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The maintenance of normal vision is dependent on preserving corneal transparency. For this to occur, this tissue must remain avascular and its stromal architecture needs to be retained. Epithelial transparency is maintained provided the uppermost stratified layers of this tissue are composed of terminally differentiated non-keratinizing cells. In addition, it is essential that the underlying stromal connective tissue remains avascular and scar-free. Keratocytes are the source of fibroblasts that are interspersed within the collagenous framework and the extracellular matrix. In addition, there are sensory nerve fibers whose lineage is possibly either neural crest or mesenchymal. Corneal wound healing studies have been undertaken to delineate the underlying pathogenic responses that result in the development of opacification following chemical injury. An alkali burn is one type of injury that can result in severe and long- lasting losses in ocular transparency. During the subsequent wound healing process, numerous different proinflammatory cytokines and proteolytic enzymes undergo upregulation. Such increases in their expression levels induce maladaptive expression of sustained stromal inflammatory fibrosis, neovascularization, and losses in the smooth optical properties of the corneal outer surface. It is becoming apparent that different transient receptor potential channel (TRP) isoforms are important players in mediating these different events underlying the wound healing process since injury upregulates both their expression levels and functional involvement. In this review, we focus on the involvement of TRPV1, TRPA1 and TRPV4 in mediating some of the responses that underlie the control of anterior ocular tissue homeostasis under normal and pathological conditions. They are expressed on both different cell types throughout this tissue and also on corneal sensory nerve endings. Their roles have been extensively studied as sensors and transducers of environmental stimuli resulting from exposure to intrinsic modulators and extrinsic ligands. These triggers include alteration of the ambient temperature and mechanical stress, etc., that can induce pathophysiological responses underlying losses in tissue transparency activated by wound healing in mice losses in tissue transparency. In this article, experimental findings are reviewed about the role of injury-induced TRP channel activation in mediating inflammatory fibrotic responses during wound healing in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Okada, Sumioka, Reinach, Miyajima and Saika.)

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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.

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.

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

Purpose: Our previous study revealed the immunomodulatory property of the secreted lymphocyte antigen (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1), abundantly expressed in the cornea and associated with the hyperkeratotic disorder Mal de Meleda. Here, we test the hypothesis that SLURP1 modulates the functions of membrane-tethered uPAR by acting as a soluble scavenger of its ligand urokinase-type plasminogen activator (uPA)., Methods: Human corneal limbal epithelial (HCLE) and mouse corneal stromal fibroblast MK/T-1 cells were employed to examine the effect of SLURP1 on cell proliferation and migration. Human corneal limbal epithelial cell clones stably expressing SLURP1 under the control of cytomegalovirus (CMV) promoter were generated using lentiviral vectors. Recombinant 6× His-mouse Slurp1 and maltose-binding protein (MBP)-mouse uPA were expressed in Escherichia coli and partially purified using nickel-ion and amylose columns, respectively. Slurp1 interaction with uPA was detected using ligand blots, ELISA, pull-down assays, and immunofluorescent staining., Results: Stable expression of SLURP1 in HCLE cells was confirmed by immunoblots and immunofluorescent staining. Human corneal limbal epithelial and MK/T-1 cell proliferation and migration rates were suppressed by exogenous SLURP1. Ligand blots, ELISA, and pull-down assays indicated that Slurp1 efficiently interacts with uPA. Immunofluorescent staining demonstrated that exogenous SLURP1 decreased the amount of cell surface-bound uPA in the leading edges of migrating cells. In gap-filling assays, wild-type HCLE cells responded to uPA by increasing their velocity and closing larger area, while the SLURP1-expressing HCLE cells failed to do so., Conclusions: SLURP1 modulates corneal homeostasis by serving as a soluble scavenger of uPA and regulating the uPA-dependent functions of uPAR., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

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., (© 2013 American College of Veterinary Ophthalmologists.)

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.

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.

