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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2009 Aug;175(2):571–579. doi: 10.2353/ajpath.2009.090112

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Sarah E Byeseda *, Alan R Burns *†, Sean Dieffenbaugher *, Rolando E Rumbaut *‡, C Wayne Smith *‡, Zhijie Li *§
PMCID: PMC2716957  PMID: 19608878

Abstract

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.

Intercellular adhesion molecule-1 (ICAM-1, CD54)1 is a conserved member of the immunoglobulin supergene family2 and is expressed by many cell types in response to stimuli such as cytokines,3,4 and oxidative and physical stress.5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium7 and epithelium,8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),12 and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.7 This capability influences functions such as leukocyte transendothelial migration7 and vascular permeability.13

Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.14,15,16,17,18,19,20,21 It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. 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.14 Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,22,23 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-1.24,25

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,14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,27 and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.

Materials and Methods

Animals

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−/− mice24,25 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.28 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.14,29

Corneal Epithelial Wounding Model

The central corneal wound was performed as previously described.30 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.

Immunohistology

Taking care to include the limbus, wounded corneas were dissected, fixed, permeabilized, and incubated with the following labeled monoclonal antibodies as described14,26,30,31: 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.14 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.

Histological Assessment of Corneal Thickness

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).

Quantitative Real-Time PCR

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.

Statistical Analysis

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.

Results

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.22,23,32 Epithelial density increases thereafter as a result of cell division.14 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.14 ICAM-1 expression extends throughout the epithelium within 12 to 18 hours of central corneal abrasion.14 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.26

Figure 1.

Figure 1

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A: Recovery of epithelial basal cell density was determined microscopically by counting 4′,6-diamidino-2-phenylindole-stained nuclei in five fields from limbus to center of the cornea at various times from immediately after wounding (0 hour) to 7 days after a 2 mm diameter central epithelial abrasion. Limbus (L), paralimbus (PL), parawound (PW), wound (W), center wound (CW). B: ICAM-1 expression in murine corneal epithelium. Limbal, paralimbal, and parawound epithelium was collected immediately (0 hour) or at 3, 6, or 18 hours after the central epithelial abrasion. Epithelial ICAM-1 mRNA levels were determined by qPCR and expressed relative to glyceraldehyde-3-phosphate dehydrogenase. Wild-type mice revealed significant (*P < 0.01, n = 6–18) increases in ICAM-1 expression above uninjured epithelium at each time point after abrasion. C: ICAM-1 expression in TCRδ−/− mice was similar to that of wild-type (C57BL/6) mice (n = 6). D: ICAM-1 expression in TCRδ−/− mice at 12 hours after central corneal epithelial abrasion. a) limbal vessel, anti-CD31-PE, showing endothelial cells (red); b) merged image with anti-ICAM-1-fluorescein isothiocyanate, showing ICAM-1 expression of cells lining the limbal vessel (arrows); c) corneal epithelial basal cells in the limbus are also positive for anti-ICAM-1-PE.

ICAM-1 and Epithelial γδ T cells

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).

Figure 2.

Figure 2

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Corneal and limbal epithelial γδ T cells in response to corneal abrasion. A: Whole mount corneal preparations were stained with PE-tagged monoclonal antibody GL3 (anti-TCRδ) at various times after central epithelial abrasion. The sum of GL3+ cells in the epithelium in nine microscopic fields across the cornea from limbus to limbus are plotted for wild-type, ICAM-1−/−, and P-selectin−/− mice. All values for the wild-type and P-selectin−/− mice are significantly different (P < 0.01, n = 6) from those of the ICAM-1−/− mice. B: Photomontage covering the limbal and paralimbal regions of wild-type (top) and ICAM-1−/− (bottom) corneas at 24 hours after central abrasion showing the density and distribution of GL3+ cells. Scale bar = 10 μm. C: GL3+ cells remained elevated in the wild-type corneas at 48 and 96 hours and 7 days after injury (P < 0.01, n = 6) for each time compared with uninjured wild-type corneas. ICAM-1−/− corneas were not significantly different from uninjured ICAM-1−/− corneas, and remained significantly less (P < 0.01, n = 6) than wild-type after injury at all time points studied. D: The distribution of GL3+ cells across the corneas of wild-type mice at various times after central epithelial abrasion is plotted (n = 6) Limbus (L), paralimbus (PL), parawound (PW), wound (W), center wound (CW). The value at 24 hours in the paralimbal (PL) region was significantly different (P < 0.05) from the other times points in the PL region.

