keratan-sulfate and Keratitis

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum(-/-)) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that invitro Lum(-/-) macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum(-/-) mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum(-/-) mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum(-/-) mice compared to wild types. Infected Lum(-/-) corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24-48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum(+/-) mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum(-/-) compared to Lum(+/-) infected corneas by 2-5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.

Topics: Animals; Base Sequence; Chondroitin Sulfate Proteoglycans; DNA Primers; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Flow Cytometry; Keratan Sulfate; Keratitis; Lipopolysaccharides; Lumican; Mice; Mice, Knockout; Polymerase Chain Reaction; Pseudomonas aeruginosa; Pseudomonas Infections

Keratocan and lumican are keratan-sulfate proteoglycans (KSPG), which have a critical role in maintaining corneal clarity. To determine whether these KSPGs have a role in corneal inflammation, we examined Kera(-/-) and Lum(-/-) mice in a model of lipopolysaccharide (LPS)-induced keratitis in which wild-type mice develop increased corneal thickness and haze due to neutrophil infiltration to the corneal stroma. Corneal thickness increases caused by LPS mice were significantly lower in Kera(-/-) and Lum(-/-) than wild-type mice. Further, LPS-injected Lum(-/-) mice had elevated corneal haze levels compared with that of Kera(-/-) and wild-type. At 24 h post-injection, total enhanced green fluorescent protein-positive bone marrow-derived inflammatory cells in chimeric mice was significantly lower in Kera(-/-) mice and Lum(-/-) mice compared with wild-type mice. Neutrophil infiltration was inhibited in Kera(-/-) and Lum(-/-) mice at 6 and 24 h post-stimulation, with Lum(-/-) corneas having the most profound defect in neutrophil migration. Reconstitution of keratocan and lumican expression in corneas of Kera(-/-) and Lum(-/-) mice using adeno-keratocan and adeno-lumican viral vectors, respectively, resulted in normal neutrophil infiltration in response to LPS. Immunoprecipitation/Western blot analysis showed that lumican and keratocan core proteins bind the CXC chemokine KC during a corneal inflammatory response, indicating that corneal KSPGs mediate neutrophil recruitment to the cornea by regulating chemokine gradient formation. Together, these data support a significant role for lumican and keratocan in a corneal inflammatory response with respect to edema, corneal clarity, and cellular infiltration.

Topics: Adenoviridae; Animals; Chemokine CXCL1; Chondroitin Sulfate Proteoglycans; Cornea; Inflammation; Keratan Sulfate; Keratitis; Lipopolysaccharides; Lumican; Mice; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Protein Binding; Proteoglycans; Time Factors; Transduction, Genetic

The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling. The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries.. Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts. Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively. Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA.. Lum-/- -injured corneas showed significantly lower caspase-3/7 activity (apoptosis). Lum-/- -wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta. The Fas protein level, before and after wounding, was dramatically lower in Lum-/- - compared with Lum+/+-injured cornea. However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level. Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican.. The data suggest that lumican binds FasL and facilitates induction of Fas. Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing.

Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Caspases; Cell Culture Techniques; Chondroitin Sulfate Proteoglycans; Corneal Stroma; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Fluorescent Antibody Technique, Indirect; Keratan Sulfate; Keratitis; Lumican; Macrophages; Membrane Glycoproteins; Mice; Mice, Knockout; Neutrophils; Peroxidase; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Wound Healing

Topics: Animals; Autoradiography; Chondroitin Sulfates; Cornea; Culture Techniques; Dermatan Sulfate; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Keratan Sulfate; Keratitis; Keratoconus; Rabbits; Time Factors