Local treatment with alpha-melanocyte stimulating hormone reduces corneal allorejection.

BACKGROUND: Corneal grafting is by far the most common form of transplantation. Many grafts suffer from immune rejection and current therapies are associated with many side effects, requiring more effective and safe therapies. alpha-Melanocyte stimulating hormone (alpha-MSH) is a neuropeptide that suppresses host inflammatory defense mechanisms. The purpose of this study was to determine the role of local therapy with alpha-MSH on corneal allograft survival, and the mechanisms by which it may influence graft outcome. METHODS: Orthotopic corneal transplantation was performed, with recipients receiving subconjunctival alpha-MSH or sham injections twice weekly. Grafts were followed up for 70 days, and graft inflammation/opacification was compared between the two groups in a masked fashion. Graft infiltration and ocular gene expression of select inflammatory cytokines was evaluated at different timepoints. Additionally, allospecific delayed-type hypersensitivity was compared among the groups 3 weeks posttransplantation. RESULTS: Results showed a significant increase in corneal graft survival in alpha-MSH-treated recipients compared with controls. Although 75% of allografts in alpha-MSH-treated hosts survived at 70 days, 43% survived in controls (P=0.04). Graft infiltration studies demonstrated a significant decrease in the number of mononuclear and polymorphonuclear cells in alpha-MSH-treated mice compared with controls at days 7 and 14 after transplantation. Furthermore, allospecific delayed-type hypersensitivity and gene expression of interferon-gamma and interleukin-2 showed a significantly reduced expression in alpha-MSH-treated mice compared with controls. CONCLUSIONS: This study provides for the first time, in vivo evidence that treatment with local alpha-MSH may significantly reduce allorejection of orthotopic transplants.

Corneal epithelial proliferation and thickness in a mouse model of dry eye.

Although several studies have previously focused on the conjunctival epithelial response to surface dryness, little is known about the effect of a dry environment on corneal epithelium, which is the most clinically significant tissue affected in dry eye. The aim of this study was to quantitatively evaluate the effect of desiccating stress on the number of proliferating corneal epithelial cells and corneal epithelial thickness in mice placed in a controlled-environment chamber (CEC) that induces dry eye. Corneal epithelial cell proliferation and thickness were studied in 8- to 12-week-old female BALB/c mice placed in the CEC (temperature: 22.3+/-0.7 degrees C; relative humidity: 22.5+/-4.5%; airflow: 15 L/min) for 7 days and compared to a control group of mice with no dry eye. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle. To detect the spatial distribution of proliferative cells, Ki-67(+) cells were counted in three areas of the epithelium: center, periphery, and limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin-eosin. Results from each experimental group were compared using the Mann-Whitney test. The number of Ki-67(+) cells observed in the corneal epithelium of mice exposed to the CEC was significantly higher in each area (center: 32.1+/-1.1; periphery: 94.2+/-5.3; limbus: 4.0+/-1.5) than in the control group (center: 13.2+/-1.0, p=0.02; periphery: 42.9+/-2.3, p=0.02; limbus: 0.0, p=0.01). In mice subjected to desiccating stress, a significant number of Ki-67(+) positive cells were detected in the basal and suprabasal cell layers (central area 46%; periphery 30.8%: limbus 0%), whereas in the control group the cells were exclusively distributed through the basal cell layer. Ki-67(+) cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was found to be significantly thicker in dry eye mice (54.94+/-6.09 microm) as compared to the controls (43.9+/-6.23 microm, p<0.0001) by a mean of 25%. These results demonstrate that desiccating stress increases corneal epithelial turnover and thickness, similar to what is observed in other chronic inflammatory states of other epithelialized surfaces. The CEC can facilitate the study of the regulation of epithelial cell function and turnover at the molecular and cellular levels under desiccating stress conditions.

Topical omega-3 and omega-6 fatty acids for treatment of dry eye.