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).

Figure 3.

Figure 3

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Epithelial γδ T cells in response to corneal abrasion. A: Whole mount corneal preparations were stained with PE-tagged monoclonal antibody GL3 (anti-TCRδ) at 24 hours after central epithelial abrasion, the peak accumulation in wild-type mice (see Figure 2). The sum of GL3+ cells in the epithelium in nine microscopic fields across the cornea are plotted for wild-type, ICAM-1−/−, CD11a−/−, and P-selectin−/− mice, as well as wild-type mice treated with either anti-ICAM-1 (30 minutes i.p. before abrasion) or anti-GP1bα (24 hours i.p. before abrasion) monoclonal antibodies. *P < 0.01, n = 6. B: Photomicrographs of wild-type corneal paralimbus at 24 hours after injury stained with both anti-TCRδ-fluorescein isothiocyanate and anti-CD11a-PE showing (a) only GL3+ cells and (b) merged image with both antibodies. Arrows indicate double positive cells. Scale bar = 10 μm.

ICAM-1 and Corneal Stromal γδ T Cells

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).

Figure 4.

Figure 4

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Stromal γδ T cells in response to corneal abrasion. A: Limbus of cornea from wild-type and ICAM-1−/− mice 24 hours after central epithelial abrasion stained with anti-TCRδ (green) and anti-CD31 (red). Scale bar = 20 μm. B: Whole mount corneal preparations were stained with antibody GL3 (anti-TCRδ) at various times after central epithelial abrasion. The sum of GL3+ cells in the stroma in nine microscopic fields across the cornea are plotted for wild-type, and ICAM-1−/− mice. *P < 0.05, as compared with wild-type at 18 and 24 hours, n = 6. C: Distribution of GL3+ cells in the stroma of the cornea from limbus to center wound region at 18 hours after wounding wild-type, and ICAM-1−/− mice. *P < 0.05, compared with wild-type limbus (L). D: GL3+cells were counted in the stroma of corneas at 24 hours following central corneal abrasion. Numbers of these cells in ICAM-1−/− and CD11a−/− mice were significantly (*P < 0.01, n = 6) greater then wild-type (WT); values for P-sel−/− and platelet depleted (anti-GP1bα) were not significantly different from wild-type.

ICAM-1 and Chemokine Expression by Corneal Epithelium

Corneal epithelium is known to express a number of chemokines that potentially attract leukocytes in response to injury or infection.33 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.

Figure 5.

Figure 5

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Epithelial chemokine expression. Expression analysis of corneal epithelium at 3 hours after central corneal abrasion in wild-type and ICAM-1−/− mice. RNA was isolated from the regions of the cornea not directly injured by abrasion. All chemokines listed were significantly increased (P < 0.05, n = 3 pools of RNA from six mice per pool) over uninjured epithelium of the same region. *P < 0.01.

ICAM-1 and Epithelial Wound Healing

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,34 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,26 both ICAM-1−/− mice (recovering ∼66%, Figure 3A) and TCRδ−/− mice (recovering ∼60%26) had significantly less epithelial height at this time (P < 0.01, n = 6 corneas from each strain). Epithelial thickness at 96 hours after injury remained significantly less in the ICAM-1−/− than in uninjured corneas in both the central region where epithelium was removed by abrasion and in the paralimbal region that was initially uninjured (Figure 7A). Two weeks after abrasion ICAM-1−/− epithelium attained a thickness equivalent to wild-type (P > 0.05, n = 6 corneas, Figure 7B), but TCRδ−/− recovery remained at 60.7% of the uninjured thickness (P < 0.05, n = 6 corneas, Figure 7B). These results indicate that ICAM-1 deficiency significantly reduces the rate of epithelial recovery after abrasion but is less detrimental than deficiency of γδ T cells.

