Polymorphonuclear neutrophils delay corneal epithelial wound healing in vitro

Using an in vitro system for measuring epithelial wound healing, we studied the effect of polymorphonuclear neutrophils (PMNs) and PMN lysate on rat corneal epithelial wound healing. After 22 hr of organ culture, epithelial defects that were originally 3 mm in diameter (7.06 mm2) measured 0.41 mm2 (range, 0.17-0.72 mm2) in control rat corneas, 2.11 mm2 (range, 0.81-3.91 mm2) in corneas incubated in medium containing 5 X 10(6) PMN/ml, and 2.49 mm2 (range, 2.21-2.76 mm2) in corneas incubated with lysate obtained from an equivalent number of PMNs. Scanning and transmission electron microscopy showed similar morphology in the three groups. PMNs selectively adhered to the leading edge of the wound in corneas incubated with PMNs. These data indicate that PMNs and PMN lysate significantly slow corneal epithelial wound healing in vitro.     

Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins

PURPOSE: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS: Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS: The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea.     

Interaction between corneal invasion of polymorphonuclear leukocytes and corneal epithelium

The interaction between corneal invasion of polymorphonuclear leukocytes (PMNs) and corneal epithelium was investigated following different types of corneal injury. Histological examination of centrally denuded corneas demonstrated that it took 18-24 hours for PMNs to infiltrate into the center of the denuded stroma, approximately synchronous with the reepithelialization. On the other hand, in the centrally denuded corneas immediately covered by glue to prevent the reepithelialization, the time required for the appearance of PMNs at the central portion of the corneas was more than 96 hours. Chemotactic activities of PMNs in the conditioned media of normal cornea, completely denuded cornea and reepithelializing cornea were examined using a Boyden chamber. The highest chemotactic activity was defected in the conditioned medium of the cornea with reepithelialization. These data suggest the possibility that the corneal epithelium, especially the re-covering epithelium, stimulates infiltration of the stroma by PMNs.     

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1^−/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1^−/− corneas was reduced to  ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1^−/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1^−/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.     

Immunohistochemical localization of EGF, TGF-alpha, TGF-beta, and their receptors in rat corneas during healing of excimer laser ablation

PURPOSE: Members of the epidermal growth factor (EGF) and transforming growth factor beta (TGF-beta) families of growth factors and receptors are known to regulate key aspects of corneal wound healing, including epithelial migration and scar formation. To further understand their roles, mRNA levels were measured and proteins were immunolocalized in rat corneas at multiple time points during healing of excimer laser ablation injury. METHODS: Excimer laser photoablation was performed to a depth of 50 microm on rat corneas. Levels of mRNAs for EGF, TGF-alpha, TGF-beta isoforms 1, 2, and 3, and their receptors (EGF-R and TGFbeta-IIR) were measured by quantitative RT-PCR on days 0, 1.5, 7, 21, 42, and 91 after ablation. Immunohistochemical localization of the growth factors and their receptors was performed on days 0, 7, and 21 in corneal sections. RESULTS: Levels of EGF mRNA remained stable in rat corneas after ablation (68 +/- 12 copies/cell, mean +/- SD), whereas levels of TGF-alpha mRNA progressively increased sixfold to a maximum at day 42 (300 copies/cell) then slightly decreased on day 91. Levels of EGF-R mRNA rapidly increased 60-fold on day 7 compared with day 0 (571 vs. 9 copies/cell) then decreased sixfold above baseline at day 91. Levels of TGF-beta1 mRNA remained stable (36 +/- 10 copies/cell), whereas levels of TGF-beta2 and TGF-beta3 mRNAs peaked on day 21 (300-fold and 25-fold increase) and remained elevated through day 91. Levels of TGFbeta-IIR mRNA showed a similar pattern. Immunostaining of all the growth factors and receptors was primarily in basal layers of epithelial cells in uninjured cornea and during healing. Intensity of immunostaining for TGF-beta1, TGFbeta-IR, and TGFbeta-IIR increased appreciably in the basal epithelial layers after ablation. CONCLUSIONS: Levels of mRNAs for several key members of the EGF and TGF-beta systems increase during corneal wound healing. In addition, the proteins are primarily localized in basal layers of epithelial cells, which suggest these cells are active in synthesizing autocrine and paracrine growth factors that modulate corneal wound healing.     