Purpose: To study the corneal development in the human fetal eye with particular emphasis on the epithelial basement membrane and Bowman's layer. Thus, immunohistochemical markers supposed to stain this region were employed., Material and Methods: 19 formalin-fixed fetal eyes and a 16-day-old newborn's cornea without any obvious irregularities of the anterior segment were investigated. The age of the fetal eyes ranged from 11 to 38 week of gestation (WoG). The eyes (including the corneal thickness) were measured and, in addition to routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, immunohistochemical labeling with antibodies to collagen IV, V, IX, and XVII was performed., Results: Analysis of the H&E stains revealed that measurements of corneal thickness correlated well with corneal development as a basic indicator for maturation. In a more detailed immunohistochemical analysis, collagen IV was expressed in the epithelial basement membrane (BM) of the cornea, conjunctiva, and Descemet's membrane in fetal eyes up to the age of 23 WoG. In fetal eyes older than 23 WoG, staining was confined to the limbal area only. With the antibody against collagen V, the corneal stroma and the BM were intensely stained. Bowman's layer (first detected at 17 WoG by light microscopy) was not labeled. Anti-collagen IX labeled predominantly the conjunctival and corneal epithelium. With anti-collagen XVII, the BM of the cornea and conjunctiva was stained in all fetal eyes, whereas intracellular expression in the epithelium increased with age., Conclusion: Our results indicate maturation-associated variations of collagen expression in the human cornea. Measurements of the corneal thickness may serve as an additional parameter to narrow down the developmental age with possible implications for pediatric pathology and forensic issues.

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-1β, 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-1β, 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.

Purpose: The peripheral cornea contains mature and immature resident dendritic cells (DCs) while the central cornea is exclusively equipped with immature DCs. There must be some factors that cause immature DCs. This study investigated whether corneal stroma cells (CSCs) inhibit DC maturation by secreting cytokines., Methods: The messenger ribonucleic acid (mRNA) and protein level of transforming growth factor beta 2 (TGF-β(2)) was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immature DCs were induced to mature in the presence of lipopolysaccharide (LPS) and with concentrations of CSC culture supernatant (containing and not containing neutralizing TGF-β(2) antibodies). Then, the DC phenotypic and functional maturation were analyzed., Results: CSCs exhibited positive expressions of TGF-β(2) mRNA and secreted high concentrations of TGF-β(2) protein. In the presence of LPS, DCs, which were treated with a CSC culture supernatant, displayed reduced expressions of cluster of differentiation 80 (CD80), CD86, and major histocompatibility complex II (MHC II) in a dose-dependent manner. Moreover, treated DCs showed lower T-cell stimulation capacity and a higher endocytosis function. However, these phenotypic and functional modifications were partially reversed after the application of neutralizing TGF-β(2) antibodies., Conclusions: This study demonstrates that CSCs can partially inhibit LPS-induced DC maturation through TGF-β(2) secretion in vitro.

TLRs are critical for host defense and innate immunity. Emerging evidence also supports a role for TLRs in many chronic inflammatory diseases, including inflammatory eye disease, known as uveitis. The activation of TLR4 by endotoxin induces a standard model of murine uveitis. How activation of additional TLRs influences the onset and/or severity of anterior uveitis has not been examined. We sought to elucidate the potential of TLRs (TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7/8, and TLR9) to trigger uveitis in mice. Directly stimulated iris/ciliary body explants demonstrated a marked increase in production of inflammatory cytokines TNF-α, IL-6, IP-10/CXCL10, MCP-1, and KC with relatively little production of IFN-γ, IL-10, IL-12p40, IL-12p70, IL-17, IL-1β, IL-4, or RANTES. The cytokine-response profiles were comparable amongst the TLR agonists, albeit some differences were noted, such as greater IP-10 production following TLR3 activation. Intra-ocular injection of TLR agonists increased leukocyte interactions with the endothelium of the iris vasculature and resulted in chemotaxis into the iris tissue. Assessment of leukocytic responses by ivt videomicroscopy and histology revealed quantitative differences amongst responses to the TLR agonists with respect to the timing and numbers of rolling, adhering, iris-infiltrating, and aqueous humor-infiltrating cells, along with cytokine levels in vivo. Our data demonstrate the eye's responsiveness to a diverse array of microbial products, which activates TLRs, and reveal differences in relative cellular response among the various TLR agonists in vivo.

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 β-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.

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.

Purpose: To investigate the effect of ex vivo culture on the expression of human leukocyte antigen (HLA) in human corneal limbal epithelial cells (LECs), and to evaluate the expression of HLA and costimulatory molecules on human corneal limbal stromal cells (LSCs)., Methods: The expression of HLA-ABC and HLA-DR were evaluated using flow cytometry on cultured human LECs and LSCs after serial passage and compared with that on freshly isolated cells. Additionally, the expression of costimulatory molecules CD80, CD86, CD40, and immunoregulatory molecule HLA-G were investigated on LSCs with or without an addition of interferon (IFN) γ (250 U/mL, 4 days)., Results: HLA-ABC and HLA-DR were expressed in 99.12 ± 1.02% and 25.86 ± 4.34% of freshly isolated LECs, respectively. After culture, the HLA-DR-expressing cells significantly decreased with serial passage, whereas the percentage of HLA-ABC-expressing cells did not change. More than 90% of LSCs expressed HLA-ABC and HLA-G, whereas the expression of HLA-DR, CD80, CD86, and CD40 was negligible. This expression profile did not change after serial passage. However, the treatment with IFN-γ induced a significant increase in HLA-DR (0.17% vs. 17.68%) and CD40 (0.19% vs. 13.19%) expression on LSCs., Conclusions: The immunogenicity of LECs can be lowered after ex vivo cultivation, whereas LSCs can be immunogenic in the presence of IFN-γ.

