting in nine high-power fields (0.07 mm2, magnification, ×400) in the wound bed per section. Among the nine fields, six fields were selected from both edges of the wound bed, and the remaining three fields were chosen from the middle of the wound bed. The epithelial gap, which represents the distance between the leading edge of migrating keratinocytes, was measured in H&E-stained sections of wounds. We identified the area that consisted of newly formed capillaries and the collection of fibroblasts and macrophages as granulation tissue. Wound sections were visualized by microscopy (BX50; Olympus, Tokyo, Japan) with images collected using a digital camera (DP70; Olympus). After this, the area of granulation tissue was gated and measured using the same system. Vessel density, defined as CD31-positive regions, was measured in the whole wound bed areas using Photoshop Elements and was expressed as a percentage of this whole. For T-cell depletion studies, 100 μg of anti-mouse Thy 1.2, monoclonal Ab (mAb) (BD Pharmingen) was injected intravenously 24 hours before wounding, as described previously.25Tomita Y. Nomoto K. Prevention of induction of unresponsiveness to class I antigens by veto activity of donor marrow in cylophosphamide-treated mice.Transplantation. 1993; 56: 1473-1480Crossref PubMed Scopus (7) Google Scholar Single-cell splenic leukocyte suspensions were generated by gentle homogenization. Immunomicrobeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to purify T-cell populations according to the manufacturer's instructions. 2.0 × 106 CD3+ T cells, CD4+ T cells, or CD8+ T cells/mouse were transferred intravenously to indicated recipient mice. Total RNAs were extracted from injured skin samples using Qiagen RNeasy spin columns (Qiagen, Crawley, UK) and digested with DNase I (Qiagen) to remove chromosomal DNA in accordance with the manufacturer's protocols. Total RNA was reverse-transcribed to cDNA using Reverse Transcription System with random hexamers (Promega, Madison, WI). Real-time quantitative RT-PCR was performed using the TaqMan system (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer's instructions. TaqMan probes and primers for IL-4, IL-6, IL-10, TNF-α, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. Relative expression of real-time PCR products was determined using the ΔΔCT technique. Briefly, each set of samples was normalized using the difference in threshold cycle (CT) between the target gene and housekeeping gene (GAPDH): ΔCT = (CT target gene − CT GAPDH). Relative mRNA levels were calculated by the expression 2−ΔΔCT, where ΔΔCT = ΔCT sample − ΔCT calibrator. Each reaction was performed a minimum of three times. Samples of whole wounds were homogenized in 600 μL of lysis buffer [10 mmol/L PBS, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 5 mmol/L ethylenediaminetetraacetic acid (EDTA)] containing a complete protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany) to extract proteins. Homogenates were centrifuged at 22,000 × g for 15 minutes at 4°C to remove debris.24Mori R. Kondo T. Nishie T. Ohshima T. Asano M. Impairment of skin wound healing in beta-1,4-galactosyltransferase-deficient mice with reduced leukocyte recruitment.Am J Pathol. 2004; 164: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar The concentrations of IL-6, IL-10, IFN-γ, TNF-α, and monocyte chemotactic protein (MCP)-1 in supernatants were determined using the BD Cytometric Bead Array mouse inflammation kit (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). Total protein in the supernatant was measured with the Bicinchoninic Acid Protein Assay kit (Thermo Fischer Scientific Inc., Waltham, MA). An optimal concentration of cytokines was applied to each wound in 20 μL of phosphate-buffered saline. Cytokines were applied to wounds immediately after wounding and every 24 hours thereafter. Recombinant cytokines used in this study were as follows: 50 pg/20 μL IL-4 (R & D systems, Minneapolis, MN) and 50 pg/20 μL IL-6 (R & D systems). Skin samples from at least three mice for each genotype were harvested at 7 days after wounding. The Mann-Whitney U-test was used for determining the level of significance of differences between samples, and Bonferroni's test was used for multiple comparisons. A P value of <0.05 was considered statistically significant. The open wound areas were measured each day after wounding to assess macroscopic healing defects (Figure 1). From day 1 to day 10, open wound areas were significantly larger in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.01) than in wild-type mice. Each mutant mouse strain showed an equivalent delay in wound healing. In wild-type mice, wounds closed completely by day 10, but wounds of mutant mice required 15 days or more. Thus, the loss of ICOS and/or ICOSL dramatically disrupted skin wound healing at an early date. Migration of keratinocytes under the eschar was assessed by microscopically measuring the epithelial gap (the distance between the migrating edges of keratinocytes) (Figure 2A). Keratinocyte migration was significantly inhibited in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice relative to wild-type mice at days 3 and 7 after wounding (P < 0.01 for both) after wounding, consistent with the defects observed in macroscopic healing (Figure 1). Granulation tissue formation is one of the most important components in wound repair; therefore, the area of granulation tissue was measured microscopically (Figure 2B). At 3 days after wounding, granulation tissue formation was reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.05) relative to wild-type controls. Deficiencies in granulation tissue formation were still present at day 7 in all knockout mice as compared to wild-type controls (P < 0.05 for ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice). Angiogenesis is an important event observed in the proliferative phase of wound healing. To assess the extent of angiogenesis, we performed immunohistochemical staining with anti-CD31 mAb (Figure 2C). At 3 days after wounding, the vascular density in the wound bed was significantly reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice relative to wild-type controls (P < 0.01). At 7 days after injury, vessel density formation was also inhibited in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.01). The