Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

) and LFA-1 (␣ L / ␤ 2 ), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.Polymorphonuclear neutrophils (PMNs) 3 play a major role in the development of inflammatory responses to host injury and infection. Their functions include destruction of invading bacteria and recruitment of macrophages and lymphocytes to the affected site (1). Circulating PMNs sense injury and pathogen signals, cross the vascular endothelium, and migrate to the target tissue; two series of events control this process. The first leads to the slowing down and adherence of circulating PMNs on the vascular endothelium followed by their transendothelial migration or extravasation and activation (2). The second controls the directional migration of PMNs to the injured site through the endothelial basement membrane, a specialized type of ECM, and subsequently the deeper interstitial ECM, along chemokine and cytokine gradients. Leukocyte-to-leukocyte and leukocyte-to-endothelium interactions are important before extravasation. These are mediated by interactions between selectins and their ligands and by ␤ 2 (MAC-1 and LFA-1) and ␤ 1 (VLA-4 -6) integrin interactions with cell adhesion proteins ICAM and PECAM (3). The directional migration of PMNs through the ECM is a complex, multistep process that involves several ␣ and ␤ integrin interactions with ECM proteins. Thus far, a few basement membrane proteins, laminins, entactin, and fibronectin have been identified as specific ligands in regulating migration of PMNs after extravasation (4 -6). Additional interstitial ECM proteins and their receptors that modulate PMN migration have yet to be identified. Here we show that the ECM protein lumican is a novel regulator of PMN migration.Lumican is a secreted collagen-binding ECM protein of the corneal, dermal, and tendon stroma, arterial wall, and the intestinal submucosa (7-9). It is a member of the small leucine-rich repeat proteoglycans (10); these were initially investigated in the context of binding collagen and regulating tissue structure and biomechanics (11,12). A body of literature is beginning to indicate that these proteoglycans interact with cytokines, growth factors, and cell surface receptors to modulate cell adhesion, proliferation, and migration (13-16). Lumican and biglycan, another member of this family of proteoglycans, have been recently shown to regulate host response to pathogenassociated molecular patterns (17, 18). Thus, lumican-deficient (Lum Ϫ/Ϫ ) mice are hyporesponsive to bacterial lipopolysaccharide (LPS) endotoxins, and Lum Ϫ/Ϫ macrophages in culture produce lower levels of pro-inflammato...

Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by toll like receptor (TLR) 4, and neutrophil chemotaxis via integrins. Here we investigated the role of lumican in the development of colitis mediated by intra-rectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum+/+ and Lum−/− mice. The TNBS-treated Lum+/+ mouse colons showed marked increases in CXCL1, TNF-α and neutrophil infiltration; while these responses were significantly dampened in the Lum−/− mice. The NF-κB transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum+/+ but not in Lum−/− colons. Also, nuclear translocation of NF-κB was delayed in LPS-stimulated Lum−/− primary peritoneal macrophages. Thus, the Lum−/− mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4−/− and MyD88−/− mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

Neonates and infants have a higher morbidity and mortality associated with lower respiratory tract illnesses compared with older children. To identify age-related and longitudinal differences in the cellular immune response to acute lung injury (ALI), neonatal and juvenile mice were given Escherichia coli LPS using a novel, minimally invasive aspiration technique. Neonatal and juvenile mice received between 3.75 and 7.5 mg/kg LPS by intrapharyngeal aspiration. Airway and lung cells were isolated and characterized by flow cytometry, cytokine/chemokine mRNA expression from lung homogenates was quantified, and lung morphometry and injury scores were performed. LPS-treated neonatal mice underwent adoptive transfer with adult T regulatory cells (Tregs). After LPS aspiration, lung monocytes isolated from neonatal mice had a predominant M2 phenotype, whereas lung monocytes from juvenile mice displayed a mixed M1/M2 phenotype. At 72 hours after LPS exposure, neonatal lungs were slower to resolve inflammation and expressed lower mRNA levels of CCL2, CCL5, CXCL10, and IL-10. Juvenile, but not neonatal, mice demonstrated a significant increase in airway Tregs after LPS exposure. Adoptive transfer of adult Tregs into LPS-challenged neonatal mice resulted in reduced lung inflammation and improved weight gain. These findings underscore several vulnerabilities in the neonatal immune response to LPS-induced ALI. Most striking was the deficiency in airway Tregs after LPS aspiration. Adoptive transfer of adult Tregs mitigated LPS-induced ALI in neonatal mice, highlighting the importance of age-related differences in Tregs and their response to ALI during early postnatal development.

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