Eugenia Diaconu

Matrix metalloproteinase-8 facilitates neutrophil migration through the corneal stromal matrix by collagen degradation and production of the chemotactic peptide Pro-Gly-Pro.

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.

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.

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

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

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.

Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness).

ABSTRACT Endosymbiotic Wolbachia bacteria are abundant in the filarial nematodes that cause onchocerciasis (river blindness), including the larvae (microfilariae) that migrate into the cornea. Using a mouse model of ocular onchocerciasis, we recently demonstrated that it is these endosymbiotic bacteria rather than the nematodes per se that induce neutrophil infiltration to the corneal stroma and loss of corneal clarity (Saint Andre et al., Science 295:1892-1895, 2002). To better understand the role of Wolbachia organisms in the pathogenesis of this disease, we examined the fate of these bacteria in the cornea by immunoelectron microscopy. Microfilariae harboring Wolbachia organisms were injected into mouse corneas, and bacteria were detected with antibody to Wolbachia surface protein. Within 18 h of injection, neutrophils completely surrounded the nematodes and were in close proximity to Wolbachia organisms. Wolbachia surface protein labeling was also prominent in neutrophil phagosomes, indicating neutrophil ingestion of Wolbachia organisms. Furthermore, the presence of numerous electron-dense granules around the phagosomes indicated that neutrophils were activated. To determine if Wolbachia organisms directly activate neutrophils, peritoneal neutrophils were incubated with either parasite extracts containing Wolbachia organisms, parasite extracts depleted of Wolbachia organisms (by antibiotic treatment of worms), or Wolbachia organisms isolated from filarial nematodes. After 18 h of incubation, we found that isolated Wolbachia organisms stimulated production of tumor necrosis factor alpha and CXC chemokines macrophage inflammatory protein 2 and KC by neutrophils in a dose-dependent manner. Similarly, these cytokines were induced by filarial extracts containing Wolbachia organisms but not by Wolbachia-depleted extracts. Taken together, these findings indicate that neutrophil activation is an important mechanism by which Wolbachia organisms contribute to the pathogenesis of ocular onchocerciasis.

Toll-Like Receptor 2 Regulates CXC Chemokine Production and Neutrophil Recruitment to the Cornea in Onchocerca volvulus/ Wolbachia-Induced Keratitis

ABSTRACT The filarial nematode Onchocerca volvulus is the causative organism of river blindness. Our previous studies demonstrated an essential role for endosymbiotic Wolbachia bacteria in corneal disease, which is characterized by neutrophil infiltration into the corneal stroma and the development of corneal haze. To determine the role of Toll-like receptors (TLRs) in neutrophil recruitment and activation, we injected a soluble extract of O. volvulus containing Wolbachia bacteria into the corneal stromata of C57BL/6, TLR2−/−, TLR4−/−, TLR2/4−/−, and TLR9−/− mice. We found an essential role for TLR2, but not TLR4 or TLR9, in neutrophil recruitment to the cornea and development of corneal haze. Furthermore, chimeric mouse bone marrow studies showed that resident bone marrow-derived cells in the cornea can initiate this response. TLR2 expression was also essential for CXC chemokine production by resident cells in the cornea, including corneal fibroblasts, and for neutrophil activation. Taken together, these findings indicate that Wolbachia activates TLR2 on resident bone marrow-derived cells in the corneal stroma to produce CXC chemokines, leading to neutrophil recruitment to the corneal stroma, and that TLR2 mediates O. volvulus/Wolbachia-induced neutrophil activation and development of corneal haze.

Wolbachia- and Onchocerca volvulus-induced keratitis (River blindness) is dependent on myeloid differentiation factor 88

ABSTRACT Endosymbiotic Wolbachia bacteria that infect the filarial nematode Onchocerca volvulus were previously found to have an essential role in the pathogenesis of river blindness. The current study demonstrates that corneal inflammation induced by Wolbachia or O. volvulus antigens containing Wolbachia is completely dependent on expression of myeloid differentiation factor 88.

Filaria/Wolbachia activation of dendritic cells and development of Th1‐associated responses is dependent on Toll‐like receptor 2 in a mouse model of ocular onchocerciasis (river blindness)

Toll‐like receptors (TLRs) regulate dendritic cell function and activate signals that mediate the nature of the adaptive immune response. The current study examined the role of TLRs in dendritic cell activation and in regulating T cell and antibody responses to antigens from the filarial parasites Onchocerca volvulus and Brugia malayi, which cause river blindness and lymphatic filariasis, respectively. Bone‐marrow‐derived CD11c+ cells from C57BL/6 and TLR4−/– mice produced high levels of IL‐6 and RANTES, and showed elevated surface CD40 expression, whereas CD11c+ cells from myeloid differentiation factor 88−/– (MyD88−/–), TLR2−/– and TLR2/4−/– mice were not activated. Similarly, IFN‐γ production by splenocytes from immunized TLR2−/– mice was significantly impaired compared with splenocytes from C57BL/6 and TLR4−/– mice. In contrast, there was no difference among these strains in Th2‐associated responses including IL‐5 production by splenocytes from immunized animals, serum IgE and IgG1, or eosinophil infiltration into the corneal stroma. Neutrophil recruitment to the cornea and CXC chemokine production was inhibited in immunized TLR2−/– mice compared with C57BL/6 and TLR4−/– mice. Taken together, these findings demonstrate an essential role for TLR2 in filaria‐induced dendritic cell activation, IFN‐γ production and neutrophil migration to the cornea, but does not affect filaria‐induced Th2‐associated responses.

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.

CD4(+) depletion selectively inhibits eosinophil recruitment to the cornea and abrogates Onchocerca volvulus keratitis (River blindness).

ABSTRACT Previous studies demonstrated that in the murine model ofOnchocerca volvulus keratitis, neutrophils and eosinophils are recruited into the cornea in a biphasic manner in response to intrastromal injection. To determine if CD4+ T cells regulate migration of neutrophils and eosinophils into the cornea, CD4+ cells were depleted using monoclonal antibody GK1.5 before intrastromal injection of parasite antigens. Depletion of CD4+ cells abrogated corneal opacification at later but not early stages of disease. Consistent with this observation, CD4 depletion significantly impaired recruitment of eosinophils to the cornea but had no effect on neutrophils. These data indicate that CD4+ T cells mediate sustained O. volvuluskeratitis by regulating eosinophil recruitment to the cornea.