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.

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.

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.

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.

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.

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.

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.

Matrix metalloproteinase (MMP)-8 and MMP-9 play several roles in inflammation, including degradation of extracellular matrix (ECM) components and regulation of cytokine activity. To determine the roles of MMP-8 and MMP-9 in a neutrophil-dependent inflammatory response, we used a murine model of corneal inflammation in which LPS is injected into the corneal stroma. In contrast to wild-type mice, we found that i) lipopolysaccharide (LPS)-injected CXCR2(-/-) corneas had impaired neutrophil infiltration and did not express either MMP-8 or MMP-9; ii) neutrophil migration through the central cornea was impaired in Mmp8(-/-), but not Mmp9(-/-), mice; iii) neutrophil migration was inhibited in collagenase-resistant mice; iv) the chemotactic Pro-Gly-Pro (PGP) tripeptide that binds CXCR2 was decreased in CXCR2(-/-) mice; v) PGP production was impaired in Mmp8(-/-) corneas; and vi) neutralizing anti-PGP antibody did not inhibit neutrophil infiltration in Mmp8(-/-) mice. We found no effects of MMP-8 on LPS-induced CXC chemokine (LIX, or CXCL5)-induced neutrophil recruitment or on LPS-induced CXC chemokine production. Together, these studies indicate that neutrophils contribute to the production of both MMP-8 and MMP-9 in LPS-injected corneas and that MMP-8 regulates neutrophil migration through the dense collagenous ECM of the corneal stroma by generating chemotactic PGP during inflammation.

Purpose: To investigate herpes stromal keratitis (HSK) immunopathology by studying HSV-1-infected corneas that fail to develop HSK., Methods: Plaque assay quantified HSV-1 in the tear film of infected mice. FACS analysis enumerated corneal leukocytic infiltrate and characterized infiltrate phenotypically after staining for activation and regulatory T cell (Treg) markers and for markers of antigen-presenting cell (APC) maturation. Treg cells were depleted in vivo using anti-CD25 mAb. Luminex analysis quantified the amount of cytokines and chemokines expressed in corneal tissue homogenate., Results: Infected corneas without HSK exhibited a pronounced leukocytic infiltrate containing a significantly higher proportion and nearly identical absolute number of activated CD4+ T cells 15 days after infection when compared with those with HSK. Moreover, the frequency and absolute number of regulatory CD4+ T cells (Tregs) was lower in nondiseased corneas, and Treg depletion did not influence HSK incidence. The frequency of mature, immunogenic DCs and the ratio of mature DCs to CD4+ T cells were nearly identical in corneas with and without HSK. The authors observed a reduced population of neutrophils and reduced expression of neutrophil chemoattractants MIP-1beta and keratinocyte chemoattractant and the neutrophil-attracting cytokine IL-6 in corneas without HSK., Conclusions: These findings demonstrate that HSV-1-infected corneas can retain clarity in the presence of a substantial secondary leukocytic infiltrate, that activated CD4+ T cells, while necessary, are not sufficient for HSK development, that susceptibility to HSK is not determined by Tregs, and that clinical disease correlates with the accumulation of a critical mass of neutrophils through chemoattraction.