OBJECTIVE: To study the efficacy of topical application of alpha-linolenic acid (ALA) and linoleic acid (LA) for dry eye treatment. METHODS: Formulations containing ALA, LA, combined ALA and LA, or vehicle alone, were applied to dry eyes induced in mice. Corneal fluorescein staining and the number and maturation of corneal CD11b(+) cells were determined by a masked observer in the different treatment groups. Real-time polymerase chain reaction was used to quantify expression of inflammatory cytokines in the cornea and conjunctiva. RESULTS: Dry eye induction significantly increased corneal fluorescein staining; CD11b(+) cell number and major histocompatibility complex Class II expression; corneal IL-1alpha and tumor necrosis factor alpha (TNF-alpha) expression; and conjunctival IL-1alpha, TNF-alpha, interferon gamma, IL-2, IL-6, and IL-10 expression. Treatment with ALA significantly decreased corneal fluorescein staining compared with both vehicle and untreated controls. Additionally, ALA treatment was associated with a significant decrease in CD11b(+) cell number, expression of corneal IL-1alpha and TNF-alpha, and conjunctival TNF-alpha. CONCLUSIONS: Topical ALA treatment led to a significant decrease in dry eye signs and inflammatory changes at both cellular and molecular levels. CLINICAL RELEVANCE: Topical application of ALA omega-3 fatty acid may be a novel therapy to treat the clinical signs and inflammatory changes accompanying dry eye syndrome.

Promotion of graft survival by vascular endothelial growth factor a neutralization after high-risk corneal transplantation.

OBJECTIVE: To evaluate whether hemangiogenesis, lymphangiogenesis, and concomitant invasion of mononuclear phagocytes occurring after high-risk corneal transplantation in already vascularized high-risk recipient corneal beds increase the risk for subsequent immune rejection. METHODS: Three intrastromal sutures were left in place for 6 weeks in the corneas of BALB/c mice, causing neovascularization. Three weeks after suture removal, keratoplasty was performed (donors C57BL/6 mice). The treatment group received a vascular endothelial growth factor A (VEGF-A)-neutralizing cytokine trap at 0, 4, 7, and 14 days postoperatively (Fc protein was used as the control treatment). Morphometry was performed in corneal flat mounts using lymphatic endothelial hyaluronan receptor-1 (a specific lymphatic endothelial marker), CD31 (a panendothelial marker), and F4/80 (a marker for mononuclear phagocytes). RESULTS: After corneal transplantation, significant additional hemangiogenesis (mean area covered by vessels [SD], 68% [18%] postoperatively vs 40% [18%] preoperatively; P = .03) and lymphangiogenesis (12% [1.3%] postoperatively vs 9% [2.8%] preoperatively; P = .03) were observed. Postoperative neutralization of VEGF-A inhibited operation-induced hemangiogenesis (35% [8%]; P = .007) and lymphangiogenesis (6% [1.6%]; P = .03) and decreased the recruitment of mononuclear phagocytes into the graft (mean [SD], 501 cells/mm(2) [152] in treated mice vs 684 cells/mm(2) [35] in Fc controls; P = .03). After 8 weeks, 23% of the treated corneas were not rejected, whereas all control corneas were rejected after 21 days (P = .007). CONCLUSIONS: Neutralization of VEGF-A after high-risk corneal transplantation effectively inhibits postoperative hemangiogenesis, lymphangiogenesis, and recruitment of antigen-presenting cells and improves corneal graft survival. CLINICAL RELEVANCE: Blocking of VEGF-A after high-risk corneal transplantation may be a novel approach to improve graft survival.

Graft failure IV. Immunologic mechanisms of corneal transplant rejection.

Corneal transplantation is the oldest and the most common form of solid tissue transplantation in humans. Immunologic graft rejection is one of the main causes of short and long-term graft failure. Rejection involves donor tissue recognition and destruction by allo-specific immune cells of the recipient. This review outlines (1) the immunobiology of transplantation, with reference to ocular immune privilege, (2) factors that confer "high-risk" status to a graft and (3) the pathophysiologic mechanisms of corneal transplant rejection.

Keratoglobus in association with posterior polymorphous dystrophy.

