Cellular Infiltrate in Rheumatoid Arthritis-associated Paracentral Corneal Ulceration.

PURPOSE: To investigate an immunopathogenesis of central and paracentral corneal ulceration associated with rheumatoid arthritis. METHODS: Sparse infiltrating cells in the ulcer area were identified by immunohistochemistry applied to archived formalin fixed, paraffin embedded tissues that had been recovered from patients undergoing penetrating keratoplasty necessitated by rheumatoid-associated central or paracentral corneal ulceration. RESULTS: Clinically, the ulcers presented as non-infiltrated lesions with a modicum of other ocular inflammation. Sparse T-lymphocytes were consistently identified in the subepithelial areas adjacent to the ulcer, with some neutrophils and macrophages in the stroma. B-lymphocytes were not detected. MHC Class II antigens reactivity was noted on some infiltrating cells and on corneal endothelium of two specimens. CONCLUSIONS: Immunohistochemistry of archival tissue facilitated detection and identification of sparse infiltrate in this infrequent corneal melting. Selective, consistent finding of T-lymphocyte infiltration in the ulcer area supports an immunopathogenesis of this clinical entity.

Novel model of innate immunity in corneal infection.

The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-µm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.

A novel association between resident tissue macrophages and nerves in the peripheral stroma of the murine cornea.

PURPOSE: To characterize the interactions between resident macrophage populations and nerves in naïve and injured corneas of the mouse eye. METHODS: Corneas from wild-type (WT) C57BL/6J, BALB/cJ, and transgenic Cx3cr1-eGFP mice were subjected to a 1-mm central epithelial debridement injury. The eyes were fixed and immunostained as flat mounts with a range of antibodies to identify macrophages, neurons, and Schwann cells. Interactions between nerves and immune cells were analyzed and quantitated using three-dimensional reconstructions of confocal microscopy images. Naïve eyes acted as controls. RESULTS: A distinctive association between resident immune cells and corneal nerves was noted in the peripheral or perilimbal stromal nerve trunks. These epineurial cells were mostly Cx3cr1(+) Iba-1(+) major histocompatibility complex (MHC) class II(+) F4/80(+) CD11b(+) macrophages. The number of nerve-associated macrophages was greater in WT BALB/c mice than in C57BL/6J mice. There were no qualitative or quantitative differences in the circumferential distribution of nerve-associated macrophages in the cornea. Sterile corneal epithelial debridement led to a dissociation of macrophages from peripheral nerve trunks as early as 2 hours postinjury, with numbers returning to baseline after 72 hours. This dissociation was Cx3cr1 dependent. CONCLUSIONS: This study is the first to highlight a direct physical association between nerves and resident immune cells in the murine cornea. Furthermore, we reveal that this association in normal eyes is responsive to central corneal epithelial injury and is partly mediated by Cx3cr1 signaling. This association may serve as an indicator of malfunctioning neuroimmune communication in disease states such as neurotrophic keratitis and peripheral neuropathy.

Equine deep stromal abscesses (51 cases - 2004-2009)--Part 2: the histopathology and immunohistochemical aspect with attention to the histopathologic diagnosis, vascular response, and infectious agents.

PURPOSE: To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF-A), pigment epithelium-derived factor (PEDF), and interleukin-1 receptor antagonist (IL-1ra). SAMPLE POPULATION: Paraffin-embedded biopsy samples from 51 DSA. The biopsies were collected from full-thickness penetrating keratoplasty or split-thickness lamellar keratoplasty surgeries at the University of Florida Veterinary Medical Center in the period from 2004 to 2009. PROCEDURE: The histopathologic and immunohistochemical findings were tested for association between each other. Prevalence calculation and test for association with qualitative data analysis was used for data evaluation. RESULTS: Fungal hyphae were found histologically in 47.1% (n = 24) of the DSA cases. Histopathologically, most fungal DSA showed suppurative keratitis (n = 34; 66.7%) and little to no stromal vascularization infiltrating the abscess (negative association, P = 0.005). All three angiogenesis-related factors were expressed to some degree in DSA tissue. A negative association between VEGF-A and PEDF when compared to the presence of fungal hyphae (P < 0.001, P = 0.023) indicated that cases positive for these two factors will most probably not have fungal hyphae present. CONCLUSION: Abnormally decreased VEGF-A expression is suggested as the reason for the slow vascularization and delayed resolution of fungal DSA, whereas PEDF and IL-ra did not seem to have any influence on the vascularization process. Clinical and histopathologic characteristics of DSA make it possible to suggest an etiology for an equine DSA with an unknown etiology.