Figure 6.

Figure 6

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Corneal epithelial wound healing. A: At 18 hours after central epithelial abrasion, the wound area was assessed microscopically in 4′,6-diamidino-2-phenylindole-stained whole mount preparations, and the area without epithelial cells was calculated. This area was significantly less (*P < 0.01, n = 5) in wild-type mice than ICAM-1−/−. B: Dividing epithelial cells were counted in nine microscopic fields across the corneas from limbus to limbus at various times after central abrasion. ICAM-1−/− mice exhibited significantly fewer (*P < 0.01, n = 6) dividing cells at 12, 18, and 24 hours after injury. C: Basal epithelial cell density was analyzed across uninjured corneas of wild-type and ICAM-1−/− mice. D: Basal epithelial cell density was significantly less (P < 0.05, n = 6) at 48 hours after abrasion in ICAM-1−/− mice.

Figure 7.

Figure 7

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Corneal epithelial wound healing. At 96 hours or 2 weeks after central epithelial abrasion, epithelial thickness was measured in fixed toluidine blue stained 0.5-μm thick plastic sections in the central region (ie, healed epithelium) and in the paralimbus (ie, epithelium that was not directly injured). A: ICAM-1−/− mice failed to attain uninjured epithelial thickness at 96 hours after injury either in the central or paralimbal regions. This is in contrast to wild-type mice.26 B: Photomicrographs of central corneal sections from wild-type, ICAM-1−/− and TCRδ−/− mice 2 weeks after central epithelial abrasion. In contrast to wild-type and ICAM-1−/− mice, TCRδ−/− epithelium was thinner and less cellular. The arrows indicate the measured height of the epithelium from the basement membrane beneath the basal cells to the outer surface of the epithelium. C: Blood platelet depletion was induced by i.p. injection of anti-GP1bα monoclonal antibody 24 hours before central corneal abrasion as previously reported.14 GL3+ cells were counted in the epithelium in designated regions of the cornea at 24 hours following abrasion.

Platelet Depletion and Epithelial γδ T Cells

Wild-type mice treated with anti-GP1bα to deplete blood platelets exhibit delays in corneal epithelial wound healing31 comparable with that seen in the ICAM-1−/− mice. In the current study, wild-type mice were given anti-GP1bα to induce prolonged platelet depletion. In contrast to ICAM-1−/− mice where GL3+ cells failed to accumulate in the healing epithelium, increases in GL3+ cells in the epithelium following platelet depletion were not different from control wild-type mice (Figures 3A and 7C). These observations demonstrate a condition that delays wound healing without significantly reducing γδ T cell accumulation in the epithelium.

Discussion

ICAM-1-deficient mice and wild-type mice treated with anti-ICAM-1 monoclonal antibody failed to show the expected accumulation of γδ T cells in the peripheral corneal epithelium following central epithelial abrasion. Since ICAM-1-deficient mice have delayed wound healing, this failure could simply reflect some aspect of wound healing other than a direct dependence on ICAM-1. Against this interpretation are three experimental observations: 1) Epithelial γδ T cells accumulated equally in wild-type and platelet-depleted mice, a condition that induces delay in wound closure31 comparable with ICAM-1-deficient mice. 2) P-selectin-deficient mice have a significant delay in wound healing31 but exhibited normal epithelial accumulation of γδ T cells. 3) Mice deficient in CD11a, the α subunit of LFA-1 and the major leukocyte adhesion molecule recognizing ICAM-1, exhibited significantly reduced accumulation of epithelial γδ T cells. These data are consistent with a direct requirement for ICAM-1 in the accumulation of γδ T cells in the corneal epithelium following central corneal abrasion.

