Transcriptional activation of mRNA of intercellular adhesion molecule 1 and induction of its cell surface expression in normal human gingival fibroblasts by Mycoplasma salivarium and Mycoplasma fermentans

Abstract

Lipoproteins in the cell membranes of both Mycoplasma salivarium and Mycoplasma fermentans were demonstrated to trigger the transcription of intercellular adhesion molecule-1 mRNA in normal fibroblasts isolated from human gingival tissue and to induce its cell surface expression by a mechanism distinct from that of Escherichia coli lipopolysaccharide. The lipid moiety of the lipoproteins was suggested to play a key role in the expression of the activity.

FIG. 1

Analysis of expression of mRNAs of β-actin, ICAM-1, and VCAM-1 in Gin-1 cells stimulated with CM of M. salivarium (salm) or M. fermentans (ferm). The confluent monolayers of Gin-1 cells in 3.5-cm-diameter culture dishes were incubated at 37°C for 6 h in the absence (None) or the presence of CM of M. salivarium (salm) or M. fermentans (ferm) at a protein concentration of 40 μg/ml. The RNAs were prepared from Gin-1 cells and analyzed by RT-PCR.

FIG. 2

Immunostaining of ICAM-1 expressed on the cell surface of Gin-1 cells stimulated with cell membranes of M. salivarium. Gin-1 cells were incubated at 37°C for 6 h in the presence (CM) or the absence (None) of cell membranes of M. salivarium. The cells were fixed in PBS containing 20% (vol/vol) acetone and 10% (wt/vol) saccharose. The fixed cells were reacted with monoclonal antibodies to ICAM-1 (BBIG-I1) and with VECTOR-ABC and -VIP kits. The stimulated cells were stained (CM), but the nonstimulated cells were not stained and were therefore visualized by differential interference microscopy (None). Magnification, ×300.

FIG. 3

Effect of polymyxin B on cell surface expression of ICAM-1 on Gin-1 cells stimulated with Lpsal or E. coli LPS. Gin-1 cells were grown in DME medium until they reached confluency. The confluent monolayers of Gin-1 cells were preincubated for 37°C for 1 h with polymyxin B (5,000 or 10,000 U/ml) in the presence of Lpsal (50 μg of protein/ml [■]) or E. coli LPS (1 μg/ml [□]) and then examined for cell surface expression of ICAM-1 on Gin-1 cells by Cell-ELISA.

FIG. 4

Analysis of expression of mRNAs of β-actin and ICAM-1 in Gin-F and PDL-F as well as Gin-1 cells stimulated with Lpsal. The confluent monolayers of Gin-F and PDL-F as well as Gin-1 cells in 3.5-cm-diameter culture dishes were incubated at 37°C for 6 h in the absence (None) or the presence of Lpsal at a protein concentration of 60 μg/ml. The expression of mRNAs of β-actin or ICAM-1 was analyzed by RT-PCR.