NDT Advance Access originally published online on September 26, 2007
Nephrology Dialysis Transplantation 2008 23(1):72-81; doi:10.1093/ndt/gfm581
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Synthesis of TNF-
by mesangial cells cultured with polymeric anionic IgA—role of MAPK and NF-
B
Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong
Correspondence to: Prof. Kar Neng Lai, Department of Medicine, University of Hong Kong, Room 409, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. Email: knlai{at}hkucc.hku.hk
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Abstract |
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Background. Deposition of polymeric IgA1 (pIgA) in kidney mesangium is the hallmark of IgA nephropathy (IgAN). Current consensus is that a fraction of IgA1 molecules in the circulation of IgAN patients exhibit aberrant structures or properties that may lead to their deposition. Our previous findings suggest that the anionic property of IgA1 may play a role in mesangial IgA1 deposition in patients with IgAN. In the present study, the functional consequences of the binding of anionic polymeric IgA1 to human mesangial cells (HMCs) were investigated.
Methods. Anionic polymeric IgA1 from IgAN patients and healthy subjects was isolated by sequential jacalin affinity chromatography, size exclusion chromatography using size exclusion and MonoQ ion exchange chromatography. HMCs were cultured with purified anionic polymeric IgA1 and the release of tumour necrosis factor-
(TNF-) and interleukin-6 (IL-6) was examined by enzyme-linked immunosorbent assay. The signalling pathways involved in anionic pIgA-mediated HMC activation were examined by immunoblotting. Standard electrophoretic mobility shift assay (EMSA) was used to further examine whether the transcriptional factor NF-
B is associated in the signalling process. To define the mechanism of TNF- and IL-6 production, HMCs were cultured with anionic pIgA in the presence or absence of p42/p44 mitogen-activated protein kinase (MAPK) inhibitor (PD98059), NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) or NF-B blocking permeable peptides.
Results. Compared with less anionic pIgA or monomeric IgA1 (mIgA), anionic pIgA from patient with IgAN significantly increased cell proliferation (P < 0.05) when cultured with HMC. These anionic pIgA significantly increased the synthesis of TNF- (P < 0.05) and IL-6 (P < 0.05) in a dose and time-dependent manner. Furthermore, the increased synthesis of IL-6 and TNF- by anionic pIgA in HMC was significantly diminished (P < 0.01) in the presence of NF-B inhibitor pyrrolidine dithiocarbamate and NF-B blocking permeable peptides SN50 (P < 0.01). The increased synthesis of IL-6 by anionic pIgA in HMC was reduced by inhibitor to NF-B or p42/p44 MAPK and was abolished by the simultaneous presence of inhibitors to p42/p44 MAPK and NF-B. The up-regulation of TNF- was partially suppressed by inhibitor to NF-B but not PD98059.
Conclusion. Our results suggest that polymeric anionic IgA1 could activate HMC and increase the synthesis of TNF- and IL-6. While both the p42/p44 MAPK and NF-B pathways are essential in regulating the anionic pIgA-induced synthesis of IL-6, TNF- synthesis mediated by anionic pIgA is partly dependent on NF-B.
Keywords: anionic IgA; human mesangial cell; IgA nephropathy; polymeric IgA
Received for publication: 14. 3.07
Accepted in revised form: 31. 7.07
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