Amnionmembrantransplantation bessert experimentelle herpetische Keratitis

PURPOSE: Transplantation of human amniotic membrane (AMT) accelerates the healing of experimental ulcerative herpetic keratitis. Here the expression and activity of matrix metalloproteinase (MMP)-9 was studied. METHODS: BALB/c mice were corneally infected with HSV-1. Whereas the infected corneas of mice in group 1 were covered with AM, tarsorrhaphies were performed in others (group 2). After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically, and the corneal PMN infiltration was studied histologically. The expression of MMP-9 in the corneas was localized by immunohistochemistry and analyzed by Western-blot technique. The MMP-9 activity in the corneas was determined by zymography. RESULTS: On day 14, the ulcerating corneas had a dense PMN infiltration, the ulcers and the majority of PMNs were highly positive for MMP-9, and the active forms of MMP-9 were detected. Gelatinolytic activity was found in these corneas by zymography. Compared with the mice of group 2, ulceration, stromal inflammation and neovascularization markedly improved clinically and histologically within 2 days in mice of group 1. This was associated with a reduced expression of MMP-9 in corneal tissue and in PMNs. The gelatinolytic activity of MMP-9 was reduced after AMT. CONCLUSIONS: These observations suggest that improvement of herpetic corneal ulcers and reduced corneal neovascularization after AMT may result from a reduced expression and activity of MMP-9.     

Inflammatory mediators in alkali-burned corneas: preliminary characterization

Leukocyte chemotactic factors (LCF) are important inflammatory mediators which activate and recruit leukocytes from the circulation into sites of tissue damage. These factors were recently detected in the tear fluid of inflamed eyes induced by alkali burn. It remains unclear, however, whether the detected LCF are released from injured corneal tissues or leaked from the circulation. Using a corneal cup model developed in our laboratory, we began examining the capability of corneal tissues to produce LCF in response to alkali injury. We also evaluated the influence of citric acid on the production of LCF from corneas preinjured by the alkali sodium hydroxide (NaOH). For these studies, the epithelial surfaces of corneas isolated from bovine and human eyes were exposed to 1N NaOH for 35 seconds at room temperature. The NaOH was then removed and the epithelial surfaces washed once with buffer and incubated with culture medium for 1, 2, 4, and 6 hours at 37 degrees C/5% CO2 atmosphere. Our results showed that (1) NaOH2 induced corneal epithelial cell injury ranging from cell discoloration and moderate damage of the upper half of the epithelium (1-4 hrs) to total destruction of the epithelium (6 hrs); (2) NaOH-injured corneas (2 hr incubation post injury) produced significant levels of chemotactic activity (via checkerboard analysis) specific for neutrophils (115% maximum chemotactic response [MCR]) and mononuclear cells (94% MCR); (3) preliminary characterization of these factors revealed that they are protease and heat sensitive, extractable by organic solvents, and possess molecular weight values greater than 100,000 daltons; and (4) incubation of NaOH-pretreated corneas with 0.01% citric acid for 2 hours markedly inhibited the production of LCF for both neutrophils (98% inhibition) and mononuclear cells (91% inhibition). Results of these studies indicate that alkali-burned bovine and human corneas generate leukocyte chemoattractants which differ in their biochemical characteristics from previously known low molecular weight chemotactic factors such as C5a, interleukin-1, or leukotriene B4.     

Prevention of stromal ulceration in the alkali-burned rabbit cornea by glued-on contact lens. Evidence for the role of polymorphonuclear leukocytes in collagen degradation.

Stromal ulceration of the alkali-burned rabbit cornea was found to be associated invariably with phagocytically active polymorphonuclear leukocytes (PMNs). A glued-on methylmethacrylate lens applied to corneas soon after burning, however, prevented re-epithelialization and also prevented PMN infiltration of the stroma and stromal ulceration. Subsequent partial detachment or complete removal of the lens resulted in epithelial resurfacing of the stroma, PMN infiltration, and stromal ulceration. Glued-on lenses applied to already ulcerating corneas arrested further ulceration by prohibiting additional PMN infiltration. Either surface debridement or glued-on methylmethacrylate rings also prevented re-epithelialization and ulceration in stromas not infiltrated by PMNs, but neither treatment was sufficient to prevent ulceration in corneas already containing numerous PMNs. The data suggest the possibility that the epithelium stimulates infiltration of the stroma by PMNs which then participate in stromal matrix degradation. Although no claim is made that only PMNs mediate matrix destruction in corneal ulceration, the efficacy of the lens would seem to be due to exclusion of the epithelium and the consequent prevention of stromal infiltration by PMNs.     