proliferation of myofibroblasts is also an important event in wound healing; therefore, we performed immunohistochemical staining of myofibroblasts with anti–α-SMA mAb (Figure 2D). The percentage of cells staining positively for α-SMA among fibroblastic cells was significantly reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.01) at day 3 after wounding, as compared with wild-type controls. Similar trends were also observed at day 7 after wounding (P < 0.05 for ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice). The numbers of neutrophils that migrated outside of blood vessels were assessed in wound tissue by immunohistochemical analysis using anti-myeloperoxidase mAb (Figure 3A). At 1 hour after wounding, extravasated neutrophil numbers were significantly reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.05) relative to wild-type controls. Neutrophil numbers remained lower in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice at 4 hours after wounding (P < 0.05) relative to wild-type controls. T-cell infiltration was assessed using immunohistochemical staining for CD3 (Figure 3, B and D). CD3-positive T-cell numbers were significantly reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice (P < 0.01) relative to wild-type controls on day 3. At 7 days after wounding, the numbers of CD3-positive T cells in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice had increased relative to corresponding day 3 values, but remained lower than the wild-type controls (P < 0.05 for all groups). Macrophage infiltration was assessed by immunohistochemistry using the F4/80 mAb (Figure 3, C and E). Macrophage numbers were significantly reduced in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice compared with wild-type mice at 3 days (P < 0.05) and 7 days (P < 0.05) after injury. To assess whether impaired T-cell function contributed to the delayed wound healing in ICOS−/−, ICOSL−/−, and ICOS−/− ICOSL−/− mice; a T cell–depleting anti-Thy1.2 mAb was administered to wild-type mice 24 hours before wounding (Figure 4A). Depletion of more than 80% of CD3+ T cells was confirmed by flow cytometry. Wild-type mice treated with anti-Thy1.2 mAb showed a significant delay in wound healing relative to untreated wild-type mice, with wound closure by day 10 in untreated mice and day 14 in treated mice (Figure 4A). ICOS−/− and ICOSL−/− mice injected with anti-Thy1.2 mAb showed no significant changes compared with untreated ICOS−/− and ICOSL−/− mice. Therefore, depletion of CD3+ T cells from wild-type mice results in an equivalent delay in wound healing compared to both ICOS−/− and ICOSL−/− mice. These findings indicate that the loss of ICOS or ICOSL may be affecting wound healing via inhibition of ICOS signaling in T cells. The role of ICOS-ICOSL signaling in wound healing was assessed using adoptive transfer experiments. Splenic T cells were purified from wild-type mice or ICOS−/− mice, and 2 × 106 cells were transferred into ICOS−/− and ICOSL−/− mice. ICOS−/− mice that received T cells from wild-type mice showed improved wound healing, with wound closure by day 13 (Figure 4B). There were no significant changes in the wound-healing process in ICOS−/− recipient mice that received ICOS-negative T cells, or in ICOSL−/− mice that received wild-type T cells. Furthermore, wound sizes were significantly smaller during days 1 to 10 in ICOS−/− recipient mice that received T cells from wild-type mice compared with ICOS−/− recipient mice that received T cells from ICOS−/− mice, or ICOSL−/− recipient mice that received wild-type T cells (P < 0.05). These findings, together with the results of our T-cell depletion studies, indicate that ICOS-ICOSL signaling is critical for wound healing. Next, we assessed whether ICOS expression on either CD4+ or CD8+ T cells is important for wound healing. CD4+ T cells and CD8+ T cells were purified from wild-type mice, and 2 × 106 cells of each cell type were transferred into ICOS−/− mice. Although CD4+ T cell transfer significantly reduced the wound size relative to unmanipulated ICOS−/− mice during day 2 to 7, wound closure did not occur until day 15, similar to what was seen in unmanipulated ICOS−/− mice. CD8+ T-cell transfer did not significantly improve wound healing in ICOS−/− mice. Thus, ICOS expression on CD4+ T cells rather than CD8+ T cells is important for wound healing. To assess the effects of ICOS and/or ICOSL loss on cytokine expression, mRNA expression levels of IL-4, IL-6, IL-10, TNF-α, CTGF, TGF-β, PDGF, and VEGF in the wounds were assessed by real-time RT-PCR on day 0, 1, 3, and 7 (Figure 5A). In addition, the concentrations of IL-6, IL-10, IFN-γ, TNF-α, and MCP-1 in wound lysates were measured by cytometric bead array analysis, and were reported as a ratio of the total protein concentration (Figure 5B). Total protein concentrations (mean ± SEM) in the wound lysates were comparable between strains (wild type: 12.9 ± 6.1 mg/mL; ICOS−/−: 11.3 ± 6.7 mg/mL; ICOSL−/−: 12.7 ± 6.4 mg/mL; ICOS−/− ICOSL−/−:11.3 ± 5.5 mg/mL). In wild-type mice, mRNA levels or protein concentrations of these cytokines and growth factors tended to increase during the process of wound healing, although mRNA levels of IL-6 and IL-10 did not increase and the protein concentration of IFN-γ actually decreased. The mRNA levels of IL-4, IL-6, and IL-10 were significantly lower in mutant mice relative to wild-type mice from day 0 to day 7 (P < 0.05, Figure 5A). mRNA of TNF-α and CTGF tended to be reduced in mutant mice relative to wild-type mice from day 1 to day 7. Expression levels of PDGF, TGF-β, and VEGF were not generally different between strains. The relative concentration (cytokine concentration/total proteins concentration in tissue lysates) of IL-6 (from day 0 to day 7) and IL-10 (from day 1 to day 3) in the wounds from mutant mice were lower than those of wild-type mice (P < 0.05, Figure 5B). The relative concentrations of IFN-γ, TNF-α, and MCP-1 were not significantly different between strains (Figure 5B). Thus, reduced IL-4, IL-6, and IL-10 expression may be associated with the impaired wound healing in mutant mice. Cytokines can promote wound repair, and are produced during the initial inflammatory phase by neutrophils, macrophages, and

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