In contrast to the epithelium, γδ T cells entered the stroma of ICAM-1-deficient mice in response to epithelial abrasion and the initial rate of increase in the area around the limbal vessels was essentially the same as that of wild-type mice. It appears that ICAM-1 is not critical to their migration from the vasculature into the stroma. Corneal epithelial cells and limbal vessel endothelial cells can express ICAM-114,15,16,17,18,19,20,21 in response to inflammatory cytokines35,36 such as tumor necrosis factor and interleukin-1β (both expressed in our model of epithelial abrasion). While ICAM-1 is known to contribute to lymphocyte adhesion and transendothelial migration, other adhesion molecules (eg, VCAM-1) also support lymphocyte transmigration.37,38,39,40 In contrast to endothelial adhesion, Iwata et al in earlier work20 found that LFA-1/ICAM-1 interaction is dominant in human lymphocyte adhesion to human corneal epithelial cells expressing ICAM-1 after stimulation with inflammatory cytokines. Our current observations confirm CD11a on wild-type murine γδ T cells, and CD11a−/− mice (ie, LFA-1-deficient) have significantly reduced γδ T cell accumulation in the corneal epithelium. Expression of ICAM-1 mRNA and the presence of ICAM-1 on limbal and paralimbal epithelial cells14 after central epithelial abrasion coincides temporally with the accumulation of γδ T cells in those corneal regions. These observations are consistent with the idea that epithelial ICAM-1 serves as a necessary adhesive ligand for LFA-1-dependent migration or retention of γδ T cells into the corneal epithelium.

The source of γδ T cells in corneal epithelium is not clear. In wild-type mice, γδ T cells increased in the stroma near the limbal vessels at 12 hours after injury and then decreased as they increased in the epithelium of the limbal and paralimbal regions over the next 12 hours. A possible route may be blood-derived γδ T cells migrating through the stroma into the epithelium. Evidence in the ICAM-1-deficient mice is consistent with this idea since there is increased accumulation of γδ T cells in the limbal stroma at 18 and 24 hours after injury without appearance in the epithelium, as if entrance into the epithelium requires ICAM-1, but migration from blood into stroma does not. Since the stromal γδ T cells in the ICAM-1-deficient mice remain essentially localized in the limbus (see Figure 4C), migration through the stroma may be deficient. A possible role for ICAM-1 in the stroma is its expression on the keratocytes41,42 serving as an adhesive site for cell locomotion. We published morphometric evidence that neutrophils interact with keratocytes in the corneal stroma and that deficiency of β2 (CD18) integrins (including LFA-1) significantly reduces this interaction.43

Locomotion of γδ T cells is influenced by chemokines, and expression analysis of the epithelium indicates numerous potential chemokines increased within the time frame of γδ T cell accumulation. CCL3, CCL4, and CCL5 are of potential interest given results with some populations of γδ T cells shown to express a relevant chemokine receptor CCR5.37,44 Another chemokine receptor of possible importance for future studies is CXCR3 given the prominence of CXCL10 in the epithelial expression profile and published evidence for its expression by some populations of γδ T cells.37,45 A third chemokine receptor of possible importance is CCR2, given increased CCL2 in the expression profile and published evidence on some γδ T cells.44 The data on chemokine expression thus far fails to identify a deficiency in the ICAM-1−/− mice that could account for the failure of γδ T cell migration into the epithelium. Our current hypothesis is that ICAM-1 is functioning as a necessary adhesive ligand for γδ T cell motility or retention in the epithelium.