Background: Corneal infection with herpes simplex virus-1 (HSV) can cause an inflammatory eye disease termed herpetic stromal keratitis (HSK). Interferon-gamma (IFN-gamma) is known to be involved in the development of this disease. In this study, antisense oligonucleotides targeting IFN-gamma mRNA (IFN-gamma-ASON) were investigated for their effects in experimental HSK., Methods: Splenic cells were used to examine the efficacy of IFN-gamma-ASON to decrease IFN-gamma- release into the cell culture supernatants as measured by ELISA. Mice were corneally infected with 10(5) PFU HSV, and IFN-gamma-ASON were given subepithelially. Alternatively, mice were infected without any further treatment, received only buffer, or received control oligonucleotides (CON) to observe substance specific effects. The animals were followed up clinically for the signs of herpetic keratitis. On days 14 and 28 post infection (p.i.), animals were sacrificed, and eyes were collected for histological analysis. On day 7 p.i., infectious virus particles in the eyes were determined by a plaque assay., Results: While IFN-gamma-ASON diminished the content of IFN-gamma in a concentration-dependent manner in vitro, CON showed no significant effects. Whereas buffer-treated and only infected mice showed severe necrotizing keratitis on day 14 p.i., this was abolished after treatment with IFN-gamma-ASON, even after 28 and 52 days. CON-treated mice also showed an improved HSK on day 14, but not on day 28. The incidence of the disease was also clearly diminished after treatment with IFN-gamma-ASON at all time points examined. The number of inflammatory cells in both the central and the peripheral cornea were strongly reduced after the application of IFN-gamma-ASON as compared to the controls. In contrast, the infectious viral particles in eyes at day 7 p.i. did not differ between the four groups., Conclusions: Topical treatment with IFN-gamma-ASON induced a long-term improvement of the course and the incidence of HSK in the murine model. IFN-gamma seems to be involved in a proinflammatory manner during the pathogenesis of HSK, while the antiviral defense against HSV was not affected by this topical cytokine inhibition. Unspecific CON induced a transient and cytokine independent improvement of HSK.

Background/aims: Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and alkali burns are associated with chronic, severe inflammation of the ocular surface that occasionally lead to corneal stem cell deficiencies. The corneal stroma in these diseases has not been studied comprehensively. The purpose of this study was to determine whether the keratocytes in the stroma were normal and whether the stroma remained inflamed in the chronic phase of these diseases., Methods: Five pathological corneas, two with SJS, two with OCP and one with an alkali burn were examined. Corneal specimens were obtained during lamellar keratoplasty and the histological sections were immunostained with antibodies against CD34 and several cell surface antigens. The level of expression of proteoglycans (lumican, keratocan, biglycan) and chemokines (monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP) 1alpha, MIP1beta) were examined by quantitative real-time RT-PCR., Results: The number of CD34-positive cells in the stroma was decreased and the expression level of biglycan increased in all of the pathological corneas. The numbers of CD45-positive and CD14-positive cells were increased in four of the five pathological corneas. The expression level of MIP1alpha and MIP1beta were markedly increased in all of the pathological corneas., Conclusions: These findings indicate that the keratocytes are abnormal and inflammation is still present in the corneal stroma in the chronic phase of SJS, OCP and alkali burns.

Purpose: To analyze age-related changes in susceptibility to experimental Staphylococcus aureus keratitis and purified alpha-toxin in rabbits., Methods: Intrastromal injection of S. aureus (100 colony-forming units [CFUs]) induced keratitis in young (6-8 weeks) and aged (approximately 30 months) New Zealand White rabbits. Bacteria and polymorphonuclear leukocytes (PMNs) per cornea were quantified. Purified alpha-toxin at 1, 10, 25, or 50 hemolytic units (HU) or heat-inactivated alpha-toxin was intrastromally injected into corneas, and pathologic changes were determined by slit lamp examination (SLE) and histopathologic analysis. alpha-Toxin hemolysis assays were performed using erythrocytes from young and aged rabbits., Results: S. aureus keratitis produced significantly higher SLE scores in young rabbits than in aged rabbits at 15, 20, and 25 hours postinfection (PI; P < or = 0.001); aged rabbits essentially recovered from S. aureus keratitis by 7 days PI. At 25 hours PI, numbers of CFUs and PMNs in corneas of young and aged rabbits were equivalent (P > or = 0.6); the bacterial burden in aged rabbits declined by 5 logs per cornea from day 1 to day 7 PI. Intrastromal injection of > or =10 HU alpha-toxin also produced significantly more disease in young than in aged rabbit corneas (P < or = 0.05), whereas 1 HU or heat-inactivated toxin yielded negligible pathologic changes in either group. Hemolysis assays of erythrocytes from young rabbits demonstrated greater susceptibility to alpha-toxin compared with those from aged rabbits., Conclusions: Corneas and erythrocytes of young rabbits, relative to aged rabbits, are significantly more susceptible to S. aureus keratitis and to alpha-toxin.