PURPOSE: To report a case of a 34-year-old woman presenting with keratoglobus and posterior polymorphous corneal dystrophy (PPMD). METHODS: Observational case report including ophthalmologic examination, topographic findings, and specular microscopy findings. RESULTS: This patient presented with corneal steepening on topography consistent with keratoglobus, as well as large areas of irregular polymorphous changes of the corneal endothelium on specular microscopy consistent with PPMD. CONCLUSIONS: We report the first case with clinical features of both keratoglobus and PPMD. This report brings forth the description of keratoglobus findings on Orbscan topography.

The function of donor versus recipient programmed death-ligand 1 in corneal allograft survival.

Programmed death-ligand (PD-L)1 and PD-L2, newer B7 superfamily members, are implicated in the negative regulation of immune responses and peripheral tolerance. To examine their function in alloimmunity, we used the murine model of orthotopic corneal transplantation. We demonstrate that PD-L1, but not PD-L2, is constitutively expressed at high levels by the corneal epithelial cells, and at low levels by corneal CD45+ cells in the stroma, whereas it is undetectable on stromal fibroblasts and corneal endothelial cells. Inflammation induces PD-L1 up-regulation by corneal epithelial cells, and infiltration of significant numbers of PD-L1+CD45+CD11b+ cells. Blockade with anti-PD-L1 mAb dramatically enhances rejection of C57BL/6 corneal allografts by BALB/c recipients. To examine the selective contribution of donor vs host PD-L1 in modulating allorejection, we used PD-L1-/- mice as hosts or donors of combined MHC and minor H-mismatched corneal grafts. BALB/c grafts placed in PD-L1-/- C57BL/6 hosts resulted in pronounced T cell priming in the draining lymph nodes, and universally underwent rapid rejection. Allografts from PD-L1-/- C57BL/6 donors were also significantly more susceptible to rejection than wild-type C57BL/6 grafts placed into BALB/c hosts, primarily as a result of increased T cell infiltration rather than enhanced priming. Taken together, our results identify differential roles for recipient vs donor PD-L1 in regulating induction vs effector of alloimmunity in corneal grafts, the most common form of tissue transplantation, and highlight the importance of peripheral tissue-derived PD-L1 in down-regulating local immune responses.

Dexamethasone inhibits interleukin-1beta-induced corneal neovascularization: role of nuclear factor-kappaB-activated stromal cells in inflammatory angiogenesis.

Dexamethasone, a synthetic corticosteroid, is widely used as a potent anti-inflammatory drug in various diseases including corneal angiogenesis. However, dexamethasone's impact on interleukin (IL)-1beta-dependent inflammatory angiogenesis is unknown. Here, we show that dexamethasone inhibits IL-1beta-induced neovascularization and the expression of the angiogenesis-related factors, vascular endothelial growth factor-A, KC, and prostaglandin E(2) in the mouse cornea 2 days after IL-1beta implantation. IL-1beta caused IkappaB-alpha phosphorylation in corneal stromal cells but not in infiltrated CD11b(+) cells 2 days after IL-1beta implantation. In contrast, both cell types were positive for phosphorylated IkappaB-alpha 4 days after IL-1beta implantation. Dexamethasone significantly inhibited IkappaB-alpha phosphorylation 2 and 4 days after IL-1beta implantation. Furthermore, dexamethasone inhibited IL-1beta-induced expression of vascular endothelial growth factor-A, KC, and prostaglandin E(2), and signaling of nuclear factor (NF)-kappaB in corneal fibroblasts in vitro. A selective NF-kappaB inhibitor attenuated IL-1beta-induced corneal angiogenesis. These findings suggest that NF-kappaB activation in the corneal stromal cells is an important early event during IL-1beta-induced corneal angiogenesis and that dexamethasone inhibits IL-1beta-induced angiogenesis partially via blocking NF-kappaB signaling.

Effect of the ocular microenvironment in regulating corneal dendritic cell maturation.