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.     

Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats

PURPOSE. The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). METHODS. Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. RESULTS. A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser(136) and Ser(112)) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. CONCLUSIONS. In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.     

The immune system in experimental Pseudomonas keratitis. Model and early effects

A model to study the immune system in Pseudomonas keratitis was developed using defined flora rats (WAG/RijMCW) that have not been exposed to Pseudomonas aeruginosa. One group of rats was made immunocompetent towards P. aeruginosa by intraperitoneal injection of phenol-killed P. aeruginosa while a second group remained naive to this organism. Corneas of both groups were scratched centrally with a 21-g needle, before inoculation with 2 X 10(8) P. aeruginosa organisms. Corneas of control animals were either only scratched or only inoculated with the bacterium. At 18 hr, the naive animals were killed. In naive rat corneas, light and electron microscopy showed bacteria throughout the cornea, polymorphonuclear leukocytes (PMNs) distributed from the limbus towards the center, and little stromal degradation. In contrast, massive corneal degradation was observed in the immunocompetent rats; PMNs were present, but no bacteria were observed free in the stroma. The total acid protease content was higher in the immunocompetent than in the naive rat corneas, a possible reason for the observed difference in corneal degradation. This difference was not due to increased numbers of PMNs since nearly equal numbers of PMNs were counted after enzymatic disaggregation of both types of corneas. Glycogen-induced peritoneal PMNs from both types of rats migrated equally well towards P. aeruginosa culture media and media of corneas incubated with this bacterium. The authors conclude that immune recognition is (1) involved in the corneal host response to P. aeruginosa and (2) required for efficient phagocytosis by PMNs but not their recruitment.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.     

Studies on the source and release of collagenase in thermally burned corneas of vitamin A-deficient and control rats

In previous studies we found that a mild thermal burn of the vitamin A-deficient rat cornea caused collagenase release into the medium of corneas placed in culture media 72 hr after applying the burn. Collagenase was released only on day 1 of culture and was not released from identically burned corneas of control rats. We now demonstrate that in deficient corneas, this collagenase release on day 1 of culture increased gradually with increasing time between burn and sacrifice, reaching a maximum at 16 hr after burning a remaining high up to 72 hr. In control rats day-1 collagenase release also increased to a maximum at 16 hr after the burn but then declined to almost zero at 72 hr. Trypsin treatment of day-1 media from both control and deficient corneas, taken at 72 hr after the burn, showed an almost complete absence of latent (inhibited) collagenase. Histologic observations revealed a close correlation between the presence of infiltrating polymorphonuclear neutrophils (PMNs) and the ulcerative lesions seen in burned, deficient corneas. When PMN infiltration was blocked by application of a tissue adhesive, no ulceration occurred and collagenase activity in the day-1 media dropped to almost zero. If burned and unburned areas of deficient corneas were separated and cultured separately, the burned area (containing most of the PMNs) was found to have 10 times the collagenase activity of the unburned area. In the controls, PMNs and some collagenase activity was detectable only 16 hr after the burn. We concluded that in the burned, vitamin A-deficient cornea there is increased attraction of PMNs to the lesion, resulting in collagenase release by these and possibly other cells, and ultimately resulting in ulceration.     

Thymosin-beta4 modulates corneal matrix metalloproteinase levels and polymorphonuclear cell infiltration after alkali injury

PURPOSE: Corneal alkali injury is highly caustic, and present clinical therapies are limited. The purpose of this study was to investigate the ability of thymosin-beta4 (Taubeta4) to promote healing in an alkali injury model and the mechanisms involved in that process. METHODS: Corneas of BALB/c mice were injured with NaOH, irrigated copiously with PBS, and treated topically with either Tbeta4 or PBS twice daily. At various time points after injury (PI), corneas from the Tbeta4- versus the PBS-treated group were examined for polymorphonuclear leukocyte (PMN) infiltration, chemokine, and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) expression. RESULTS: Tbeta4-treated corneas demonstrated improved corneal clarity at day 7 PI. Whereas Tbeta4 decreased corneal MMP-2 and -9 and MT6-MMP levels after alkali injury, no change in TIMP-1 and -2 expression was detected. Tbeta4 treatment also decreased corneal KC (CXCL1) and macrophage inflammatory protein (MIP)-2 chemokine expression and PMN infiltration. Immunohistochemistry studies demonstrated MMP-9 expression at the leading edge of the epithelial wound, in the the limbus (containing stem cells), and in stromal PMNs. CONCLUSIONS: Tbeta4 treatment decreases corneal inflammation and modulates the MMP/TIMP balance and thereby promotes corneal wound repair and clarity after alkali injury. These results suggest that Tbeta4 may be useful clinically to treat severe inflammation-mediated corneal injuries.     