Purpose: To study the phenotypes, distribution, and morphologies of different antigen-presenting cells (APCs) in the murine cornea., Methods: Intravitreal injection of fluorescently tagged ovalbumin (OVA) or antibodies to MHC-II (I-A(d)), F4/80, CD11c, B7-1, and B7-2 was performed to label cells in the murine cornea. Light and transmission electron microscopy were used to examine corneal histology. Intravital microscopy, epifluorescence microscopy, and confocal microscopy were used to evaluate the labeled cells. In vitro staining was performed to validate the in vivo staining and localize the labeled cells. Three-dimensional rotatable images were taken to evaluate relationships between two differently labeled cells., Results: Histological examination revealed no observable change in the cornea following intravitreal injection. In vivo staining showed that OVA+ cells and cells positive for MHC-II, F4/80, CD11c, B7-1, or B7-2 were noted throughout the cornea with a decreasing density from limbus toward the central cornea. Two populations with distinct morphological features were identified among these APCs. Labeled cells were found beneath the epithelium or in the shallow stroma in the central and paracentral cornea, but in all layers in the peripheral cornea. A number of F4/80+ and CD11c+ cells were also positive for OVA, MHC-II, B7-1, or B7-2. Rotatable images showed a close contact between two differently labeled cells., Conclusions: Intravitreal injection of labeled antibodies can be adapted to visualize labeled cells in the cornea. APCs with distinct morphologies, phenotypes, and distribution may contribute to the immunologically privileged feature of the cornea.

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.

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.

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.

Early defects due to the afflux of monocytic phagocytes into the inflammatory focus, rapid resorption of detritis and normalisation of correlation between contents of matrix proteinases and their inhibitors, stimulation of stromal cornea keratinocytes migration and their proliferation characterize repair of postburn defects in transplantation of the stromal equivalent. Repair against the background of stromal equivalent transplantation is of a histotypical character: collagen gel organizes and serves the basis for migration of the cornea fibroblasts and temporary matrix for growing new epithelium, while restored basal membrane is a derivative of cornea epithelium.

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.

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.

Purpose: Toll-like receptors (TLRs), which recognize microbial products, have an important role in the host innate immune response. The purpose of the present study was to determine whether activation of these receptors leads to development of keratitis and to assess the role of the common adaptor molecule myeloid differentiation factor-88 (MyD88)., Methods: Corneal epithelium of C57BL/6, TLR2(-/-), TLR9(-/-), and MyD88(-/-) mice was abraded and treated with Pam(3)Cys, LPS, or CpG DNA, which bind TLR2, -4, and -9, respectively, and neutrophil recruitment to the corneal stroma, development of corneal haze, and chemokine production were measured., Results: Activation of TLR2 and -9 stimulated neutrophil recruitment to the corneal stroma of C57BL/6 mice, but not TLR2(-/-) or -9(-/-) mice, respectively. In marked contrast, neutrophil migration to the corneal stroma of MyD88(-/-) mice challenged with Pam(3)Cys, LPS, or CpG DNA was completely ablated. Activation of TLR2, -4, and -9 also caused a significant increase in corneal thickness and haze, indicative of disruption of corneal clarity; however, this response was ablated in MyD88(-/-) mice, which were not significantly different from untreated corneas. Production of CXC chemokines MIP-2 and KC, which mediate neutrophil recruitment to the corneal stroma, was elevated in the corneal epithelium and stroma of control, but not MyD88(-/-) mice., Conclusions: Together, these findings demonstrate that the corneal epithelium has functional TLR2 and -9, and that TLR2, -4, and -9 signal through MyD88. This pathway is therefore likely to have an important role in the early events leading to microbial keratitis.

Herpetic stromal keratitis (HSK) results in postinfection with Herpes simplex virus type 1 (HSV-1). The pathogenesis involves tissue damage by the host immune system, classifying HSK as an immunopathological disease. The crucial disease orchestrating cells is thought to be the T lymphocytes. The present study elucidates pathogenic and protective T cell subsets involved in the development of HSK using the gBT mice, which possess a monoclonal population of CD8+ T cells reactive to a HSV immunodominant epitope. Results show that HSV-reactive CD8+ T cells enter infected corneas during the acute but not the chronic phase of the disease during which the predominant population is CD4+ T cells. Adoptive transfer experiments in T and B cell-deficient recombination-activating gene knockout mice revealed that HSV-reactive CD8+ T cells are capable of ocular virus clearance, possibly through a combination of corneal and peripheral nervous system antiviral effects, but are not involved in lesion development. CD4+ T cells of the virus-specific or nonspecific species emerged as the pathogenic T cells capable of precipitating disease. These observations have the potential to yield important treatment strategies by targeting specific cell types in HSK.