OBJECTIVE: To determine whether the ocular anterior segment (aqueous humor and cornea) actively inhibits dendritic cell (DC) maturation. METHODS: Dendritic cells were injected into syngeneic corneas or conjunctivae, and their surface major histocompatibility complex class II expression in response to the local milieu was assessed using confocal microscopy. Immature DCs were cocultured with corneal supernatant or with aqueous humor to evaluate their regulation of DC phenotypic and functional maturity. RESULTS: In contrast to conjunctivally injected DCs, DCs injected into the cornea resisted up-regulation in expression of surface major histocompatibility complex class II. Corneal supernatant-treated and aqueous humor-treated DCs retained their immaturity, as reflected by high antigen uptake but low costimulatory molecule (CD80 and CD86) expression and poor T-cell stimulation. Anti-transforming growth factor beta(2) treatment of aqueous humor and of corneal supernatant led to complete and partial blockade of their inhibition of DC maturation, respectively. However, alpha-melanocyte-stimulating hormone and calcitonin gene-related peptide had no demonstrable effect on DC maturation. CONCLUSION: Cornea and aqueous humor, principally through transforming growth factor beta(2,) promote generation of phenotypically and functionally immature DCs. Clinical Relevance Our results indicate that relative immune quiescence in the cornea and in the anterior segment is actively maintained in part by the inhibitory effect of transforming growth factor beta(2) on resident DCs and by their suppression of T-cell-mediated immune and inflammatory responses.

Very late antigen 1 blockade markedly promotes survival of corneal allografts.

OBJECTIVE: To investigate the role of very late antigen 1 (VLA-1) (also known as integrin receptor alpha(1)beta(1)) in corneal transplantation inflammation and allograft survival. METHODS: Cell infiltration and vasculogenesis (both angiogenesis and lymphangiogenesis) associated with allodisparate corneal transplantation were assessed in VLA-1-deficient conditions and controls by immunofluorescent microscopic studies. Corneal allograft survival was also assessed after anti-VLA-1 antibody treatment and in VLA-1 knockout recipient mice. RESULTS: Anti-VLA-1 antibody treatment leads to a profound reduction in the granulocytic, monocytic, and T-cell infiltration after corneal transplantation. In addition, corneal angiogenesis and lymphangiogenesis were both significantly suppressed in VLA-1 knockout mice. Remarkably, universal graft survival was observed in both anti-VLA-1 antibody treatment and knockout mice. CONCLUSIONS: Very late antigen 1 blockade markedly reduces inflammation and inflammation-induced tissue responses, including vasculogenic responses, associated with corneal transplantation and promotes allograft survival. CLINICAL RELEVANCE: These studies offer insights into important integrin-mediated mechanisms of corneal transplant-related inflammation and provide possible new integrin-based immunotherapies for transplant rejection.

The chemokine receptor CCR7 mediates corneal antigen-presenting cell trafficking.

PURPOSE: Trafficking of corneal antigen-presenting cells (APC) to draining lymph nodes (LN) is critical in triggering immune responses. However, very little is known about the molecular regulation of this pathway. We investigated the expression and function of the chemokine receptor CCR7 in mediating corneal APC migration in inflammation. METHODS: Expression of CCR7 and its ligands, CCL21 and CCL19, in the normal and inflamed corneas was analyzed by RT-PCR and immunofluorescence staining. The phenotype of CCR7-expressing cells was identified by double-staining with different cell surface markers. To trace the trafficking of APC to draining LN, we injected corneal grafts with Alexa488-conjugated ovalbumin (OVA) and transplanted to syngeneic recipients. CCR7 expression on the Alexa488-conjugated OVA+ cells in the ipsilateral draining LN was analyzed by flow cytometry. To determine the functional role of CCR7, we injected anti-CCL21 neutralizing antibody subconjunctivally after corneal transplantation and analyzed changes in numbers of OVA+ cells in the draining LN. Each experiment was repeated at least three times. RESULTS: Both CCR7 and its ligand CCL21 were significantly upregulated in inflamed corneas as measured by RT-PCR and immunofluorescence staining. CCR7+ cells were detected especially in the corneal periphery near LYVE-1+ lymphatic vessels. CCR7+ cells were universally CD11b+CD11c+, and a majority were major histocompatibility complex class II positive, suggesting a monocytic dendritic cell lineage and a relative state of maturation. Forty-eight h after syngeneic transplantation with OVA-loaded grafts, CCR7 expression was detected on the OVA+ cells in both the host corneal beds and the draining LN. Local administration of anti-CCL21 led to a significant suppression in the flow of OVA+CD11c+ cells to the draining LN. CONCLUSIONS: These data suggest that in inflammation, APC expressing CCR7 on their cell surface interact with CCL21 to facilitate their migration from the cornea to draining LN via afferent lymphatics.

