ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing
2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.090112
ISSN1525-2191
AutoresS. Byeseda, Alan R. Burns, S. Dieffenbaugher, Rolando E. Rumbaut, C. Wayne Smith, Zhijie Li,
Tópico(s)T-cell and B-cell Immunology
ResumoWound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium. Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium. 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Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,22Lu L Reinach PS Kao WW Corneal epithelial wound healing.Exp Biol Med (Maywood). 2001; 226: 653-664Crossref PubMed Scopus (326) Google Scholar, 23Nagasaki T Zhao J Centripetal movement of corneal epithelial cells in the normal adult mouse.Invest Ophthalmol Vis Sci. 2003; 44: 558-566Crossref PubMed Scopus (116) Google Scholar it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.24Robker RL Collins RG Beaudet AL Mersmann HJ Smith CW Leukocyte migration in adipose tissue of mice null for icam-1 and mac-1 adhesion receptors.Obes Res. 2004; 12: 936-940Crossref PubMed Scopus (56) Google Scholar, 25Brake DK Smith EO Mersmann H Smith CW Robker RL ICAM-1 expression in adipose tissue: effects of diet-induced obesity in mice.Am J Physiol Cell Physiol. 2006; 291: C1232-C1239Crossref PubMed Scopus (109) Google Scholar As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Li Z Burns AR Rumbaut RE Smith CW Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion.Am J Pathol. 2007; 171: 838-845Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,27Liu Z Guo B Lopez RD Expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human gammadelta-T cells: implications in the design of gammadelta-T-cell-based immunotherapies for pancreatic cancer.J Gastroenterol Hepatol. 2008; 24: 900-911Crossref PubMed Scopus (26) Google Scholar and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,20Iwata M Sawada S Sawa M Thoft RA Mechanisms of lymphocyte adhesion to cultured human corneal epithelial cells.Curr Eye Res. 1997; 16: 751-760Crossref PubMed Scopus (19) Google Scholar, 27Liu Z Guo B Lopez RD Expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human gammadelta-T cells: implications in the design of gammadelta-T-cell-based immunotherapies for pancreatic cancer.J Gastroenterol Hepatol. 2008; 24: 900-911Crossref PubMed Scopus (26) Google Scholar it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1. T cell receptor (TCR)δ−/− mice on the C57BL/6 background and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). ICAM-1−/− mice24Robker RL Collins RG Beaudet AL Mersmann HJ Smith CW Leukocyte migration in adipose tissue of mice null for icam-1 and mac-1 adhesion receptors.Obes Res. 2004; 12: 936-940Crossref PubMed Scopus (56) Google Scholar, 25Brake DK Smith EO Mersmann H Smith CW Robker RL ICAM-1 expression in adipose tissue: effects of diet-induced obesity in mice.Am J Physiol Cell Physiol. 2006; 291: C1232-C1239Crossref PubMed Scopus (109) Google Scholar were backcrossed as previously described at least 10 generations with C57BL/6 mice. CD11a−/− and P-selectin-deficient (P-sel−/−) mice were prepared as described.28Ding ZM Babensee JE Simon SI Lu H Perrard JL Bullard DC Dai XY Bromley SK Dustin ML Entman ML Smith CW Ballantyne CM Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.J Immunol. 1999; 163: 5029-5038PubMed Google Scholar All animals were bred and housed in our facility according to the guidelines described in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Vision and Ophthalmic Research and Baylor College of Medicine Animal Care and Use Committee policy. Mice were controlled for sex (female) and age (12 to 15 weeks). To examine the contribution of adhesion molecule ICAM-1 and platelets to γδ T cell migration to wounded corneas, some mice were injected intraperitoneally with anti-mouse ICAM-1 mAb (YN1 clone; ATCC) or anti-mouse GPIbα (Emfret Analytics, Würzburg, Germany) with 0.2 mg in 200 μl PBS 1 or 24 hours before wounding, respectively.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 29Bowden RA Ding ZM Donnachie EM Petersen TK Michael LH Ballantyne CM Burns AR Role of α4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.Circ Res. 2002; 90: 562-569Crossref PubMed Scopus (94) Google Scholar The central corneal wound was performed as previously described.30Li Z Rivera CA Burns AR Smith CW Hindlimb unloading depresses corneal epithelial wound healing in mice.J Appl Physiol. 