Corneal epithelial wound healing is delayed in ICAM-1-deficient mice. The rate of wound closure, the level of epithelial cell division within the first day following injury, the return of epithelial cell density, and thickness of epithelial stratification in the wounded region, were all significantly delayed in ICAM-1−/− mice. ICAM-1 has also been found necessary for efficient epidermal healing,46,47 linked to emigration of neutrophils and macrophages, a well-established role for this adhesion molecule at sites of inflammation.7,46 In the current study, we raise that possibility that ICAM-1 contribution to corneal epithelial healing may be linked to lymphocytes entering the epithelium. Elevated numbers of γδ T cells occur during the phase of re-epithelialization and remain in the epithelium for at least 7 days following injury. Their contribution to corneal healing is indicated by the finding that epithelial recovery is significantly delayed at least 2 weeks following injury in TCRδ−/− mice. γδ T cells have been reported necessary for efficient epidermal healing,48,49 and the healing epidermis contains γδ T cells.50 Potential direct contributions of resident epidermal γδ T cells include the production of keratinocyte growth factor and insulin-like growth factor-1,51 as well as the induction of hyaluronan.48 Whether the γδ T cells that migrate into the cornea following epithelial wounding also produce these factors remains to be determined, but it cannot be assumed, since we have preliminary evidence (Smith et al unpublished) that the corneal γδ T cells are a different subset from the Vγ3Vδ1 dendritic epidermal T cells found in mouse skin.

In summary, we have demonstrated in the current studies that γδ T cells accumulate in the corneal epithelium within 18 hours following central epithelial abrasion and remain at significantly elevated levels for at least 7 days, a time well past full recovery of epithelial density and thickness. Further, γδ T cell deficiency results in failure to recover full epithelial density and thickness even 2 weeks after abrasion. ICAM-1 deficiency results in significant delay in corneal epithelial wound healing, and the expected accumulation of γδ T cells in the epithelium fails to occur. CD11a-deficiency also significantly reduces γδ T cells in the epithelium suggesting that normal migration of γδ T cells into the epithelium requires LFA-1 (CD11a/CD18) interactions with ICAM-1.

Footnotes

Address reprint requests to Zhijie Li, Ph.D., Section of Leukocyte Biology, Children’s Nutrition Research Center, Room 6014, 1100 Bates, Houston, Texas 77030. E-mail: zhijiel@bcm.tmc.edu.

Supported by National Institutes of Health grants EY018239, EY017120, HL079368, and National Natural Science Foundation of China Grants 39970250 and 30672287.

