Soluble CD14 mediates lipopolysaccharide-induced intercellular adhesion molecule 1 expression in cultured human gingival fibroblasts

Abstract

Intercellular adhesion molecule 1 (ICAM-1) is involved in the accumulation and activation of leukocytes in inflammatory diseases such as periodontitis. As reported previously, ICAM-1 is up-regulated on cultured human gingival fibroblasts (HGF) by exposure to lipopolysaccharide (LPS), suggesting a specific LPS recognition mechanism. We therefore investigated the role of CD14, an LPS receptor, in stimulation of HGF by LPS. Cell surface CD14 antigen was not observed on HGF by flow cytometric analysis. In addition, expression of CD14 mRNA in HGF was not detected by reverse transcription-PCR analysis. Since HGF did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) in ICAM-1 induction on HGF by LPS. The serum-dependent ICAM-1 induction by LPS was observed in HGF. In medium containing human serum, anti-CD14 antibody inhibited ICAM-1 induction on HGF by LPS. Depletion of sCD14 from human serum markedly reduced ICAM-1 expression on HGF in response to LPS. Supplementation of the serum-free medium with sCD14 alone restored the capacity of HGF to respond to LPS. These results show that induction of ICAM-1 in HGF by LPS does not involve binding to cell surface CD14 but is mediated by serum-derived sCD14.

Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide

“…One study showed that HGF express CD14 and that the LPS-induced signal transducing pathway is mediated by mCD14 (37). Another study found that HGF express CD14 neither on the surface nor in the mRNA and that ICAM-1 expression by HGF is up-regulated by serum-derived sCD14 (15). Our data support both sets of observations.…”

“…Watanabe et al (37) reported that HGF express mCD14 and that the LPS-induced signal-transducing pathway is mediated by mCD14. In contrast, Hayashi et al (15) found that HGF expressed CD14 neither on the surface nor in an mRNA analysis but were activated with LPS in a serum-derived sCD14dependent manner.…”

“…Reverse transcription of the RNA samples to cDNA was done with Moloney murine leukemia virus reverse transcriptase (GIBCO BRL, Grand Island, N.Y.) and oligo(dT) [12][13][14][15][16][17][18] primer (GIBCO BRL). To transcribe the total RNA into cDNA, 2.5 g of RNA, 0.25 g of oligo(dT) [12][13][14][15][16][17][18] primer, First-Strand Buffer (GIBCO BRL), 0.2 mM deoxynucleoside triphosphates, 500 U of Moloney murine leukemia virus reverse transcriptase, and 10 mM dithiothreitol were added to a total volume of 50 l. The reaction mixture was incubated for 1 h at 37°C followed by 5 min at 95°C. The primers used for PCR had the following sequences: CD14, forward 5Ј-CT CAACCTAGAGCCGTTTCT-3Ј and reverse 5Ј-CAGGATTGTCAGACAGGT CT-3Ј; and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5Ј-TGAAGGTCGGAGTCAACGGATTTGGT-3Ј and reverse 5Ј-CATG TGGGCCATGAGGTCCACCAC-3Ј.…”

Lipopolysaccharide Enhances the Production of Vascular Endothelial Growth Factor by Human Pulp Cells in Culture

“…A soluble form of CD14 (sCD14) lacking the glycosylphosphatidylinositol anchor is also present in serum (1), and sCD14 participates in the LPS-induced activation of endothelial and epithelial cells that normally do not express mCD14 (20). Hayashi et al (9) indicated that although human gingival fibro- blasts (HGF) expressed neither CD14 mRNA nor mCD14 on the cell surface, HGF were activated with LPS in a manner dependent on serum-derived sCD14 molecules. In contrast, Watanabe et al (36) reported mCD14 expression by HGF.…”

“…Hayashi et al (9) reported that the induction of intercellular adhesion molecule 1 by LPS on human gingival fibroblasts is mediated by serum-derived sCD14. To clarify the participation of sCD14 in FBS in the enhanced production of VEGF by HPC stimulated with LPS, we added neutralizing anti-CD14 MAb MY4 to HPC cultures stimulated with LPS.…”