2004; 97: 641-647Crossref PubMed Scopus (27) Google Scholar In brief, mice were anesthetized by i.p. injection of pentobarbital (50 mg/kg body weight), and the central corneal epithelium was demarcated with a 2-mm trephine and then removed using a diamond blade for refractive surgery (Accutome, Malvern, PA) under a dissecting microscope. Care was taken to minimize injury to the epithelial basement membrane and stroma. While under anesthesia, ocular surfaces were protected from drying by topical administration of sterile saline immediately following injury, and 0.1 mg/kg subcutaneous buprenorphine was administered at the time of surgery and every 6 to 12 hours thereafter, as needed, for pain control. At various times following injury, corneal tissue including the limbus was excised and processed for immunohistology, or mRNA isolation. Taking care to include the limbus, wounded corneas were dissected, fixed, permeabilized, and incubated with the following labeled monoclonal antibodies as described14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Li Z Burns AR Rumbaut RE Smith CW Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion.Am J Pathol. 2007; 171: 838-845Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 30Li Z Rivera CA Burns AR Smith CW Hindlimb unloading depresses corneal epithelial wound healing in mice.J Appl Physiol. 2004; 97: 641-647Crossref PubMed Scopus (27) Google Scholar, 31Li Z Rumbaut RE Burns AR Smith CW Platelet response to corneal abrasion is necessary for acute inflammation and efficient re-epithelialization.Invest Ophthalmol Vis Sci. 2006; 47: 4794-4802Crossref PubMed Scopus (48) Google Scholar: anti-TCRδ-phycoerythrin (PE) colors (clone GL3), anti-PECAM-1-fluorescein isothiocyanate, and anti-ICAM-1-PE, which were selected for staining γδ T cells, endothelial cells of the limbal vessels, and ICAM-1, respectively. Radial cuts were made in the cornea so that it could be flattened by a coverslip and the cornea was mounted in Airvol (Celenase, Ltd., Dallas, TX), containing 1 μmol/L 4′,6-diamidino-2-phenylindole (Sigma Chemical, St. Louis, MO) to assess nuclear morphology. Digital images were captured and saved for computer analysis (Delta Vision; Applied Precision, Issaquah, WA). A standard pattern for morphometric analysis was used throughout the study as we described before.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Whole mounts were evaluated using a ×40 oil immersion lens to assess each field of view across the cornea from limbus to limbus. The limbus was defined as most peripheral field containing limbal vessels. All images selected were representative images of at least six corneas. The graphical values plotted represent the total number of a selected cell type across a diameter of a cornea. This value was obtained by counting the total number of cells within a selected corneal layer in each of nine ×40 fields of view comprising the diameter of a cornea. Four diameters per cornea were counted and averaged. Six corneas were analyzed. Enucleated eyes were fixed overnight at 4°C in 0.1 M/L sodium cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde. The cornea was then excised, taking care to include the limbal tissue, and postfixed in 1% osmium tetroxide for 1 hour at room temperature, dehydrated through an ethanol series and embedded in resin (LX 112; Polysciences, Warrington, PA). Thick (0.5 μm) sections were cut on an ultramicrotome (RMC 7000; Venana Medical Systems, Tucscon, AZ) equipped with a diamond knife. Sections were stained with toluidine blue O and viewed on an inverted microscope (DeltaVision Spectris; Applied Precision, Issaquah, WA) using a ×20 objective, and transverse measurements of the central epithelial thickness were made using the calibrated linear measurement tool contained in the supplied imaging software (SoftWorx). Total RNA was isolated from corneal epithelium with the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Quantity and quality of the extracted RNA were verified using a Nanodrop-1000 spectrophotometer (Nanodrop Technology, Wilmington, DE). Purified RNA was stored at −80°C until analysis. First-strand cDNA synthesis was performed with the TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA) using 2 μg total RNA, per the manufacturer’s recommendations. The resulting cDNA was stored at −20°C until further analysis. For