References

  1. Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol. 1986;137:1270–1274. [PubMed] [Google Scholar]
  2. Staunton DE, Marlin SD, Stratowa C, Dustin ML, Springer TA. Primary structure of intercellular adhesion molecule 1 (ICAM-1) demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell. 1988;52:925–933. doi: 10.1016/0092-8674(88)90434-5. [DOI] [PubMed] [Google Scholar]
  3. Smith CW. Endothelial adhesion molecules and their role in inflammation. Can J Physiol Pharmacol. 1993;71:76–87. doi: 10.1139/y93-012. [DOI] [PubMed] [Google Scholar]
  4. Mickelson JK, Kukielka G, Bravenec JS, Mainolfi E, Rothlein R, Hawkins HK, Kelly JH, Smith CW. Differential expression and release of CD54 induced by cytokines. Hepatology. 1995;22:866–875. [PubMed] [Google Scholar]
  5. Sung HJ, Yee A, Eskin SG, McIntire LV. Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types. Am J Physiol Cell Physiol. 2007;293:C87–C94. doi: 10.1152/ajpcell.00585.2006. [DOI] [PubMed] [Google Scholar]
  6. Hu X, Zhang Y, Cheng D, Ding Y, Yang D, Jiang F, Zhou C, Ying B, Wen F. Mechanical stress upregulates intercellular adhesion molecule-1 in pulmonary epithelial cells. Respiration. 2008;76:344–350. doi: 10.1159/000137509. [DOI] [PubMed] [Google Scholar]
  7. Rahman A, Fazal F. Hug tightly and say goodbye: role of endothelial ICAM-1 in leukocyte transmigration. Antioxid Redox Signal. 2008;11:823–839. doi: 10.1089/ars.2008.2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burns AR, Smith CW, Walker DC. Unique structural features that influence neutrophil emigration into the lung. Physiol Rev. 2003;83:309–336. doi: 10.1152/physrev.00023.2002. [DOI] [PubMed] [Google Scholar]
  9. Porter JC, Hall A. Epithelial ICAM-1 and ICAM-2 regulate the egression of human T cells across the bronchial epithelium. FASEB J. 2009;23:492–502. doi: 10.1096/fj.08-115899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Uchida R, Ashihara E, Sato K, Kimura S, Kuroda J, Takeuchi M, Kawata E, Taniguchi K, Okamoto M, Shimura K, Kiyono Y, Shimazaki C, Taniwaki M, Maekawa T. Gamma delta T cells kill myeloma cells by sensing mevalonate metabolites and ICAM-1 molecules on cell surface. Biochem Biophys Res Comm. 2007;354:613–618. doi: 10.1016/j.bbrc.2007.01.031. [DOI] [PubMed] [Google Scholar]
  11. Kato Y, Tanaka Y, Tanaka H, Yamashita S, Minato N. Requirement of species-specific interactions for the activation of human gamma delta T cells by pamidronate. J Immunol. 2003;170:3608–3613. doi: 10.4049/jimmunol.170.7.3608. [DOI] [PubMed] [Google Scholar]
  12. Staunton DE, Dustin ML, Erickson HP, Springer TA. The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell. 1990;61:243–254. doi: 10.1016/0092-8674(90)90805-o. [DOI] [PubMed] [Google Scholar]
  13. Sumagin R, Lomakina E, Sarelius IH. Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling. Am J Physiol Heart Circ Physiol. 2008;295:H969–H977. doi: 10.1152/ajpheart.00400.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Li Z, Burns AR, Smith CW. Lymphocyte function-associated antigen-1-dependent inhibition of corneal wound healing. Am J Pathol. 2006;169:1590–1600. doi: 10.2353/ajpath.2006.060415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zhu SN, Dana MR. Expression of cell adhesion molecules on limbal and neovascular endothelium in corneal inflammatory neovascularization. Invest Ophthalmol Vis Sci. 1999;40:1427–1434. [PubMed] [Google Scholar]
  16. Berger RB, Blackwell NM, Lass JH, Diaconu E, Pearlman E. IL-4 and IL-13 regulation of ICAM-1 expression and eosinophil recruitment in Onchocerca volvulus keratitis. Invest Ophthalmol Vis Sci. 2002;43:2992–2997. [PubMed] [Google Scholar]
  17. Pereira HA, Ruan X, Gonzalez ML, Tsyshevskaya-Hoover I, Chodosh J. Modulation of corneal epithelial cell functions by the neutrophil-derived inflammatory mediator CAP37. Invest Ophthalmol Vis Sci. 2004;45:4284–4292. doi: 10.1167/iovs.03-1052. [DOI] [PubMed] [Google Scholar]
