Effects of anti-DNA antibodies on pleural mesothelial cells : in vitro studies to explore the pathogenetic mechanism of pulmonary lupus
Effects of Anti-DNA Antibodies on Pleural Mesothelial Cells: in vitro Studies to Explore the Pathogenetic Mechanism of Pulmonary Lupus
For the degree of Master of Philosophy at the University of Hong Kong
in August 2003
Pulmonary abnormalities, including pleuritis, are common in SLE. The pathogenic effects of autoantibodies in pulmonary lupus remain unclear. This study has investigated in vitro the pathophysiologic effect of anti-DNA containing IgG from lupus patients on pleural mesothelial cells and the related mechanisms. Our in vivo study has explored the pulmonary expression of inflammatory cytokines in lupus-prone mice and has evaluated the potential therapeutic effect of mycophenolate mofetil (MMF) on the release of cytokines.
In in vitro experiments, IgG from 28 lupus patients and 13 healthy controls was purified by protein-G affinity chromatography. The amount of anti-dsDNA, anti-histone and anti-nucleohistone IgG were determined with ELISA. The lupus patients were divided into an active or an inactive group based on the SLE Disease Activity Index. The binding oflgG to a human pleural mesothelial cell-line (MeT-5A) under different conditions including pretreatment with DNase, pre-incubation with exogenous histone, DNA or nucleohistone was examined using flow cytometry and cellular ELISA. Gene expression and protein synthesis for IL-l f3, MCP-l and
TGF-f31 in MeT-5A cells were determined using RT-PCR and ELISA.
We demonstrated that the binding oflgG to MeT-5A cells was higher in the active lupus group than that in the inactive lupus group or controls. The binding decreased in the lupus group following the pretreatment of MeT-5A cells with DNase. The binding of IgG to MeT-5A cells was increased by 112% in the active lupus group following pre-incubation with histone, but not with DNA or nucleohistone. Gene expression and protein synthesis of MCP-1, TGF-f31, and IL-1f3 in MeT-5A cells were significantly enhanced following incubation with IgG from patients with active lupus when compared with the inactive lupus and control group. The concentration ofanti-dsDNA antibodies correlated with the binding ofIgG to MeT-5A cells as well as with cytokine synthesis by MeT-5A cells. In addition, the anti-histone antibody in the active lupus group was higher than that in the inactive lupus group and correlated with the cell binding or the synthesis ofMCP-l.
In our in vivo study, eight-week-01d MRL/1pr mice were treated with MMF in vehicle for 8 or 12 weeks, while the control MRL/1pr and MRL mice received vehicle alone. The synthesis of MCP-1, TGF-f31, and IL-1f3 in the lungs were determined by R T - PCR and Western blot. We observed that gene expression of cytokines was significantly increased in MRL/1pr lupus mice as compared with the MRL controls. Gene expression of these cytokines and protein synthesis of TGF-f31 in the lung of MMF-treated MRL/1pr mice were significantly reduced following 8 or 12 weeks of treatment as compared with the untreated MRL/1pr mice.
In summary, we have demonstrated that the in vitro binding of autoantibodies from active lupus patients to MeT -5A cells is closely associated with the disease activity. The binding of antibodies to MeT -5A cells is mediated via DNA and histone.
Anti-dsDNA antibodies exert a direct pathogenic effect in lupus pleuritis by binding directly to the pleural mesothelial cells and inducing the synthesis of proinflammatory cytokines. We have also provided in vivo evidence that the pathogenesis of pulmonary lupus is associated with the over-production of inflammatory cytokines. MMF has a beneficial role in lupus by inhibiting the production of intra-pulmonary inflammatory cytokines.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:dna antibodies lupus genetic aspects cytokines immunosuppressive agents
Date of Publication:01/01/2003