Herpetic stromal keratitis (HSK), resulting from corneal HSV-1 infection, represents a T cell-mediated immunopathologic lesion. In T cell transgenic mice on a SCID or RAG knockout background, the T cells mediating lesions are unreactive to viral Ags. In these bystander models, animals develop ocular lesions but are unable to control infection. Transfer of HSV-immune cells into a CD8(+) T cell bystander model resulted in clearance of virus from eyes, animals survived, and lesions developed to greater severity. However, the adoptively transferred CD8(+) T cells were not evident in lesions, although they were readily detectable in the lymphoid tissues as well as in the peripheral and CNS. Our results indicate that viral-induced tissue damage can be caused by bystander cells, but these fail to control infection. Immune CD8(+) T cells trigger clearance of virus from the eye, but this appears to result by the T cells acting at sites distal to the cornea. A case is made that CD8(+) T cell control is expressed in the trigeminal ganglion, serving to curtail a source of virus to the cornea.

Herpetic stromal keratitis (HSK) is a T cell-orchestrated, immunoinflammatory lesion that results from corneal Herpes simplex virus infection. Previous reports indicate an essential role for proinflammatory cytokine interleukin (IL)-1 in HSK pathogenesis. The present study evaluates the efficacy of IL-1 receptor antagonist (IL-1 ra) protein in the management of HSK. Mice receiving IL-1 ra had diminished disease severity. The administration of IL-1 ra was shown to reduce the influx into the cornea of cells of the innate and adaptive immune response. In addition, the treatment diminished corneal vascular endothelial growth factor levels, resulting in reduced angiogenic response. Our results show the importance of targeting early proinflammatory molecules such as IL-1 to counteract HSK and advocate IL-1 ra as an effective agent to achieve this.

Background: This study was performed to investigate the influence of fumaric acid esters on the course of herpes stromal keratitis (HSK)., Methods: The corneas of BALB/c mice were inoculated with 105 plaque-forming units of herpes simplex virus 1 (HSV-1, KOS strain). Groups of mice were treated intraperitoneally with phosphate buffered saline (PBS) (control mice), or with dimethylfumarate (DMF) at 15 mg/kg of body weight dissolved in PBS daily for 28 days pre-infection and for 14 days post-infection. The course of HSV-1 keratitis was studied clinically. Corneal sections were examined for inflammatory cell infiltration. The numbers of CD3, GR-1, CD11b and F4/80-expressing cells infiltrating the corneas were analyzed by immunohistochemistry., Results: On day 14 after HSV infection, 72% of the mice in the control group had severe HSK. The development of HSK was reduced by DMF treatment in the DMF group (22%) (P=0.004). The total number of inflammatory cells and infiltration of polymorphonuclear-neutrophils (PMNs) were reduced in the corneas of DMF-treated mice. Compared to the PBS-treated mice, numbers of CD3, CD11b, GR-1 and F4/80-positive cells were reduced in the DMF group of mice., Conclusions: The course of experimental herpes stromal keratitis can be improved with systemic fumaric acid ester treatment. The improvement of keratitis correlates with a reduced corneal infiltration of T cells and mononuclear cells.

The eye has been described as an immunologically privileged site where immunity is purely expressed. It has been demonstrated that administration of antigen into the eye induces only a weak immune response. However, the anterior part of the eye represents an important protective barrier against pathogens and other harmful invaders from the outer environment. Therefore, effective immune mechanisms, which operate locally, need to be present there. Because the cornea has been shown to be a potent producer of various cytokines and other molecules with immunomodulatory properties, we investigated a possible regulatory role for the individual corneal cell types on cytokine production by activated T cells. Mouse spleen cells were stimulated with the T cell mitogen concanavalin A in the presence of either corneal explants or cells of corneal epithelial or endothelial cell lines and the production of T helper 1 (Th1) or Th2 cytokines was determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR). We found that the cornea possesses the ability to inhibit, in a dose-dependent manner, production of the inhibitory and anti-inflammatory cytokines interleukin (IL)-4 and IL-10 by activated T cells. The production of cytokines associated with protective immunity [IL-2, IL-1beta, interferon (IFN)-gamma ] was not inhibited under the same conditions. Corneal explants deprived of epithelial and endothelial cells retained the ability to suppress production of anti-inflammatory cytokines. This suppression was mediated by a factor produced by corneal stromal cells and occurred at the level of cytokine gene expression. We suggest that by this mechanism the cornea can potentiate a local expression of protective immune reactions in the anterior segment of the eye.

