Differential cytokine patterns in mouse macrophages and gingival fibroblasts after stimulation with porphyromonas gingivalis or Escherichia coli lipopolysaccharide

Abstract

Background: A major cause of chronic inflammatory periodontal disease is Porphyromonas gingivalis, a non-motile, Gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known.

Methods: The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli. The expression of immune response molecules was quantified by real-time polymerase chain reaction and enzyme-linked immunoassay.

Results: AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide, and monocyte chemotactic protein-1 expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1β, IL-6, and monocyte chemotactic protein-1, relative to stimulation by E. coli LPS.

Conclusion: These findings demonstrate that E. coli LPS induces a stronger cytokine and chemokine response in gingival fibroblasts, whereas P. gingivalis LPS induces a stronger response in macrophages.

Figure 1

Phenotypic characterization of ESK-1 cells. ESK-1 cells were non-reactive with (A) G8.8 mAb but were highly-reactive with (B-C) anti-vimentin antibody, indicating fibroblast origin of those cells. The lack of expression of the (D) CD45-LCA antigen indicates that ESK-1 cells were not of hematopoietic origin. ESK-1 cells were further confirmed by flow cytometry to be fibroblasts based (E) a lack of G8.8 staining, and (F) reactivity with anti-vimentin antibody. (G) Two-thirds of the ESK-1 cells expressed the CD14 co-receptor of TLR4, and also expressed the (H) TLR2 marker. AM cells expressed (I) CD45-LCA, (K) vimentin, (L) CD14, and (M) TLR2, but did not express (J) G8.8.

Figure 2

EIA analyses of IL-6 and MCP-1 production by AM and ESK-1 cells confirm, at the protein level, the differential effects of P. gingivalis vs. E. coli LPS stimulation as shown in the gene expression experiments described in Table 2 and 3.