Exposure to a dry environment induces strain-specific responses in mice.

Most current animal models of dry eye have a single causative mechanism and do not take into consideration the influence of environmental conditions on tear secretion and associated ocular surface signs. Since immunity and inflammation have been implicated in dry eye pathogenesis, and different mouse strains are known to have differentially biased immune responses, we conducted the present study to test the hypothesis that strains with specifically polarized T cell responses (T helper-1 [Th1] vs. T helper-2 [Th2]) develop differential signs of dry eye when exposed to a controlled low humidity setting. Eight to 12-week-old BALB/c (Th2 biased) and C57BL/6 (Th1 biased) mice were placed in a controlled environment chamber (CEC) where relative humidity (RH), temperature (T), and air flow (AF) were continuously regulated and monitored. Mice were exposed to specific environmental controlled conditions (RH=15.5+/-3.8%, AF=15 l/min, T=21-23 degrees C) for 3 to 7 days. Aqueous tear production by means of the cotton thread test, corneal fluorescein staining (NEI grading scheme, score 0-15) and goblet cell density in the superior and inferior conjunctivae were measured by a masked observer. No statistically significant differences between the groups were found at baseline. Statistically significant decreases in tear secretion were seen after exposure to the CEC environment. Mean cotton thread wetting was 1.9+/-0.2 (baseline), 1.4+/-0.3 (day 3), and 0.9+/-0.2 mm (day 7) for BALB/c mice, and 1.7+/-0.3 (baseline), 0.9+/-0.3 (day 3), and 0.4+/-0.2 mm (day 7) for C57BL/6 mice. These mice showed reduced tear secretion as compared to BALB/C at each time point tested (P<.005, t-test). Both BALB/c and C57BL/6 mice showed a significant increase in corneal fluorescein staining at both day 3 and day 7 as compared to baseline. With exposure to the CEC goblet cell density significantly decreased in the superior and inferior conjunctivae in BALB/c mice, while it remained unchanged in C57BL/6 mice. This study indicates that exposure of non-pharmacologically modified mice to a low humidity environment in the CEC can lead to significant alterations in tear secretion, goblet cell density, and acquisition of dry eye-related ocular surface signs which are strain-specific.

Deletion of the chemokine receptor CCR1 prolongs corneal allograft survival.

PURPOSE: Many corneal grafts undergo immune rejection, and current therapies are associated with many side effects. The purpose of this study was to identify critical chemokine pathways involved in generating the alloimmune response to corneal transplants. METHODS: Orthotopic corneal transplantation was performed in fully mismatched strains. Cytokine and chemokine receptor gene expression was determined by the RNase protection assay. Knockout (KO) strains for chemokine-chemokine receptors that are upregulated after transplantation underwent corneal transplantation. Results derived from KO murine hosts were compared with cyclosporine (Cy) therapy. In addition to graft survival, graft infiltration, allospecific delayed-type hypersensitivity (DTH), and cytokine expression were compared among the recipient groups. RESULTS: Initial experiments revealed gene upregulation of the chemokine receptors CCR1, -2, and -5 after corneal allorejection. Although CCR1 KO hosts showed a significant increase in graft survival compared with wild-type (WT) hosts, allografts in CCR5, CCR2/CCL3(MIP-1alpha), CXCR3, CXCL10/IP-10, and CCL3/MIP-1alpha KO mice did not show a significant improvement in graft survival. Further, CCR1 KO hosts showed a significantly higher survival rate than with systemic Cy therapy in WT hosts. Moreover, graft infiltration by leukocytes and gene expression of proinflammatory cytokines were reduced in CCR1 KO mice compared with both Cy treated and untreated WT mice, as was the induction of allospecific DTH. CONCLUSIONS: These studies provide, for the first time, evidence that targeting of specific chemokine pathways can significantly promote survival of corneal transplants, and suggest that select deletion or suppression of CCR1 can be a useful therapeutic target in corneal transplant immunity.

