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VIP Inhibits Porphyromonas gingivalis LPS-induced Immune Responses in Human Monocytes

Oral Microbiology and Host Responses Group, School of Dental Sciences, University of Newcastle Upon Tyne, NE2 4BW, UK;

View larger version (14K): [in a new window]   Figure 1. CD14 expression and flow cytometric analysis of VitD3-treated THP1 cells. (A) FACS analysis of CD14 expression on THP1 surface; arrows show CD14 expression on the surfaces of pro-monocytic THP1 cells and VitD3-treated monocytic THP1, compared with an isotype control. The histogram is representative of results repeated on at least 5 separate occasions. (B) Population of cells gated prior to FACS analysis (R1) with characteristics of high forward scatter (FSC) (largest cells in population) and low (< 102) side-scatter (non-granular cells).

View larger version (19K): [in a new window]   Figure 2. TNF- production by THP1 monocytes. (A) Cells were incubated in medium alone or stimulated with increasing concentrations of Pg LPS for 6 and 24 hrs. All treatments induced significant increases (P < 0.05) in TNF- production compared with that in unstimulated (control) cells. (B) LPS (100 ng/mL)-stimulated cells incubated with and without VIP (10–8 M). TNF- levels were measured by ELISA and compared with unstimulated (control) cells or cells stimulated with phorbol myristate acetate (positive control). Data are presented as means ± SD (n = 3). *P < 0.05. Similar data were obtained from 3 separate experiments.

View larger version (83K): [in a new window]   Figure 3. Subcellular location of transcription factors (NFB or c-Jun) in THP1 monocytes. Cells were analyzed after incubation in medium alone (A,D), following treatment with 100 ng/mL Pg W50 LPS (B,E), and with 100 ng/mL Pg LPS with VIP (10–8 M).(C,F) Fluorescently labeled antibodies were used to detect NFB (green fluorescence) and c-Jun (red fluorescence), and sections were analyzed by confocal laser scanning microscopy. Nuclei were counter-stained with DAPI (blue fluorescence). Each image was taken between 6 and 8 µm through the cells, so that the nuclei could be clearly determined. Scale bar in bottom left corner = 30 µm. Each result was replicated on at least 6 separate occasions.

View larger version (16K): [in a new window]   Figure 4. CD14 expression on THP-1 monocytes following exposure to LPS. (A) Isotype control (IgG2a. PE). (B) Unstimulated THP1 cells (negative control). (C) THP1 cells stimulated with Pg LPS (100 ng/mL) for 6 hrs. (D) THP1 cells stimulated with LPS and VIP (10–8 M). CD14 expression was determined by flow cytometry. The upper-right-quadrant highly granular cells express CD14, and the upper-left-quadrant low-granular cells express CD14. The figures in the dot-blots represent the proportion of the total cell number in each of these upper quadrants. This experiment is representative of similar results obtained on at least 8 separate occasions.