Eric Pearlman

CXC Chemokine Receptor 2 But Not C-C Chemokine Receptor 1 Expression Is Essential for Neutrophil Recruitment to the Cornea in Helminth-Mediated Keratitis (River Blindness)

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2−/− and CCR1−/− mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1α was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2−/− mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2−/− mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2−/− mice, which was similar to BALB/c mice. Furthermore, although MIP-1α production was lower in CXCR2−/− mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.

Toll-like receptors at the ocular surface.

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. we present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation.

Distinct roles for PECAM-1, ICAM-1, and VCAM-1 in recruitment of neutrophils and eosinophils to the cornea in ocular onchocerciasis (river blindness).

Infiltration of granulocytes into the transparent mammalian cornea can result in loss of corneal clarity and severe visual impairment. Since the cornea is an avascular tissue, recruitment of granulocytes such as neutrophils and eosinophils into the corneal stroma is initiated from peripheral (limbal) vessels. To determine the role of vascular adhesion molecules in this process, expression of platelet endothelial cell adhesion molecule 1 (PECAM-1), ICAM-1, and VCAM-1 on limbal vessels was determined in a murine model of ocular onchocerciasis in which Ags from the parasitic worm Onchocerca volvulus are injected into the corneal stroma. Expression of each of these molecules was elevated after injection of parasite Ags; however, PECAM-1 and ICAM-1 expression remained elevated from 12 h after injection until 7 days, whereas VCAM-1 expression was more transient, with peak expression at 72 h. Subconjunctival injection of Ab to PECAM-1 significantly inhibited neutrophil recruitment to the cornea compared with eyes injected with control Ab (p = 0.012). Consistent with this finding, corneal opacification was significantly diminished (p < 0.0001). There was no significant reduction in eosinophils. Conversely, subconjunctival injection of Ab to ICAM-1 did not impair neutrophil recruitment, but significantly inhibited eosinophil recruitment (p = 0.0032). Injection of Ab to VCAM-1 did not significantly inhibit infiltration of either cell type to the cornea. Taken together, these results demonstrate important regulatory roles for PECAM-1 and ICAM-1 in recruitment of neutrophils and eosinophils, respectively, to the cornea, and may indicate a selective approach to immune intervention.

Expression of Innate and Adaptive Immune Mediators in Human Corneal Tissue Infected With Aspergillus or Fusarium

BACKGROUNDnFilamentous fungi of the genera Aspergillus and Fusarium are major causes of corneal ulcers in the United States and in the developing world and result in significant visual impairment and blindness.nnnMETHODSnRNA was extracted from 110 patients with corneal ulcers in southern India within 1 week of infection with either Fusarium solani or Aspergillus flavus, and gene expression was determined by quantitative polymerase chain reaction. Posttransplant corneas from later stage disease (>2 weeks after infection) were also examined.nnnRESULTSnExpression of Dectin-1, Toll-like receptor 2 (TLR2), TLR4, TLR9, and NOD-like receptor protein (NLRP)3 messenger RNA was elevated >1000-fold compared with uninfected donor corneas, whereas Dectin-2 was constitutively expressed in uninfected corneas. Furthermore, interleukin 1β (IL-1β) expression was elevated >1000-fold, whereas IL-1α expression was not increased. Expression of IL-8, IL-17, and tumor necrosis factor α was also elevated. CD3(+)and CD4(+) T cells were detected in infected posttransplant corneas. Expression of IL-17 and interferon γ was elevated but not that of IL-4. There were no significant differences in the host response between Aspergillus- and Fusarium-infected corneas at any time point.nnnCONCLUSIONSnThere is a common innate and adaptive immune response to these filamentous fungi, which includes the generation of T-helper 1 and T-helper 17 cells.

Inhibition of corneal inflammation by liposomal delivery of short-chain, C-6 ceramide

Ceramide is recognized as an antiproliferative and proapoptotic sphingolipid metabolite; however, the role of ceramide in inflammation is not well understood. To determine the role of C6‐ceramide in regulating inflammatory responses, human corneal epithelial cells were treated with C6‐ceramide in 80 nm diameter nanoliposome bilayer formulation (Lip‐C6) prior to stimulation with UV‐killed Staphylococcus aureus. Lip‐C6 (5 μM) inhibited the phosphorylation of proinflammatory and proapoptotic MAP kinases JNK and p38 and production of neutrophil chemotactic cytokines CXCL1, CXCL5, and CXCL8. Lip‐C6 also blocked CXC chemokine production by human and murine neutrophils. To determine the effect of Lip‐C6 in vivo, a murine model of corneal inflammation was used in which LPS or S. aureus added to the abraded corneal surface induces neutrophil infiltration to the corneal stroma, resulting in increased corneal haze. Mice were treated topically with 2 nMoles (811 ng) Lip‐C6 or with control liposomes prior to, or following, LPS or S. aureus stimulation. We found that corneal inflammation was significantly inhibited by Lip‐C6 but not control liposomes given prior to, or following, activation by LPS or S. aureus. Furthermore, Lip‐C6 did not induce apoptosis of corneal epithelial cells in vitro or in vivo, nor did it inhibit corneal wound healing. Together, these findings demonstrate a novel, anti‐inflammatory, nontoxic, therapeutic role for liposomally delivered short‐chain ceramide.

A Dominant Role for Fcγ Receptors in Antibody-Dependent Corneal Inflammation

Although production of specific Ab is a critical element of host defense, the presence of Ab in tissues leads to formation of immune complexes, which can trigger a type III Arthus reaction. Our studies on a mouse model of river blindness showed that Ab production is essential for recruitment of neutrophils and eosinophils to the cornea and for development of corneal opacification. In the current study, we determined the relative contribution of complement and FcγR interactions in triggering immune complex-mediated corneal disease. FcγR−/− mice, C3−/− mice, and immunocompetent control (B6/129Sj) mice were immunized s.c. and injected intrastromally with Onchocerca volvulus Ags. Slit lamp examination showed that control mice, C3−/− mice, and control mice injected with cobra venom factor developed pronounced corneal opacification, whereas corneas of FcγR−/− mice remained completely clear. Furthermore, recruitment of neutrophils and eosinophils to the corneal stroma was significantly impaired in FcγR−/− mice, but not in C3−/− mice or cobra venom factor-treated mice. We therefore conclude that FcγR-mediated cell activation, rather than complement activation, is the dominant pathway of immune complex disease in the cornea. These findings demonstrate a novel role for FcγR interactions in mediating ocular inflammation.

Onchocerca volvulus keratitis (river blindness) is exacerbated in BALB/c IL-4 gene knockout mice

To determine the outcome of Onchocerca volvulus keratitis in IL-4(-/-) BALB/c mice, animals were immunized subcutaneously and injected into the corneal stroma with soluble O. volvulus antigens. IL-4(-/-) BALB/c mice had a deviated cellular response, with decreased serum IgE and IgG1 and elevated IgG2a compared with control BALB/c mice. In marked contrast to control BALB/c, C57BL/6, and IL-4(-/-) C57BL/6 mice, IL-4(-/-) BALB/c mice developed severe corneal opacification and neovascularization that was associated with a pronounced neutrophil infiltrate to the corneal stroma. STAT-6(-/-) BALB/c mice had the same phenotype as IL-4(-/-) BALB/c mice, and complement depletion had no effect on the severity of O. volvulus keratitis in these mice. These findings indicate that on a BALB/c background, IL-4 has a critical role in regulating neutrophil recruitment to the cornea and development of O. volvulus keratitis.