IL1beta, IL6 and IL8 gene polymorphisms involvement in recurrent corneal erosion in patients with hereditary stromal corneal dystrophies.

TGFBI gene mutations cause corneal stromal dystrophies of autosomal dominant inheritance. The most frequent complication of stromal dystrophies is recurrent corneal erosion with varying degree of accompanying inflammation. IL-1beta, IL-6 and IL-8 are main cytokines involved in corneal erosion healing. This study aimed to investigate the association between IL1B gene -511C/T, IL6 gene -174G/C and IL8 gene -781C/T polymorphisms and risk of recurrent erosion development in patients with hereditary corneal stromal dystrophies. A trend to decrease of IL1B gene -511TT genotype frequency in group with erosion (3.7%) comparing to control (6.7%) was observed. IL6 gene -174C allele carriers frequency in control group (65.9%) was significantly (P < 0.05) lower comparing to patients with erosion (80.5%). Frequency of IL8 -781TT genotype was significantly (P < 0.05) lower in the group with erosion (10.7%) comparing to patients without erosion (30.8%) and control (25%). IL6 gene -174C allele may be considered as genetic marker of corneal erosion risk in patients with hereditary stromal corneal dystrophies, whereas IL8 -781TT genotype is associated with negative recurrent erosion prognosis in such patients.

Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.

Purpose. To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED). Methods. Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1ß, IL-6, TNF, and CCL2), corneal infiltration of CD11b(+) antigen-presenting cells, and lymph node frequency of mature MHC-II(hi) CD11b(+) cells were assessed. Results. The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1ß, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b(+) cells and the lymph node frequency of MHC-II(hi) CD11b(+) cells. Conclusions. These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Substance P in the corneal stroma regulates the severity of herpetic stromal keratitis lesions.

PURPOSE: To determine whether substance P (SP) in herpes simplex virus-1 (HSV-1) infected cornea regulates the severity of herpetic stromal keratitis (HSK) lesions in a mouse model. METHODS: C57BL/6 mice were infected ocularly with HSV-1 (RE). The corneas with HSK lesions, on Day 15 postinfection, were grouped on the basis of the corneal opacity as mild (≤2) or severe (>2). The amount of SP was determined in the corneas with mild or severe HSK lesions by enzyme immunosorbent assay (EIA) and confocal microscopy. Subconjunctival inoculation of spantide I, SP receptor antagonist, was carried out during the clinical phase of HSK. ELISA and flow cytometry were used to determine the level of cytokines, chemokines, and influx of immune cell types in the corneal lesions. RESULTS: The authors determined a significantly higher level of SP in the corneas with severe HSK lesions in comparison with mild lesions. The corneas with a higher level of SP also exhibited higher amounts of proinflammatory cytokines (IL-6, IFN-γ) and chemokines (CCL3, CXCL2) when compared with the corneas with a lower level of SP. SP receptor NK1R expression was determined in CD45- and CD45+ cells in infected cornea. SP present in the corneal stroma of the eyes with severe HSK lesions colocalized with ß-III tubulin(+) and IA(b+) cell types. Subconjunctival inoculation of spantide I during the clinical phase of HSK resulted in significant reduction in the corneal opacity and angiogenesis. CONCLUSIONS: Collectively, these results demonstrate the relative contribution of substance P in regulating the clinical severity of HSK lesions in a mouse model.

Long-term follow-up of a supradescemetic keratoprosthesis in rabbits: an immunofluorescence study.

