Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells

Abstract

Background: Both interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic cytokines that have been implicated in the development of glomerular and tubular injury in various forms of immune-mediated renal disease, including glomerulonephritis. Although TNF-alpha has been shown to stimulate IL-6 production in renal cells in culture, the signaling mechanisms that regulate IL-6 production are not fully understood. The aim of this study was to examine the role of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways in regulating TNF-alpha-mediated IL-6 production from both primary human mesangial cells (HMCs) and human proximal tubular (HPT) cells.

Methods: Primary mesangial and proximal tubular cells were prepared from nephrectomized human kidney tissue. Cells were treated for 24 hours with TNF-alpha in the presence and absence of the specific p38 and ERK1,2 MAPK inhibitors SB203580 and PD98059, respectively, either alone or in combination. IL-6 levels in the cell culture media were measured by enzyme-linked immunosorbent assay. MAPK activation was demonstrated by immunoblot for the active kinase (tyrosine/threonine phosphorylated) in whole cell extracts using phospho-specific antibodies. p38 MAPK activity in HPT cells was measured using an in vitro immunokinase assay using ATF2 as the substrate.

Results: TNF-alpha (0.1 to 100 ng/ml) stimulated a dose-dependent increase in IL-6 production in both renal cell types. The activation of the p38 and the ERK1,2 MAPKs occurred following TNF-alpha stimulation. The role of these activations in IL-6 production was confirmed by the ability of both inhibitors SB203580 (1 to 30 microM) and PD98059 (0.01 to 10 microM) to inhibit basal and TNF-alpha-stimulated IL-6 production in both cell types. The addition of both inhibitors in combination caused greater decreases in IL-6 production compared with either inhibitor alone. Pretreatment with SB203580 (10 microM) had no effect on basal or TNF-alpha-stimulated phosphorylation of p38 MAPK but completely abolished TNF-alpha-stimulated p38 MAPK activity. PD98059 decreased both basal and TNF-alpha-stimulated phosphorylation of ERK1,2.

Conclusions: This study provides evidence that both the p38 and ERK MAPK pathways are important for the regulation of the production of IL-6 from the proximal tubular and glomerular mesangial regions of the nephron. In response to TNF-alpha, the activation of both pathways leads to IL-6 production. These findings could aid in an understanding of the cellular mechanisms that regulate IL-6 production and could provide insights into possible pharmacological strategies in inflammatory renal disease.