Characterization of the response of GM-CSF supplemented THP-1 human monocytes to LPS of oral microorganisms

Abstract

The effect of granulocyte macrophage colony stimulating factor (GM-CSF) on the differentiation and activation of a human monocyte cell line (THP-1) in the presence of lipopolysaccharide (LPS) from oral organisms has not been investigated. It was hypothesized that GM-CSF treated THP-1 cells are immunologically and functionally hyperactivated in the presence of LPS of oral microorganisms. A study was undertaken to elucidate the immunological expression of activation antigens and production of cytokines by THP-1 cells after treatment with GM-CSF and in response to LPS of the putative periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). LPS of F. nucleatum and P. gingivalis was prepared, characterized by SDS PAGE, standardized by protein concentration and tested for endotoxin content. Morphological changes in THP-1 cells were observed with light, immunofluorescence (IF) and transmission electron microscopy (TEM), following treatment with LPS/PMA (phorbol-12-myristate-13 acetate) and/or GM-CSF at various concentrations and time intervals. The expression of seven different activation antigens namely, CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR, in THP-1 cells was evaluated in this experimental model. Direct labeling of THP-1 cell activation antigens was performed using the Vectastain ABC-AP staining kit with light microscopy. Alternatively, LPS/PMA and/or GM-CSF stimulated and unstimulated cells were stained with FITC labelled antibodies for IF and gold-labelled antibodies were used for TEM. To evaluate chemotaxis as a functional variation in THP-1 cells, an assay was performed using a microchemotaxis chamber and polyvinylpyrolidone free polycarbonate membrane filters. To determine phagocytic activity in the experimental model, an assay was performed using FITC labelled S. cereviseae. Phagocytic uptake of cells was determined with the use of a fluorescence microscope. Production of extracellular cytokines, TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12, by THP-1 cells in the experimental model, was measured by ELISA. Reverse transcription polymerase chain reaction (RT-PCR) of the of TNF-alpha, IL-1 beta and IL-6 cytokines was performed to correlate the cytokine gene transcription with cytokine gene translation (ELISA). Three percent of untreated THP-1 cells expressed HLA-DR; 9%, CD-11b; 8%, CD-11c; 22%, CD-14; 9%, CD-35 and 7%, CD-68 antigens. CD-71 was not expressed in untreated THP-1 cells. Treatment with LPS of F. nucleatum and P. gingivalis and PMA increased the expression of activation antigens. Following treatment with combined GM-CSF and LPS of P. gingivalis and F. nucleatum there was a significant (p<0.05) up-regulation and expression of HLA-DR, CD-11b, CD-11c, CD-35 and CD-71 activation antigens over baseline values. Expression of the LPS receptor, CD-14, was significantly (p<0.05) down-regulated by this treatment for 1-2 d and then up-regulated at 2-4 d. Antigens important in phagocytosis, CD-11b and CD-35, were significantly (p<0.05) up-regulated by GM-CSF. The up-regulation was further demonstrated in phagocytosis functional assays. Stimulation with combined GM-CSF and oral LPS resulted in a significant (p<0.05) escalation in phagocytosis by the THP-1 cells. There was a two-fold increase in chemotactic response with GM-CSF treatment by 4 d, which decreased after 7 d. RT-PCR data indicated that TNF-alpha transcripts were constituitively produced in the THP-1 cell but that translation to a high level of production of functional cytokines required the LPS/GM-CSF stimulus. Gene transcription for IL-6 was detected as early as 5 min post stimulation. (Abstract shortened by UMI.)