OBJECTIVE: To evaluate the long-term clinical and immunohistological outcome of two different non-penetrating keratoprosthesis (KPro) implanted in non-injured rabbit corneas. MATERIALS AND METHODS: Three rabbits underwent implantation of a pHEMA-MMA(34) synthetic cornea in the supradescemetic space, and PMMA synthetic corneas in the supradescemetic space and within the central stroma. Animals were followed for at least 24 months before euthanasia. Periodic evaluation was performed with slit-lamp examination and photography. At the end of the follow-up, histological examination including hematoxylin eosin staining and immunocharacterization against collagen IV, alpha smooth muscle actin (α-SMA) and macrophages was performed. RESULTS: The pHEMA-MMA(34) implant was not extruded, and remained transparent until the end of follow-up. This material did not induce any cell infiltration, corneal scarring or tissue remodeling in the surrounding stroma as shown by immunofluorescence. In contrast, synthetic corneas made of PMMA-induced myofibroblast differentiation, stromal remodeling and macrophage infiltration. This reaction was even more significant in the rabbit with the PMMA implant within the corneal stroma. CONCLUSION: pHEMA-MMA(34) was clinically biocompatible, and did not induce any inflammatory reaction or scarring when implanted in the supradescemetic space. This material showed more promising biocompatibility results than for PMMA, whether implanted within the central cornea stroma or in the supradescemetic space.

Immunotactoid microtubular corneal deposits in bilateral paraprotein crystalline keratopathy.

PURPOSE: To report an ultrastructurally distinct form of paraprotein crystalline keratopathy. METHODS: Three corneas submitted from a single patient including one native cornea from each eye and a failed corneal graft from the right eye. Light microscopy, immunohistochemistry, immunofluorescence, and transmission electron microscopic examination were performed. RESULTS: The transmission electron microscopy showed "immunotactoid" extracellular microtubular deposits measuring >30 nm in diameter with a central lucent core. CONCLUSIONS: Immunotactoid keratopathy is a distinct type of paraprotein crystalline keratopathy associated with a monocolonal immunoglobulin G kappa light chain (IgGk) protein. Larger case series are needed to determine the clinical significance of this entity.

Amniotic membrane transplantation induces apoptosis in T lymphocytes in murine corneas with experimental herpetic stromal keratitis.

PURPOSE: To investigate the effect of human amniotic membrane transplantation (AMT) on T-cell immune response in murine corneas with herpetic stromal keratitis (HSK). METHODS: Herpes simplex virus (HSV)-1-infected BALB/c mice with necrotizing HSK were treated with AMT. CD3(+) cell apoptosis was determined in treated corneas and in vitro by flow cytometric analysis using the annexin V/7-AAD system. The effect of interleukin (IL)-2, cyclosporine, rapamycin, or Fas on T-cell survival was measured. Activation phenotype was measured by (3)H-thymidine uptake and flow cytometry (CD25, CD69, major histocompatibility complex class II). Cytokine/chemokine secretion from amniotic membrane (AM)-treated corneas or draining lymph node cells was measured. The immune-modulating capacity of long-term AMT treatment and adoptive transfer of AM-treated splenocytes was tested. RESULTS: After AMT, HSK and corneal inflammatory cell infiltration improved, and T-lymphocyte apoptosis occurred. T-cell apoptosis was also induced in vitro, independently of rIL-2, cyclosporine, rapamycin, or Fas. AMT-treated corneas and cultured lymphocytes had reduced IL-2, IL-10, IL-12, CRG-2, and CCL-2 content. Long-term AMT treatment decreased the proliferative response and type 1 helper T-cell cytokine level in draining lymph node cells. The improvement in HSK did not persist. Delayed-type hypersensitivity or HSV-1-specific cytotoxicity was not altered CONCLUSIONS: The results suggest that murine HSK improves after AMT through reduced local T-helper cell immune responses by inducing apoptosis in T lymphocytes, independently of passive apoptosis or activation-induced cell death. AM also reduces local T-helper cytokine and chemokine levels but does not result in immune deviation. Immunologic memory against HSV-1 is not affected by AMT, and long-term protection or tolerance is not induced.

The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold.