Ocular infection with HSV may result in the blinding immunoinflammatory lesion stromal keratitis (SK). This represents a CD4+ T cell-mediated immunopathologic lesion in both humans and a mouse model. Early events in the pathogenesis that set the stage for SK are poorly understood. The present study evaluates the role of IL-1 using a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein. Such transgenic mice were markedly resistant to SK compared with IL-1ra(-/-) and C57BL/6 control animals. The resistance was shown to be the consequence of reduced expression of molecules such as IL-6, macrophage-inflammatory protein-2, and vascular endothelial growth factor, normally up-regulated directly or indirectly by IL-1. A critical event impaired in IL-1ra transgenic mice was vascular endothelial growth factor production with a consequent marked reduction in angiogenesis, an essential step in SK pathogenesis. Targeting IL-1 could prove to be a worthwhile therapeutic approach to control SK, an important cause of human blindness.

Purpose: To investigate the feasibility of corneal reconstruction utilizing cultured human corneal endothelial cells (HCECs)., Methods: We cultivated HCECs using culture dishes pre-coated with bovine corneal endothelial extracellular matrix. The effect of donor age on HCECs was investigated. We reconstructed corneas using cultured HCECs and human corneal stroma, then examined their functioning. The possibility of porcine corneal stroma as a carrier of cultured HCECs was investigated., Results: The older the donor, the more frequently large senescent cells appeared in the passaged HCECs. The density of HCECs on the reconstructed cornea reached 2500 cells/mm2. The potential difference in the reconstructed and normal corneas was 0.30 mV and 0.40 mV, respectively; this indicates that the pump function of the reconstructed corneas is 75% of that of normal corneas. Porcine corneal stroma expressing little xenosugar antigen alpha-gal epitope induced no superacute rejection but mild cellular rejection when transplanted into corneas of animals possessing natural antibody to alpha-gal epitope., Conclusions: To reconstruct corneas that are the same as, or superior to, normal corneas, innovation is necessary in the methods used for culturing and seeding HCECs. Porcine corneal stroma is promising as a carrier of HCECs instead of human corneal stroma, the supply of which is limited. The validity of porcine corneal stroma, acellularized to prevent retrovirus infection, should be evaluated.

Background: The normal corneal stroma is endowed with large numbers of resident dendritic cells (DCs). The purpose of this study was to examine the phenotype and distribution of these cells in inflammation., Methods: Normal and inflamed murine corneas were excised at different time points and immunofluorescence staining with multiple antibodies was performed by confocal microscopy on whole-mounted corneal stromas to characterize and evaluate the distribution of DCs in inflammation., Results: CD11c+CD11b+ myeloid DCs were present throughout the anterior stroma. In the periphery of the normal cornea, nearly one half were major histocompatibility complex (MHC) class II+CD80+CD86+, while they were uniformly MHC class II-CD80-CD86- in the center. In inflammation, in addition to a significant increase in the number of DCs, a majority up-regulated their expression of MHC class II, CD80, and CD86, indicating their state of maturation. The up-regulation of MHC class II and costimulatory molecules on DCs was seen as early as 24 hours after induction of inflammation or transplantation. In addition to the CD11c+DCs in the anterior stroma, a CD11c-CD11b+ population of monocytes/macrophages was present almost exclusively in the posterior stroma of the cornea. These cells were found throughout all layers of the stroma, and in increased numbers, after induction of inflammation., Conclusion: The present study demonstrates, for the first time, the phenotypic changes and distribution of resident stromal DCs in corneal inflammation. Clinical Relevance These novel data suggest that the cornea is capable of participating in immune and inflammatory responses by virtue of its own heterogeneous population of DCs.

Purpose: The immune mediated HSV-1 stromal keratitis (HSK) rapidly improves after amniotic membrane transplantation (AMT). This study investigated whether AMT modulates the T cell response and whether the anti-inflammatory action of AMT is due to local or systemic effects., Methods: Corneas of BALB/c mice were infected with 10(5) (PFU) of HSV-1. Animals with ulcerating keratitis on day 14 post-infection were divided into 4 groups: group 1 ( n=12): right eye AMT;group 2 ( n=12): right eye tarsorrhaphy; group 3 ( n=8): right eye tarsorrhaphy, left eye AMT;group 4 ( n=8): both eyes tarsorrhaphy. The mice were examined for clinical signs of HSV keratitis after 2 days. Corneal sections were studied histologically and the inflammatory cell infiltration was studied by immunohistochemical staining. DTH response and the HSV-specific 3H-thymidin-uptake were compared between the groups., Results: Compared to group 2, ulceration and stromal inflammation was profoundly improved in group 1 ( p<0.01). The corneas in the AMT mice had fewer inflammatory cells, CD3+,CD4+ and CD8+ cells than the control mice ( p<0.01). There were no significant differences between groups 1 and 2 with respect to the delayed type hypersensitivity reaction (DTH response) and the HSV-specific 3H-thymidin uptake. AMT or tarsorrhaphy on the left eyes in groups 3 and 4 had no influence on the course of keratitis or the T cell response., Conclusions: Ulcerating herpetic keratitis markedly improves after AMT. Our observations indicate that this is caused predominantly by local and not by systemic AMT-related effects.