the amplification of target genes, 5 μl cDNA was added to a corresponding 20× TaqMan MGB probe primer set for each message, multiplexed with primers for glyceraldehyde-3-phosphate dehydrogenase and 2× TaqMan Universal PCR master mix (Applied Biosystems). PCR was performed in a 7500 real-time PCR system (Applied Biosystems) using the manufacturer’s suggested thermal settings. Relative mRNA expression was calculated using the Δ comparative threshold (Ct) method. glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Each experiment was repeated three times. Data analysis was performed using analysis of variance and pairwise multiple comparisons using Tukey’s test. A P value of <0.05 was considered significant. Data are expressed as means ± SEM. The corneal abrasion re-epithelialized within 24 hours and basal epithelial cell density increased over the next 7 days. Figure 1A shows at time 0 the wound (W) and center wound (CW) fields were within the area of abrasion and devoid of epithelial cells. By 18 hours, epithelial cells were evident within the original wound area, and the density of basal cells in the limbus (L), paralimbus (PL), and parawound (PW) fields was reduced, consistent with previous studies showing that initial wound closure results from centripetal epithelial migration.22Lu L Reinach PS Kao WW Corneal epithelial wound healing.Exp Biol Med (Maywood). 2001; 226: 653-664Crossref PubMed Scopus (326) Google Scholar, 23Nagasaki T Zhao J Centripetal movement of corneal epithelial cells in the normal adult mouse.Invest Ophthalmol Vis Sci. 2003; 44: 558-566Crossref PubMed Scopus (116) Google Scholar, 32Maurice DM Zhao J Nagasaki T A novel microscope system for time-lapse observation of corneal cells in a living mouse.Exp Eye Res. 2004; 78: 591-597Crossref PubMed Scopus (6) Google Scholar Epithelial density increases thereafter as a result of cell division.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Uninjured murine corneal epithelium exhibits little binding of anti-ICAM-1 monoclonal antibody, but staining becomes evident in the unwounded limbal and paralimbal epithelium within 6 hours after central epithelial abrasion.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar ICAM-1 expression extends throughout the epithelium within 12 to 18 hours of central corneal abrasion.14Li Z Burns AR Smith CW Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing.Am J Pathol. 2006; 169: 1590-1600Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Limbal and paralimbal epithelium revealed a prominent increase in ICAM-1 mRNA levels 3 hours after central corneal epithelial abrasion (Figure 1B). ICAM-1 was evident in the epithelium and in limbal vessels of TCRδ−/− mice (Figures 1, C and D), indicating that γδ T cells are not necessary for ICAM-1 expression although they are necessary for efficient wound healing of the corneal epithelium.26Li Z Burns AR Rumbaut RE Smith CW Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion.Am J Pathol. 2007; 171: 838-845Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar GL3+ cells are rare in the corneal epithelium of wild-type mice, although they are found in the limbal epithelium26 and adjacent conjunctival epithelium. They increase significantly in the corneal epithelium after central epithelial abrasion. The possible contribution of ICAM-1 to migration of γδ T cells following corneal abrasion was assessed in ICAM-1−/− mice. ICAM-1−/− mice had fewer GL3+ cells in the limbus of uninjured corneas (Figure 2A), as compared with wild-type mice. GL3+ cells increased in the limbal and paralimbal epithelium of wild-type mice and reached peak levels at 24 hours, extending their distribution into the corneal epithelium near the original wound edge (Figure 2, A and B). ICAM-1−/− mice failed to show this increase (Figure 2, A and B) in contrast to P-sel−/−, which were not distinguishable from wild-type. GL3+ cells remained elevated in the epithelium of wild-type mice for at least 7 days but at significantly lower levels in the ICAM-1−/− mice (Figure 2C). Their distribution in wild-type mice was mostly limited to the limbal and paralimbal regions (Figure 2D). Wild-type mice receiving anti-ICAM-1 blocking antibody i.p., 30 minutes before central corneal epithelial injury, had low levels of GL3+ cells in the limbal and corneal epithelium (Figure 3A). Given that ICAM-1 is a known ligand for CD11a/CD18 (LFA-1), a