Stromal degradation in vitamin A-deficient rat cornea. Comparison of epithelial abrasion and stromal incision

Epithelial abrasions (3-mm diameter) and linear stromal incisions (50-75% depth) were made in vitamin A-deficient (A-) rat corneas to investigate what are contributory factors to the development of keratomalacia. Sixty-four rats were killed at various times after injury, and the corneas were histologically examined. In pair-fed control corneas, wounds healed without stromal degradation. Although abraded A- corneas were infiltrated by numerous polymorphonuclear neutrophil leukocytes (PMNs), reepithelialization eventually occurred and severe stromal degradation was not evident, suggesting that PMN infiltration alone cannot cause keratomalacia. In incised A- corneas, six (20%) exhibited marked stromal degradation, of which three (10%) were infected. Two other corneas studied shortly after incision were in the initial stages of infection. The results suggest that stromal injury importantly contributes to the development of keratomalacia. Bacterial infection might also be contributory. Cytochrome oxidase staining showed that metabolic functional levels of incised A- corneas were accelerated only in the wound zone, whereas abraded A- corneas were metabolically accelerated throughout.     

The relationship of corneal Langerhans cells to herpes simplex antigens during dendritic keratitis

The corneal migration and topographic distribution of Langerhans cells (LC) in relation to herpes simplex virus antigens was studied during the course of dendritic keratitis in inbred mice. Corneal epithelial sheets from infected mice at selected time points were "double stained" for Ia-positive Langerhans cells and HSV antigens, using a sequential avidin biotin immunoperoxidase and glucose oxidase technique. The amount of HSV antigen was maximum at day 2 paralleling the clinical time course, with most corneal epithelium HSV antigen negative by day 8. LC were seen in peripheral corneas by day 2 and in paracentral and central cornea by day 8, with peak numbers detected between days 8 and 11 post-infection. Although HSV antigens and LC were simultaneously detected within corneal epithelium, LC were not observed in anatomic juxtaposition to HSV antigens, even after reinoculation of infected corneas with HSV on day 14 following the primary infection. These data suggest that local factors in the corneal epithelium other than HSV antigens per se may be chemotactic for LC during the course of dendritic keratitis.     

The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40 mg kg(-1) day(-1)or 80 mg kg(-1) day(-1), or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0 mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12 hr. To determine the number of cells in S phase, entire corneas were labelled with (3)H-thymidine and subjected to autoradiography at 0, 12, 24 and 48 hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24 hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P < 0.05). In the cornea before wounding (0 hr) and at 48 hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P < 0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.     

The effects of citrate on fMLP-induced polymorphonuclear leukocyte stimulation and locomotion

Citrate reduces the incidence of corneal ulceration and perforation in alkali burned eyes and prevents polymorphonuclear leukocyte (PMN) accumulation in certain inflamed ocular tissues. Chelation of Ca2+ and Mg2+ by citrate appears to be the mechanism causing strong inhibition of in vitro PMN stimulation by opsonized zymosan. It is important to know if other activating agents of PMNs, with differing sensitivity to divalent cations, are inhibited by citrate. Citrate (12 mM) partially inhibits fMLP stimulation of the respiratory burst (50%) and degranulation (65%) of PMNs in a divalent cation free media, while having no effect or only a small effect in the presence of 1 mm Ca2+. Citrate also caused significant inhibition of fMLP (12 mM = 50%) induced locomotion of PMN when incubated in media containing 500 microM Ca2+ and 600 microM Mg2+ in a modified Boyden chamber. When used together, Ca2+ (6 mM) and Mg2+ (6 mM) reduced this inhibition to only 20%. Citrate apparently inhibits fMLP-induced stimulation in cation free media by chelating CA2+ effluxed from intracellular storage sites. In the chemotactic studies, citrate probably chelates extracellular Ca2+ and Mg2+. The divalent cation requirements of the activating agents and/or the PMN function may determine the degree of inhibition by citrate. It is therefore important to identify the mediators in alkali burned corneas as well as other inflammatory conditions.