Objective: To investigate the inhibition by CTLA-4Ig on herpetic stromal keratitis (HSK)., Methods: BALB/c mice infected with HSV-1 Mckrae strain by corneal scarification were injected intraperitoneally with murine CTLA-4Ig given on days 0, +2, +4 after the infection. The effects of CTLA-4Ig on HSK were evaluated., Results: As measured by flow cytometry, in the mice treated with CTLA-4Ig, 81.6% of CD4(+)T cells and 67.9% of CD8(+)T cells were reduced. CTLA-4Ig halted the onset of HSK, reduced the corneal stromal opacification of HSK, prevented extensive cellular infiltration in cornea, impaired the delayed type hypersensitivity and inhibited splenocytes producing Th1 cytokines., Conclusions: The results provide evidence that blockade of B7:CD28/CTLA-4 costimulation by CTLA-4Ig can inhibit the proliferation of T cells and Th1 response and impair onset and severity of HSK.

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.

Purpose: To evaluate the efficacy of DNA immunization using herpes simplex virus (HSV)-1 gD or gD-IL-2 (chimeric gene of gD and human IL-2) with reference to their immune responses and preventive effect on the development of murine herpetic stromal keratitis, based on our previous work., Methods: Plasmids containing gD (pHSDneol) or gD-IL-2 (pHDLneol) were constructed, and gD or gD-IL-2 peptides were purified. BALB/c mice were immunized by hypodermal injection, subconjunctival injection, or topical eye drop twice at 7-day intervals. Attained anti-HSV immune reaction was estimated, including neutralizing antibody, delayed-type hypersensitivity (DTH) measured by the swelling of the ear pinna, and cytotoxic T lymphocyte (CTL) measured by 51Cr release from MHC-matched target cells by splenic and/or local lymph node cells. The corneas of a group of the immunized mice were challenged with CHR3 strain of HSV-1 (10 microL of 3 x 10(6) PFU/mL) 3 weeks after the last immunization. Clinical signs (stromal and epithelial keratitis) were scored. Results. All gD- or gD-lL-2-immunized mice developed specific neutralizing antibody. Delayed-type hypersensitivity and CTL were obtained in gD DNA- or gD-IL-2 DNA-immunized mice. Chimeric DNA vaccine gD-IL-2 elicited higher DTH and more vigorous CTL activity. The scores of stromal keratitis for all gD- or gD-IL-2-immunized mice were significantly suppressed, although those of epithelial keratitis were not. Conclusion. Immunization with gD or gD-IL-2 was effective against herpetic stromal keratitis. By focusing specifically on high induction of cell-mediated immunity, gD-IL-2 DNA could have possible clinical applications against HSV-1 in the future.

Purpose: The presence of eosinophilic granulocytes in ocular tissue is a hallmark of the host response to environmental and parasite allergens. Using a mouse model of Onchocerca volvulus-mediated keratitis (river blindness), the present study examined the role of the cytokines interleukin (IL)-4 and IL-13 in regulating recruitment of eosinophils to the cornea through expression of intercellular cell adhesion molecule (ICAM)-1., Methods: C57BL/6 mice received an intrastromal injection of recombinant IL-4 and IL-13 (rIL-4 and IL-13) or were immunized by subcutaneous injection prior to receiving an intrastromal injection of a soluble O. volvulus extract. Expression of ICAM-1 and recruitment of eosinophils to the cornea were monitored by immunohistochemistry., Results: Expression of ICAM-1 was elevated after injection of rIL-4 or IL-13 together with recombinant tumor necrosis factor (rTNF)-alpha. Conversely, expression of ICAM-1 in O. volvulus-mediated keratitis was significantly reduced after subconjunctival injection of a monoclonal antibody (mAb) to IL-4 or IL-13. In addition, combined in vivo neutralization of IL-4 and IL-13 inhibited recruitment of eosinophils, but not of neutrophils, to the corneal stroma., Conclusions: These findings demonstrate that expression of ICAM-1 and recruitment of eosinophils to the cornea are tightly regulated by IL-4 and IL-13, and indicate that these cytokines are a potential target for immune intervention in ocular allergy and parasitic infections of the eye.