This study was to develop a method using phospholipase A(2) (PLA(2)) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200 U/ml PLA(2) and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA(2) and SD were 0.35+/-0.04 U/mg dry weight and 4.3+/-0.8 ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8+/-2 days, and their transparency was restored in 84+/-11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.

Gamma interferon and interleukin-1 receptor 1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis.

Toll-like receptor 2 (TLR2) is an essential mediator of corneal inflammation induced by the filarial nematode Onchocerca volvulus, which harbors endosymbiotic Wolbachia bacteria. TLR2 is also required for dendritic cell activation, gamma interferon (IFN-gamma) production, and neutrophil recruitment to the cornea. To examine the role of IFN-gamma in O. volvulus keratitis, C57BL/6 and IFN-gamma(-/-) mice were immunized subcutaneously, and a soluble antigen extract from O. volvulus adult worms (OvAg) was injected into the corneal stroma of each animal. We found that, in the absence of IFN-gamma, neutrophil recruitment to the cornea was significantly impaired, whereas there was no effect on eosinophil infiltration. Since the cornea contains resident macrophages and fibroblasts and our previous studies showed that CXC chemokines mediate neutrophil recruitment, we examined the role of recombinant IFN-gamma (rIFN-gamma) on each cell type. We found no effect of rIFN-gamma on CXC chemokine production by macrophages or corneal fibroblasts, either alone or with filarial extracts; in contrast, rIFN-gamma was found to enhance OvAg-induced tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-1alpha, and IL-1beta in macrophages. Furthermore, we found that rTNF-alpha, rIL-1alpha, or rIL-1beta induced CXC chemokine production by corneal fibroblasts but not by macrophages. To determine the relative contributions of endogenous cytokines, we injected OvAg into the corneal stroma of C57BL/6, IL-1 receptor 1(-/-) (IL-1R1(-/-)), and TNF-alphaR1/2(-/-) mice and examined neutrophil recruitment. We found that neutrophil infiltration was impaired in IL-1R1(-/-) mice but not in TNF-alphaR1/2(-/-) mice. IFN-gamma therefore appears to regulate neutrophil recruitment to the corneal stroma by enhancing TLR2 expression and OvAg-induced IL-1alpha and IL-1beta production by macrophages in the cornea, which then induce IL-1R1-dependent production of CXC chemokine by resident corneal fibroblasts.

MCP-1 derived from stromal keratocyte induces corneal infiltration of CD4+ T cells in herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is an inflammatory disorder induced by HSV-1 infection and characterized by T cell-dependent destruction of corneal tissues. It is not known what triggers CD4(+) T cell migration into the stroma of HSV-1-infected corneas. The keratocyte is a fibroblast-like cell that can function as an antigen-presenting cell in the mouse cornea by expressing MHC class II and costimulatory molecules after HSV-1 infection. We hypothesized that chemokines produced by stromal keratocytes are involved in CD4(+) T cell infiltration into the cornea. We found that keratocytes produce several cytokines and chemokines, including MCP-1, RANTES, and T cell activation (TCA)-3. HSV-1 infection increased the production of MCP-1 and RANTES by keratocytes, and these acted as chemoattractants for HSV-1-primed CD4(+) T cells expressing CCR2 and CCR5. Expression of MCP-1 in the corneal stroma was confirmed in vivo. Finally, when HSV-1-primed CD4(+) T cells were adoptively transferred into wild type and MCP-1-deficient mice that had been sublethally irradiated to minimize chemokine production from immune cells, infiltration of CD4(+) T cells was markedly reduced in the MCP-1-deficient mice, suggesting that it is the MCP-1 from HSV-1-infected keratocytes that attracts CD4(+) T cells into the cornea.

Turnover of bone marrow-derived cells in the irradiated mouse cornea.

In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1(gfp) transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1(+/gfp) recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP(+) cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1(+/gfp) cornea was 366 cells/mm(2). In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP(+) cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation.

[Biocompatibility of acellular corneal stroma and transplantation of tissue-engineered corneal epithelium].