member of the β2 integrin family, mice deficient in CD11a were analyzed at the peak time of accumulation of these cells in wild-type mice. CD11a−/− mice exhibited significant reductions in GL3+ cell numbers (Figure 3A). Immunocytology revealed staining of GL3+ cells with anti-CD11a (Figure 3B). GL3+ cells were sparse in the limbal stroma and absent in the corneal stroma of unwounded wild-type mice. GL3+ cells increased at 6 and 12 hours after central epithelial abrasion and decreased somewhat thereafter (Figure 4, A and B). In contrast to the epithelium, stromal GL3+ cells in ICAM-1−/− mice increased parallel with wild-type mice in the first 12 hours, and increased significantly beyond that of wild-type mice at 18 and 24 hours (Figures 4B). The accumulation of GL3+ cells in the ICAM-1−/− mice was primarily in the limbus (Figure 4C). The apparent compartmentalization of γδ T cells into epithelium and stroma was evident, though to a lesser extent, when CD11a−/− mice were compared with wild-type mice. CD11a-deficient GL3+ cells accumulated significantly less than wild-type in the epithelium and significantly more in the stroma (Figures 3A and 4D). Corneal epithelium is known to express a number of chemokines that potentially attract leukocytes in response to injury or infection.33Yoon KC de Paiva CS Qi H Chen Z Farley WJ Li DQ Pflugfelder SC Expression of Th-1 chemokines and chemokine receptors on the ocular surface of C57BL/6 mice: effects of desiccating stress.Invest Ophthalmol Vis Sci. 2007; 48: 2561-2569Crossref PubMed Scopus (137) Google Scholar To investigate the possibility that ICAM-1 is necessary for expression of chemokines that attract γδ T cells, we assessed epithelial mRNA levels for an array of chemokines reported to be chemotactic for γδ T cells. Limbal and paralimbal epithelium (ie, the regions not directly injured during abrasion) was collected from wild-type and ICAM-1−/− mice, either uninjured or 3 hours after central epithelial abrasion. mRNA levels for the chemokines listed in Figure 5 were significantly increased in abraded wild-type and ICAM-1−/− mice compared with unwounded epithelium. Values for CCL3, CCL4, and CXCL10 were significantly higher in ICAM-1−/− mice than in controls. Two pro-inflammatory cytokines, tumor necrosis factor and interleukin-1β, were evaluated as well, and both were significantly increased in wild-type and ICAM-1−/−. In wild-type, interleukin-1β increased 4.1-fold (P < 0.004) and tumor necrosis factor increased 1.5-fold (P < 0.004); in ICAM-1−/−, interleukin-1β increased 6.6-fold (P < 0.001), tumor necrosis factor, 2.0-fold (P < 0.008). These data contain no obvious deficits in chemokine expression to possibly account for the significantly reduced levels of γδ T cells in the epithelium of ICAM-1−/− mice. Epithelial wound closure was delayed in the ICAM-1−/− mice. Open wound area was significantly larger at 18 hours after a 2-mm central epithelial abrasion in ICAM-1−/− mice than wild-type mice (Figure 6A). Cell division in the unwounded regions of the epithelium (limbal and paralimbal) reached a peak at 18 hours after central epithelial abrasion in wild-type mice,34Li Z Burns AR Smith CW Two waves of neutrophil emigration in response to corneal epithelial abrasion: distinct adhesion molecule requirements.Invest Ophthalmol Vis Sci. 2006; 47: 1947-1955Crossref PubMed Scopus (101) Google Scholar but was significantly delayed in ICAM-1−/− mice (Figure 6B). Basal cell density across the uninjured cornea of ICAM-1−/− mice was not different from that of wild-type (Figure 6C), but recovery after injury in the center of the cornea at 48 hours was significantly reduced in the ICAM-1−/− mice (Figure 6D). The height of the uninjured central corneal epithelium was not significantly different among the wild-type, ICAM-1−/−, and TCRδ−/− mice. Though the wild-type mice recovered normal epithelial thickness within 96 hours following central corneal epithelial abrasion,26Li Z Burns AR Rumbaut RE Smith CW Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion.Am J Pathol. 2007; 171: 838-845Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar both ICAM-1−/− mice (recovering ∼66%, Figure 3A) and TCRδ−/− mice (recovering ∼60%26Li Z Burns AR Rumbaut RE Smith CW Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion.Am J Pathol. 2007; 171: 838-845Abstract Fu
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