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. 2016 May;13(5):3886-94.
doi: 10.3892/mmr.2016.5012. Epub 2016 Mar 18.

IL‑6 and IL‑8 enhance factor H binding to the cell membranes

Sylwia Popek  1 , Lucyna Kapka-Skrzypczak  2 , Krzysztof Sawicki  1 , Ewa Wolińska  3 , Maciej Skrzypczak  4 , Magdalena Czajka  1
Affiliations

IL‑6 and IL‑8 enhance factor H binding to the cell membranes

Sylwia Popek et al. Mol Med Rep. 2016 May.

Abstract

The aim of the present study was to assess the role of interleukin (IL)‑6 and IL‑8 on the expression of fluid‑phase complement inhibitor, factor H (FH), and FH‑like protein 1 (FHL‑1), in the A2780 ovarian carcinoma cell line. This cell line does not normally produce IL‑6, however, is IL‑6 responsive due to the presence of receptor for IL‑6. The presence of FH and FHL‑1 in the cell lysates was confirmed by western blotting. The levels of FH and FHL‑1 in the medium were determined by enzyme‑linked immunosorbent assay. To evaluate gene expression, reverse transcription‑quantitative polymerase chain reaction was performed. The cellular localization of FH and FHL‑1 in ovarian cancer cells was assessed by immunofluorescence. The present study revealed that FH, contrary to FHL‑1, was secreted by ovarian cancer cells, however, this process was independent of IL stimulation. No significant differences were observed in the concentration of FH in the control cells, when compared with the samples treated with IL‑6/IL‑8. The results of western blotting revealed that the protein expression levels of FH and FHL‑1 were not regulated by IL‑6 and IL‑8 in a dose‑dependent manner. Immunofluorescence analysis confirmed that the A2780 ovarian cancer cell line expressed both membrane bound and intracellular forms of FH and FHL‑1. The present data revealed that the A2780 cells expressed and secreted FH protein and are also able to bind FH and FHL‑1. This may influence the efficiency of complement mediated immunotherapy.

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Figures

Figure 1
Figure 1
Secretion of FH by A2780 ovarian cancer cells following stimulation with various concentrations of (A) IL-6 and (B) IL-6/IL-8. The results are presented as a percentage of the control untreated cells and the data are presented as the mean ± standard deviation (n=3). FH, factor H; IL, interleukin.
Figure 2
Figure 2
Western blot analysis of the expression levels of FH following incubation with various concentrations of (A) IL-6 and (B) IL-6/IL-8. The graphs show the densitometric analysis, normalized against β-actin. The data are presented as a percentage of the control untreated cells and the data are presented as the mean ± standard deviation (n=3). (C) Representative western blot of FH and β-actin proteins following stimulation with IL-6 (left) or IL-6/IL-8 (right). FH, factor H; IL, interleukin.
Figure 3
Figure 3
Western blot analysis of the expression of FHL-1 following incubation with various concentrations of (A) IL-6 and (B) IL-6/IL-8. The graphs show the densitometric analysis, normalized against β-actin. The data are presented as a percentage of the control untreated cells and the data are presented as the mean ± standard deviation (n=3). (C) Representative western blot of FHL-1 and β-actin proteins following stimulation with IL-6 (left) or IL-6/IL-8 (right). FHL-1, factor H-like protein 1; IL, interleukin.
Figure 4
Figure 4
Quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) FH and (B) FHL-1. The graph presents the fold change calculated for the samples incubated with IL-6 and IL-6/IL-8. Sample without interleukin treatment was used as an untreated control and β-actin was used as a reference gene for normalization. The data are presented as the fold change against the control untreated samples and the data are presented as the mean ± standard deviation (n=3). CFH, complement factor H; CFHR, CFH-related protein 1; IL, interleukin.
Figure 5
Figure 5
Immunofluorescence staining of factor H protein in the A2780 ovarian cancer cell line after 24 h incubation with various concentrations of IL-6. Representative images of cells treated with (A) 0, (B) 1, (C) 10 and (D) 100 ng/ml IL-6 (magnification, ×400). IL, interleukin.
Figure 6
Figure 6
Immunofluorescence staining of factor H protein in the A2780 ovarian cancer cell line after 24 h incubation with various concentrations of IL-6/IL-8. Representative images of cells treated with (A) 0, (B) 1, (C) 10 and (D) 100 ng/ml IL-6 and IL-8 (magnification, ×400). IL, interleukin.
Figure 7
Figure 7
Immunofluorescence staining of factor H-like protein 1 in the A2780 ovarian cancer cell line after 24 h incubation with various concentrations of IL-6. Representative images of cells treated with (A) 0, (B) 1, (C) 10 and (D) 100 ng/ml IL-6 (magnification, ×400). IL, interleukin.
Figure 8
Figure 8
Immunofluorescence staining of factor H-like protein 1 in the A2780 ovarian cancer cell line after 24 h incubation with various concentrations of IL-6/IL-8. Representative images of cells treated with (A) 0, (B) 1, (C) 10, (D) 100 ng/ml IL-6 and IL-8, and (E) control with goat immunoglobulin G. (magnification, ×400). IL, interleukin.
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