Corneal antigen-presenting cells.

Corneal antigen-presenting cells (APCs) were thought to reside exclusively in the peripheral cornea. However, recent evidence demonstrates that the central cornea is also endowed with a heterogeneous population of bone marrow-derived cells, including epithelial Langerhans cells (LCs) and anterior stromal dendritic cells (DCs), which under certain conditions can function as APCs. While the corneal periphery contains mature and immature resident bone marrow-derived DCs, the central cornea is endowed exclusively with highly immature/precursor-type DCs. During inflammation, a majority of resident DCs undergo maturation by acquiring high expression of major histocompatibility complex class II antigens and B7 (CD80/CD86) and CD40 costimulatory molecules. Further, macrophages are present in the posterior corneal stroma. In transplantation, donor-derived DCs migrate to host cervical lymph nodes and activate host T cells via the direct pathway when allografts are placed in inflamed, but not normal uninflamed, host beds. Migration of DCs to cervical lymph nodes is, in part, regulated by the vascular endothelial growth factor receptor-3 (VEGFR-3) that is expressed on corneal DCs. Blockade of the VEGFR-3 signaling significantly suppresses corneal DC trafficking to draining lymph nodes and rejection of corneal transplants. Much remains unknown about the function of these cells including their role in innate responses as well as in tolerance. Regardless, these data revise the tenet that the cornea is immune privileged due to a lack of resident lymphoreticular cells per se, but suggest that the cornea is capable of actively participating in the immune response to foreign antigens and autoantigens, rather than being a passive bystander. Additionally, one important aspect of immune privilege is likely the ocular 'imposition' of the immature phenotype on its resident bone marrow-derived cells.

Dry eye syndromes.

Over the past 20 years it has become clear that dry eye syndrome (DES) or keratoconjunctivitis sicca (KCS) is a complex multifactorial disease characterized by an immune and inflammatory process that affects the lacrimal glands and ocular surface. In this paradigm, inflammation is seen as both the cause and consequence of conjunctival and corneal cell damage. In this chapter, we identify the unique characteristics of the lacrimal gland, the role of epithelial cells, regulatory T cells, and cytokines in maintaining ocular surface homeostasis and tear secretion function. We analyze the factors inducing loss of the lacrimal gland homeostasis and its consequences, and in so doing hope to provide a picture of the role of the immune system in the pathophysiology of KCS and useful information to help understand the complexity of DES.

Cicatrizing and autoimmune diseases.

Autoimmune disorders of the ocular surface represent a clinically heterogeneous group of conditions where acute and chronic autoreactive mechanisms can cause significant damage to the eye. When severe and affecting the epithelium and substantia propria of the conjunctiva, cicatrization can ensue, leading to significant mechanical alterations as a result of the fibrosis. These conditions, though generally infrequent, can be the cause of profound pathology and visual disability, and often need systemic immune modulation for therapy.

Influence of aging on the polar and neutral lipid profiles in human meibomian gland secretions.

OBJECTIVE: To determine whether aging is associated with significant alterations in the polar and neutral lipid profiles in human meibomian gland secretions. METHODS: Meibomian gland secretions were collected from both eyes of younger and older men and women. Samples were processed for high-performance liquid chromatography or mass spectrometry and for the analysis of associated spectra of fragment ions. Subjects also underwent slitlamp evaluations of the eyelid. RESULTS: Aging is associated with numerous significant alterations in the lipid profiles of human meibomian gland secretions. Analysis of polar and neutral lipid patterns identified ions that were significantly different in secretions of younger vs older men and women, as well as ions that varied significantly only between men and women. Correlation coefficients within, but not between, groups were high. Aging was accompanied by increased opacity of meibomian gland secretions and by eyelid and eyelid margin changes. CONCLUSION: Aging is associated with significant sex-related alterations in the polar and neutral lipid profiles of human meibomian gland secretions. CLINICAL RELEVANCE: The observed changes may contribute to the age-related increase in the prevalence of dry eye syndromes.