Cleavage of CD14 on Human Gingival Fibroblasts Cocultured with Activated Neutrophils Is Mediated by Human Leukocyte Elastase Resulting in Down-Regulation of Lipopolysaccharide-Induced IL-8 Production

Abstract

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 μg/ml HLE treatment for 30–60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1α, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.

Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8

“…This negative feedback mechanism reported in this and other studies is to be assumed to result in the inhibition of further PMN recruitment and activation, and contributes to the control of inflammatory functions and excessive tissue damage in vivo. Generation of IL-8 from PMN in other species was previously shown to be regulated by a wide variety of soluble agonists such as IL-4, IL-10, IL-13 and INF-γ [16,22,26,45] and IL-1 and TNF-α receptors [13].…”

Neutrophil serine proteases fine-tune the inflammatory response

“…Recognition of LPS occurs mainly through the TLR4-MD2-CD14 complex (Miller et al, 2005). CG and NE released by neutrophils cleave CD14 from the cell surface in a time-and concentration-dependent manner (Le-Barillec et al, 1999;Le-Barillec et al, 2000;Nemoto et al, 2000). Proteolysis of CD14 suppresses LPS-induced cell activation and CXCL8 production, suggesting that these neutrophil serine proteases might provide a negative feedback mechanism to downmodulate the inflammatory response.…”

Porphyromonas gingivalis Lipopolysaccharide Signaling in Gingival Fibroblasts–CD14 and Toll-like Receptors

“…Under inflammatory conditions, gingival fibroblasts contributed to the inactivation of circulating TNF-␣through the preferential induction and shedding of TNF receptor (Ohe et al, 2000). Gingival fibroblast-lymphocyte interactions up-regulate the cytokine network in inflammatory gingival tissues (Murakami and Okada, 1997;Nemoto et al, 2000). On the genetic level, P. gingivalis DNA and its palindromic internal CpG motifs stimulate IL-6 expression in gingival fibroblasts by stimulating NF-B binding, and this bacterial DNA may function as a virulence factor of the organism in periodontal disease (Takeshita et al, 1999).…”