Potentiation of Lipopolysaccharide-Induced Chemokine and Adhesion Molecule Expression in Corneal Fibroblasts by Soluble CD14 or LPS-Binding Protein

Abstract

PURPOSE. The detection of bacterial lipopolysaccharide (LPS) by human cells is facilitated by LPS-binding protein (LBP) and soluble (s)CD14. The effects of these proteins on chemokine release and adhesion molecule expression in cultured human corneal fibroblasts were examined. METHODS. The release of chemokines into culture supernatants and the expression of the intercellular adhesion molecule (ICAM)-1 on the cell surface were determined by enzymelinked immunosorbent assays. The intracellular abundance of chemokine and ICAM-1 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analyses. The phosphorylation and degradation of IB-␣ and the subcellular localization of NF-B were examined by immunoblot and immunofluorescence analyses, respectively. RESULTS. Neither sCD14 nor LBP alone affected the expression of chemokines or ICAM-1 in cultured human corneal fibroblasts. However, sCD14 or LBP enhanced the LPS-induced upregulation of ICAM-1 and the chemokines interleukin-8 and monocyte chemoattractant protein (MCP)-1 in these cells at the protein and mRNA levels. B acterial corneal ulcer is a major cause of loss of vision in developed and developing countries. This condition results from the destruction of collagen fibrils in the corneal stroma by collagenolytic enzymes produced as a consequence of bacterial infection.1 Pathologic examination has revealed the presence of many infiltrating leukocytes, including neutrophils and macrophages, in or surrounding corneal ulcers. 1 We have shown that the interaction of resident corneal fibroblasts with infiltrating leukocytes very likely promotes collagen degradation.2 Whereas corneal fibroblasts alone in culture degrades collagen fibrils to a small extent, neutrophils alone do not. However, the addition of either neutrophils or neutrophilconditioned medium to corneal fibroblasts results in a marked increase in the amount of collagen degraded by the fibroblasts. We have also demonstrated an interaction between bacteria and corneal fibroblasts in culture, in that elastase produced by Pseudomonas aeruginosa both degrades collagen directly and activates the collagen-degrading matrix metalloproteinases produced by corneal fibroblasts.3 In addition, we found that corneal fibroblasts are able to detect the presence of lipopolysaccharide (LPS), a component of the cell membrane of Gramnegative bacteria, and that they upregulate the expression of the chemokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 and that of intercellular adhesion molecule (ICAM)-1 in response.4 Such a response in vivo would be expected to trigger the local infiltration of leukocytes. Corneal fibroblasts may thus function as sentinel and effector cells in the defense of the cornea against bacterial infection.LPS is an amphipathic molecule with a large hydrophobic component and a small hydrophilic domain. 5 The lipophilic portion of LPS, known as lipid A, is the active component. 6 In an aqueous environment, LPS exists as polymeric aggregates, with the hydro...

Cytokine, Chemokine, and Adhesion Molecule Expression Mediated by MAPKs in Human Corneal Fibroblasts Exposed to Poly(I:C)

“…Given that NF-B signaling is implicated in the induction of genes for chemokines and adhesion molecules in several cell types, 11,15 we examined whether poly(I:C) affected the phosphorylation or abundance of the endogenous NF-B inhibitor IB-␣ in corneal fibroblasts. Immunoblot analysis revealed that poly(I:C) (10 g/mL) induced a time-dependent increase in the phosphorylation of IB-␣ that was first apparent at 30 minutes and was maximal at 90 minutes after the onset of stimulation; it also induced the degradation of this protein, with the amount of IB-␣ greatly reduced at 30 minutes but somewhat recovered by 120 minutes (Fig.…”

PAPep, a small peptide derived from human pancreatitis-associated protein, attenuates corneal inflammation in vivo and in vitro through the IKKα/β/IκBα/NF–κB signaling pathway

“…Meanwhile, PAPep effectively inhibited the release of IL-6, IL-8 and MCP-1 in the cornea tissue. These chemokines serve as chemoattractants that attract the migration and infiltration of leukocytes to the injured sites so they are regarded as destructive factors during keratitis [12,17]. As…”

“…Once exposed to a stimulus such as LPS, they become activated and result in inflammation by producing various cytokines, up-regulating the expression of cell adhesion molecule and promoting leukocyte accumulation [17,19,20]. To evaluate the biochemical mechanism underlying the inhibition effect of PAPep on keratitis, we examined the activation of NF-κB pathway in the corneal fibroblasts.…”

Aqueous Nanomicellar Formulation for Topical Delivery of Biotinylated Lipid Prodrug of Acyclovir: Formulation Development and Ocular Biocompatibility

“…Induction of multiple proinflammatory cytokines by LPS is due to its ability to bind to intramembranal complex of CD-14 and Toll-like receptors. 54,[59][60][61][62] LPS caused significant upregulation of IL-1b, IL-6, IL-8, IL-17, TNF-a, and IFN-g expression in HCECs. However, incubation of HCECs with blank and prodrug-encapsulated nanomicelles groups did not significantly alter the expression levels of IL-1b, IL-6, IL-8, IL-17, TNF-a, and IFN-g. All the in vitro ocular biocompatibility tests revealed that nanomicelle formulations are biocompatible and suitable for topical ocular application.…”