  18. Iwata M, Fushimi N, Suzuki Y, Suzuki M, Sakimoto T, Sawa M. Intercellular adhesion molecule-1 expression on human corneal epithelial outgrowth from limbal explant in culture. Br J Ophthalmol. 2003;87:203–207. doi: 10.1136/bjo.87.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kaifi JT, Diaconu E, Pearlman E. Distinct roles for PECAM-1. ICAM-1, and VCAM-1 in recruitment of neutrophils and eosinophils to the cornea in ocular onchocerciasis (river blindness) J Immunol. 2001;166:6795–6801. doi: 10.4049/jimmunol.166.11.6795. [DOI] [PubMed] [Google Scholar]
  20. Iwata M, Sawada S, Sawa M, Thoft RA. Mechanisms of lymphocyte adhesion to cultured human corneal epithelial cells. Curr Eye Res. 1997;16:751–760. doi: 10.1076/ceyr.16.8.751.8983. [DOI] [PubMed] [Google Scholar]
  21. Nemeth G, Felszeghy S, Kenyeres A, Szentmary N, Berta A, Suveges I, Modis L. Cell adhesion molecules in stromal corneal dystrophies. Histol Histopathol. 2008;23:945–952. doi: 10.14670/HH-23.945. [DOI] [PubMed] [Google Scholar]
  22. Lu L, Reinach PS, Kao WW. Corneal epithelial wound healing. Exp Biol Med (Maywood) 2001;226:653–664. doi: 10.1177/153537020222600711. [DOI] [PubMed] [Google Scholar]
  23. Nagasaki T, Zhao J. Centripetal movement of corneal epithelial cells in the normal adult mouse. Invest Ophthalmol Vis Sci. 2003;44:558–566. doi: 10.1167/iovs.02-0705. [DOI] [PubMed] [Google Scholar]
  24. Robker 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;12:936–940. doi: 10.1038/oby.2004.114. [DOI] [PubMed] [Google Scholar]
  25. Brake 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–C1239. doi: 10.1152/ajpcell.00008.2006. [DOI] [PubMed] [Google Scholar]
  26. Li 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;171:838–845. doi: 10.2353/ajpath.2007.070008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liu 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;24:900–911. doi: 10.1111/j.1440-1746.2008.05668.x. [DOI] [PubMed] [Google Scholar]
  28. Ding 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;163:5029–5038. [PubMed] [Google Scholar]
  29. Bowden 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;90:562–569. doi: 10.1161/01.res.0000013835.53611.97. [DOI] [PubMed] [Google Scholar]
  30. Li Z, Rivera CA, Burns AR, Smith CW. Hindlimb unloading depresses corneal epithelial wound healing in mice. J Appl Physiol. 2004;97:641–647. doi: 10.1152/japplphysiol.00200.2004. [DOI] [PubMed] [Google Scholar]
  31. Li 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;47:4794–4802. doi: 10.1167/iovs.06-0381. [DOI] [PubMed] [Google Scholar]
  32. Maurice DM, Zhao J, Nagasaki T. A novel microscope system for time-lapse observation of corneal cells in a living mouse. Exp Eye Res. 2004;78:591–597. doi: 10.1016/s0014-4835(03)00210-0. [DOI] [PubMed] [Google Scholar]
  33. Yoon 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;48:2561–2569. doi: 10.1167/iovs.07-0002. [DOI] [PubMed] [Google Scholar]
  34. Li 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;47:1947–1955. doi: 10.1167/iovs.05-1193. [DOI] [PubMed] [Google Scholar]
  35. Yannariello-brown J, Hallberg CK, Haberle H, Brysk MM, Jiang Z, Patel JA, Ernst PB, Trocme SD. Cytokine modulation of human corneal epithelial cell ICAM-1 (CD54) expression. Exp Eye Res. 1998;67:383–393. doi: 10.1006/exer.1998.0514. [DOI] [PubMed] [Google Scholar]
  36. Bouchard CS, Lasky JB, Cundiff JE, Smith BS. Ocular surface upregulation of intercellular adhesive molecule-1 (ICAM-1) by local interferon-gamma (IFN-gamma) in the rat. Curr Eye Res. 1996;15:203–208. doi: 10.3109/02713689608997414. [DOI] [PubMed] [Google Scholar]
