CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Maurizio Cutolo, Stefano Soldano, Paola Montagna, Alberto Sulli, Bruno Seriolo, Barbara Villaggio, Pierfranco Triolo, Paolo Clerico, Lamberto Felli, Renata Brizzolara, Maurizio Cutolo, Stefano Soldano, Paola Montagna, Alberto Sulli, Bruno Seriolo, Barbara Villaggio, Pierfranco Triolo, Paolo Clerico, Lamberto Felli, Renata Brizzolara

Abstract

Introduction: Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.

Methods: All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 microg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) alpha, IL-1beta, transforming growth factor (TGF) beta) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.

Results: Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 microg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFalpha, IL-1beta and TGFbeta). Data were confirmed by WB and RT-PCR analysis.

Conclusions: Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNFalpha, IL1-beta and TGFbeta. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.

Figures

Figure 1
Figure 1
B7.2 expression on macrophages by immunofluorescence analysis. B7.2 expression by immunofluorescence analysis (a) on primary cultures of rheumatoid arthritis synovial macrophages (RA SM) untreated, (b) on macrophages differentiated from THP-1 untreated, (c) on SM pre-treated with lipopolysaccharide and (d) on EBV+ B lymphocytes (positive control cell line).
Figure 2
Figure 2
B7.2 expression in macrophages after CTLA4-IG treatment by immunofluorescence analysis. B7.2 expression by immunofluorescence analysis on primary cultures of rheumatoid arthritis synovial macrophages (RA SM) (a) untreated, (b) treated with CTLA4-Ig 10 μg/ml, (c) treated with CTLA4-Ig 100 μg/ml and (d) treated with CTLA4-Ig 500 μg/ml; (e) on macrophages differentiated from THP-1 untreated, (f) treated with CTLA4-Ig 10 μg/ml, (g) CTLA4-Ig 100 μg/ml and (h) CTLA4-Ig 500 μg/ml.
Figure 3
Figure 3
Immunocytochemistry for IL-6, TNFα, IL-1β and TGFβ expression in co-cultures of macrophage/Jurkat. Immunocytochemistry in co-cultures of macrophage/Jurkat, (a) untreated (controls) and (a1) treated with CTLA4-Ig 500 μg/ml for IL-6, (b) untreated (controls) and (b1) treated with TNFalpha, (c) untreated (controls) and (c1) treated with IL-1β and (d) untreated (controls) and (d1) treated with TGFβ. Quantification (mean value ± standard deviation) of immunocytochemistry (ICC-QWin) for (e) IL-6, (f) TNFα, (g) IL-1β and (h) TGFβ expression in co-cultures of macrophage/Jurkat, untreated (controls) and treated with CTLA4-Ig (10, 100, 500 μg/ml). * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 4
Figure 4
Evaluation of cytokine production by ELISA assay. Evaluation by ELISA assay of IL-6 and IL-1β production in supernatants of co-cultured macrophage/Jurkat untreated (controls (cnt)) and treated with CTLA4-Ig (10, 100, 500 μg/ml). Results are expressed as mean value ± standard deviation from three experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 5
Figure 5
Reverse transcriptase-polymerase chain reaction and western blot analysis for IL-6, TNFα and TGFβ expression in co-cultures of macrophage/Jurkat. Reverse transcriptase-polymerase chain reaction assay in co-cultures of macrophage/Jurkat, untreated (line 1: control) and treated with CTLA4-Ig 10 μg/ml (line 2), CTLA4-Ig 100 μg/ml (line 3), CTLA4-Ig 500 μg/ml (line 4), for (a) β-actin (internal positive control), (b) IL-6, (c) TNFα and (d) TGFβ. Line 5: molecular weight. Densitometry analysis of western blot in co-cultures of macrophage/Jurkat, untreated (line 2: control) and treated with CTLA4-Ig 10 μg/ml (line 3), CTLA4-Ig 100 μg/ml (line 4), CTLA4-Ig 500 μg/ml (line 5) for (e) IL-6 and (f) TNFα protein expression in co-cultures of macrophage/Jurkat. Line 1: molecular weight.
Figure 6
Figure 6
Immunocytochemistry for IL-6 and TNFα expression in co-cultures of RA SM/Jurkat. Immunocytochemistry in co-cultures of rheumatoid arthritis synovial macrophages (RA SM)/Jurkat, (a) untreated (controls) and (a1) treated with CTLA4-Ig 500 μg/ml for IL-6 and (b) untreated (controls) and (b1) treated with CTLA4-Ig 500 μg/ml for TNFα. Quantification (mean value ± standard deviation) of immunocytochemistry (ICC-QWin) for (c) IL-6 and (d) TNFα. * P < 0.05; ** P < 0.01; *** P < 0.001.

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