OBJECTIVE: To evaluate the biocompatibility of xenogeneic acellular corneal stroma, the feasibility of tissue engineered corneal epithelial transplantation, and verify the long term survival of epithelial allograft. METHODS: It was a experimental study. Porcine corneal stroma was treated by dispase followed by Triton-X-100 detergent. Treated porcine corneal stroma (group A) or fresh corneal stroma (group B) were put into the sac of rabbit cornea. Rabbit cornea without implantation of porcine corneal stroma was used as the control group (group C). Immunological rejection was evaluated in morphology, histopathology and immunohistochemistry in month 1, 3, 6. Female rabbit underwent lamellar keratoplasty (LK) using male tissue engineered corneal epithelium as donors, and immunological rejection after LK was analyzed. The corneas were collected at day 1, 3, 7, 15 and 30 after LK and evaluated by histopathology, immunohistochemistry and sex-determining region of Y-chromosome (SRY)-PCR analysis. RESULTS: All corneas became transparent gradually after the transplantation of treated porcine corneal stroma and were not rejected. The epithelium, stroma, endothelium, Bowman's and Descemet's membrane were preserved in all corneas of group A and B in histological observations, collagen fibers were parallel, a few keratocytes presented in the acellular and fresh corneal stromas. The corneas of group C were normal in histological sections. No significant immune rejection was noted in any of the corneas. The corneas in the study of transplantation of tissue engineered cornea epithelium recovered smoothly in 3 or 4 days, turned transparent in 15 or 20 days after surgery and were not stained by fluorescein. Well-differentiated corneal epithelium were recognized at 15 and 30 days after LK. Many keratocytes infiltrated into the scaffold. SRY-PCR analysis showed that allogenic donor corneal epithelium cells could survive in recipients after a long-term observation. CONCLUSIONS: Acellular porcine corneal stroma shows a satisfying biocompatibility. Tissue engineered corneal epithelium using acellular porcine corneal stroma as carrier could be used as donors for LK with satisfactory results. Donor cells have the potential to survive in recipients after long-term observation.

CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis.

The severity of corneal inflammation depends on the activity of infiltrating neutrophils responding to chemotactic factors such as CXC chemokines. This study examines the relative contribution of CXCL1/keratinocyte-derived chemokine (KC), CXCL2/monocyte-inhibitory protein-2 (MIP-2), and CXCL5/LPS-induced chemokine (LIX) in neutrophil recruitment to the corneal stroma during LPS keratitis, where neutrophils infiltrate the corneal stroma at 6 h after LPS injection and peak at 24 h. Consistent with this timeframe, KC was detected after 3 h, reached peak levels at 24 h, and decreased thereafter. In contrast, LIX production was not detected until 8 h after injection and peaked at 24 h. MIP-2 was detected at 3 h but did not reach the levels of KC and LIX. Cell types associated with corneal inflammation produced markedly different chemokines in vitro: Murine corneal fibroblasts (MK/T-1) produced LIX and KC in response to LPS but did not produce MIP-2, whereas peritoneal macrophages and neutrophils produced MIP-2 and KC but did not produce LIX. To determine the role of these chemokines in neutrophil recruitment to the cornea, anti-LIX, anti-KC, or anti-MIP-2 was injected into the corneal stroma of enhanced GFP chimeric mice prior to LPS, and total cell and neutrophil infiltration was examined. Antibody to LIX and KC, injected individually or in combination, significantly inhibited neutrophil recruitment to the cornea, whereas anti-MIP-2 had no inhibitory effect. Together, these findings demonstrate cell-specific production of CXC chemokines and show that LIX and KC mediate neutrophil recruitment into the cornea during LPS keratitis.

Characterisation of rat corneal cells that take up soluble antigen: an in vivo and in vitro study.

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 microl (30 microg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag+ cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24h after Ag injections or topical application and the distribution and phenotype of Ag+ cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag+ cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag+ cells within the corneal stroma. The distribution of Ag+ cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68+, CD172+ but rarely MHC class II+, a profile characteristic of macrophages. Occasional Ag+ keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163+ and CD11b+ macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II(-). In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.

Differences in leukocyte phenotype and interferon-gamma expression in stroma and endothelium during corneal graft rejection.

