THP-1 monocytes up-regulate intercellular adhesion molecule 1 in response to pneumolysin from Streptococcus pneumoniae

Abstract

Pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae that elicits a variety of proinflammatory responses from cells of the host immune system. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli in extravascular sites. In this study, we evaluated the effect of PLY on expression of ICAM-1 in THP-1 monocytic cells exposed to S. pneumoniae. Exposure of cells to PLY-expressing S. pneumoniae strain WU2 for 6 h led to significantly higher levels of ICAM-1 message than those in cells exposed to either medium alone or DeltaPLY1, a PLY-negative isogenic mutant of WU2. Cells exposed to purified recombinant PLY also showed a dose-dependent increase in ICAM-1 mRNA compared to cells exposed to medium alone. Exposure to recombinant PLY containing a single amino acid substitution (Trp433-->Phe) that decreases cytolytic activity did not increase ICAM-1 mRNA to levels seen with wild-type PLY. In addition, THP-1 cells exposed to wild-type strain WU2 or D39 had increased ICAM-1 on their surface compared to cells exposed to medium alone or their PLY-negative isogenic mutants DeltaPLY1 and DeltaPLY2, respectively. These data indicate that PLY induces transcription and production of a cell adhesion molecule involved in the inflammatory response that may play a role in pneumococcal infection.

FIG. 1.

Real-time analysis of ICAM-1 message levels in THP-1 cells exposed to S. pneumoniae strain WU2 (black bars) and its PLY-negative isogenic mutant, ΔPLY1 (gray bars). THP-1 cells were exposed to either medium alone, WU2, or ΔPLY1 for 1, 3, and 6 h of incubation. Data represent fold induction relative to cells receiving medium alone. The experiment was repeated three times with each quantitative PCR being done in triplicate. Bars represent the means of all experiments with error bars representing standard errors of the means. Statistical analysis was performed by using analysis of variance with Tukey's correction for multiple comparisons (P < 0.05). *, significantly different from exposure to ΔPLY1 and medium alone.

FIG. 2.

Increased expression of ICAM-1 protein on the surface of THP-1 cells exposed to S. pneumoniae. THP-1 cells were exposed for 10 h to either medium alone (Mock), D39, or PLY-deficient ΔPLY2 (A) or medium alone (Mock), WU2, or ΔPLY1 (B). Dotted line, mock; thick line, wild type; thin line, PLY mutant. At 3 h for D39 and ΔPLY2 and at 4.5 h for WU2 and ΔPLY1, chloramphenicol (2 μg/ml) was added to slow bacterial growth to allow time for protein expression. Mock controls were also treated with chloramphenicol. Representative data are shown as fluorescence intensity of the total cell population, and the geometric mean of each peak is indicated.

FIG. 3.

Real-time analysis of ICAM-1 message levels in THP-1 cells exposed to purified recombinant PLY and ΨPLY. THP-1 cells were exposed for 6 h to recombinant PLY in RPMI containing (white bars) or lacking (black bars) 10% FBS or to recombinant ΨPLY protein in RPMI lacking 10% FBS (gray bars) at concentrations of 200 ng/ml, 500 ng/ml, and 1,000 ng/ml. Data represent fold induction relative to that of cells receiving medium alone. The experiment was repeated three times with each quantitative PCR being done in triplicate. Bars represent the means of all experiments with error bars representing standard errors of the means.