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-alpha (TNF-alpha) 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-kappaB is associated in the signalling process. To define the mechanism of TNF-alpha 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-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) or NF-kappaB 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-alpha (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-alpha by anionic pIgA in HMC was significantly diminished (P<0.01) in the presence of NF-kappaB inhibitor pyrrolidine dithiocarbamate and NF-kappaB blocking permeable peptides SN50 (P<0.01). The increased synthesis of IL-6 by anionic pIgA in HMC was reduced by inhibitor to NF-kappaB or p42/p44 MAPK and was abolished by the simultaneous presence of inhibitors to p42/p44 MAPK and NF-kappaB. The up-regulation of TNF-alpha was partially suppressed by inhibitor to NF-kappaB but not PD98059.
Conclusion: Our results suggest that polymeric anionic IgA1 could activate HMC and increase the synthesis of TNF-alpha and IL-6. While both the p42/p44 MAPK and NF-kappaB pathways are essential in regulating the anionic pIgA-induced synthesis of IL-6, TNF-alpha synthesis mediated by anionic pIgA is partly dependent on NF-kappaB.