Critical to the success of human corneal transplants is prevention of corneal endothelial rejection, yet little is known about the endothelial infiltrate. To examine the endothelium, a method for removal and processing this layer as a flat sheet was used and the infiltrate was compared with stroma and epithelium. LEW or PVG strain rat corneas were transplanted to PVG strain recipients. Clinical changes after transplantation were monitored by slit lamp and animals sacrificed at a range of time points during rejection. Clinically defined rejection, accompanied by an epithelial rejection line and endothelial cell infiltration, occurred between days 10 and 15. There was some infiltration of leukocytes in the stroma of isografts at these time points, but significantly more in allografts (p<0.003 for all subsets). There was no infiltration of isograft endothelium at any time and no infiltration of allograft endothelium on day 10. On day 15, there were similar numbers of all major subsets except B cells in the stroma, while on the endothelium macrophages, MHC class II(+) cells and CD8(+) cells predominated (p<0.001 CD4(+) vs CD8(+) cells). T cells and NK cells predominated in the epithelial rejection line. While TNF-alpha and IFN-gamma-producing cells were numerous in stroma and epithelium, no IFN-gamma-producing cells were found on endothelium. Distinct differences in infiltrative profile within layers of the cornea suggest that the mechanisms of rejection may also differ. The restricted endothelial cell profile and lack of IFN-gamma suggests that the anti-endothelial response may be modulated by the anterior chamber environment.

Visualization and characterization of inflammatory cell recruitment and migration through the corneal stroma in endotoxin-induced keratitis.

PURPOSE: The infiltration of inflammatory cells into the cornea is a major determinant in the outcome of keratitis. The purpose of this study was to use enhanced green fluorescence protein (EGFP) bone marrow chimeric mice to visualize and characterize the inflammatory cells that migrate to the corneal stroma during endotoxin-induced keratitis and to explore the mechanisms underlying this process. METHODS: Keratitis was induced by injecting endotoxin into the corneas of EGFP chimeric mice. In vivo fluorescence stereomicroscopy was used to visualize in real time the recruitment of EGFP-positive cells at different time points. Immunohistochemistry and three-dimensional (3D) confocal analysis of whole-mount corneas was used for histologic characterization. Macrophage inflammatory protein-2 (MIP-2) chemokine was neutralized in vivo to determine its contribution to this process. RESULTS: Recruitment of EGFP-positive inflammatory cells in the corneal stroma can be detected in vivo by 6 hours after the injection of endotoxin, and these were mainly neutrophils. Full-thickness whole corneal mount confocal image analysis showed a distinct pattern of migration of EGFP inflammatory cells through the anterior corneal stroma. Moreover, inflammatory cells did not colocalize with the injected lipopolysaccharide (LPS) deposits in the stroma but moved from all directions toward LPS, partially in response to the production of the chemokine MIP-2. CONCLUSIONS: EGFP chimeric mice and ex vivo 3D analysis of whole-mount corneas provides unique information on the interaction of infiltrating inflammatory cells in the cornea. These findings demonstrate that a chemotactic gradient triggered in part by MIP-2 is responsible for directing inflammatory cell migration through the corneal stroma.

Stromal architecture and immune tolerance in additive corneal xenografts in rodents.

PURPOSE: To examine immune tolerance and corneal ultrastructure following additive corneal xenografts in rodents. METHODS: We carried out surgical implantation of excised BALB/c mouse corneal tissue, either freshly isolated (n=6) or after storage at--20 degrees C for 1 week (n=7), into the corneas of Wistar rats at approximately mid-stromal depth. Corneal opacity and neovascularization were evaluated postoperatively, and stromal ultrastructure was observed by transmission electron microscopy. RESULTS: Corneal opacification and neovascularization in the weeks after surgery were less prevalent in grafts of frozen-then-thawed tissue than in grafts of fresh tissue. In a well tolerated frozen-then-thawed xenograft, the matrix architecture was normal throughout most of the recipient and donor tissue, but pronounced fibrillar disorganization was evident adjacent to Descemet's membrane. CONCLUSION: We attribute the improved tolerance of frozen-then-thawed xenografts over fresh xenografts to a reduced cellular immune response.