  37. Poggi A, Zancolli M, Catellani S, Borsellino G, Battistini L, Zocchi MR. Migratory pathways of gammadelta T cells and response to CXCR3 and CXCR4 ligands: adhesion molecules involved and implications for multiple sclerosis pathogenesis. Ann NY Acad Sci. 2007;1107:68–78. doi: 10.1196/annals.1381.008. [DOI] [PubMed] [Google Scholar]
  38. Schulz B, Pruessmeyer J, Maretzky T, Ludwig A, Blobel CP, Saftig P, Reiss K. ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin. Circ Res. 2008;102:1192–1201. doi: 10.1161/CIRCRESAHA.107.169805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Costa MF, Nihei J, Mengel J, Henriques MG, Penido C. Requirement of L-selectin for gammadelta T lymphocyte activation and migration during allergic pleurisy: Co-relation with eosinophil accumulation. Int Immunopharmacol. 2009;9:303–312. doi: 10.1016/j.intimp.2008.12.004. [DOI] [PubMed] [Google Scholar]
  40. Sixt M, Bauer M, Lammermann T, Fassler R. Beta1 integrins: zip codes and signaling relay for blood cells. Curr Opin Cell Biol. 2006;18:482–490. doi: 10.1016/j.ceb.2006.08.007. [DOI] [PubMed] [Google Scholar]
  41. Seo SK, Gebhardt BM, Lim HY, Kang SW, Higaki S, Varnell ED, Hill JM, Kaufman HE, Kwon BS. Murine keratocytes function as antigen-presenting cells. European J Immunol. 2001;31:3318–3328. doi: 10.1002/1521-4141(200111)31:11<3318::aid-immu3318>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  42. Kumagai N, Fukuda K, Fujitsu Y, Nishida T. Expression of functional ICAM-1 on cultured human keratocytes induced by tumor necrosis factor-alpha. Jpn J Ophthalmol. 2003;47:134–141. doi: 10.1016/s0021-5155(02)00686-x. [DOI] [PubMed] [Google Scholar]
  43. Petrescu MS, Larry CL, Bowden RA, Williams GW, Gagen D, Li Z, Smith CW, Burns AR. Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins. Invest Ophthalmol Vis Sci. 2007;48:5023–5029. doi: 10.1167/iovs.07-0562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Penido C, Costa MF, Souza MC, Costa KA, Candea AL, Benjamim CF, Henriques MG. Involvement of CC chemokines in gammadelta T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway. Int Immunol. 2008;20:129–139. doi: 10.1093/intimm/dxm128. [DOI] [PubMed] [Google Scholar]
  45. Ajuebor MN, Jin Y, Gremillion GL, Strieter RM, Chen Q, Adegboyega PA. GammadeltaT cells initiate acute inflammation and injury in adenovirus-infected liver via cytokine-chemokine cross talk. J Virol. 2008;82:9564–9576. doi: 10.1128/JVI.00927-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nagaoka T, Kaburagi Y, Hamaguchi Y, Hasegawa M, Takehara K, Steeber DA, Tedder TF, Sato S. Delayed wound healing in the absence of intercellular adhesion molecule- 1 or L-selectin expression. Am J Pathol. 2000;157:237–247. doi: 10.1016/S0002-9440(10)64534-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yukami T, Hasegawa M, Matsushita Y, Fujita T, Matsushita T, Horikawa M, Komura K, Yanaba K, Hamaguchi Y, Nagaoka T, Ogawa F, Fujimoto M, Steeber DA, Tedder TF, Takehara K, Sato S. Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1. J Leukoc Biol. 2007;82:519–531. doi: 10.1189/jlb.0307152. [DOI] [PubMed] [Google Scholar]
  48. Jameson JM, Cauvi G, Sharp LL, Witherden DA, Havran WL. Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation. J Exp Med. 2005;201:1269–1279. doi: 10.1084/jem.20042057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Jameson J, Havran WL. Skin gammadelta T-cell functions in homeostasis and wound healing. Immunol Rev. 2007;215:114–122. doi: 10.1111/j.1600-065X.2006.00483.x. [DOI] [PubMed] [Google Scholar]
  50. Jameson J, Ugarte K, Chen N, Yachi P, Fuchs E, Boismenu R, Havran WL. A role for skin gammadelta T cells in wound repair. Science. 2002;296:747–749. doi: 10.1126/science.1069639. [DOI] [PubMed] [Google Scholar]
  51. Havran WL. A role for epithelial gammadelta T cells in tissue repair. Immunol Res. 2000;21:63–69. doi: 10.1385/IR:21:2-3:63. [DOI] [PubMed] [Google Scholar]

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