CD40 ligation stimulates MCP-1 and IL-8 production, TRAF6 recruitment, and MAPK activation in proximal tubule cells

Abstract

The mechanism of CD40/CD154-induced chemokine production and its potential role in renal inflammatory disease were explored. Human proximal tubule cells maintained in primary culture were used as the experimental model. With the use of immunocytochemistry, confocal microscopy, and a cell fractionation assay, the CD40 receptor was found to be expressed in the cell membrane of the epithelial cell, and, on engagement by CD154, its cognate ligand, translocated to the cytoplasmic compartment. Engagement of CD40 by CD154 stimulated interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production, which proceeded via receptor activation of the extracellular signal-regulated kinase (ERK)1/2, stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways. CD40 ligation also engaged tumor necrosis factor receptor-activating factor 6 (TRAF6), as evidenced by colocalization of the activated receptor with TRAF6 in the cytoplasmic compartment, translocation of both proteins from the insoluble to the soluble cell fraction, and coimmunoprecipitation of the two proteins only under ligand-stimulated conditions. Furthermore, an antisense oligodeoxyribonucleotide targeted against TRAF6 mRNA blunted p38 and SAPK/JNK but not ERK1/2 MAPK activities, as well as IL-8 and MCP-1 production, arguing that TRAF6 is an upstream activator. The zinc chelator TPEN, but not the calcium chelator BAPTA, obliterated CD154-evoked MAPK activity and chemokine production, providing indirect evidence for protein-protein interactions playing a critical role in CD40 signaling in these cells. We conclude that in human proximal tubule cells, CD40 and TRAF6 reside in separate low-density, detergent-insoluble membrane microdomains, or rafts, and on activation translocate and associate with one another probably via zinc-finger domains in the soluble or cytoplasmic compartment. TRAF6, in turn, activates SAPK/JNK and p38 MAPK phosphorylation, which in turn stimulates IL-8 and MCP-1 production in these cells.

Protective effect of baicalein on renal ischemia/reperfusion injury in the rat

“…Oxygen free radicals, cellular adhesion molecules and inflammatory cell infiltration are thought to contribute to the pathogenicity of RIRI, [2][3][4] and Bai has previously been implicated in antioxidant and free radical scavenging activities. [4][5][6][7][8] Oxygen free radicals can not only directly inhibit the reperfusion of renal tissue; but more importantly, they can induce and amplify inflammatory responses via oxidative stress, thereby increasing renal injury and apoptosis.…”

“…The mechanisms underlying the acute tubular necrosis induced by renal ischemia-reperfusion have not yet been fully elucidated. To date, no drugs have been demonstrated to effectively prevent or alleviate AKI, 1 however the role of oxygen free radicals, inflammatory cytokines (including intercellular adhesion molecule-1, ICAM-1; and monocyte chemoattractant protein-1, MCP-1) 2,3 and inflammatory cell infiltration 4 in the development of RIRI has been recognized. Baicalein (Bai, 5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, has demonstrated anti-oxidant and anti-inflammatory effects.…”

CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island

“…The expression of CD40 is prominent on many cell types in pathological processes known to be associated with high levels of VEGF, including renal EC and tubular epithelial cells at sites of inflammation (16,37,51,54,66). CD40L-CD40 interactions result in several intracellular signals in EC that Fig.…”

“…CD40, a type I transmembrane-glycoprotein member of the tumor necrosis factor receptor (TNF-R) gene family, is expressed by many cell types, including renal epithelial cells and EC (1,24,37,65,66,68). CD40L is expressed as a cell-surface molecule by activated CD4 ϩ T cells and platelets (23,47) and also exists in plasma in a soluble circulating form of unknown function.…”

How epithelial cells detect danger: aiding the immune response

“…The cytoplasmic TIR domain of the receptor forms a platform for the recruitment and activation of the adaptor molecule MyD88 and the kinases of the IL-1R-associated kinase (IRAK) family (23)(24)(25). Following IRAK-4 autophosphorylation and activation of the IRAK-1/2-complex (26), the TRAF6 adaptor protein interacts with a second kinase complex with TAK1 that ultimately leads to the inactivation of the NF-jB inhibitor IjB (27)(28)(29)(30). After activation of NF-jB, the transcription factor is no longer retained in the cytoplasm and is able to translocate to the nucleus where it activates transcription of a variety of cytokines/ chemokines (TNF-a, CXCL10, IFN-c and IL-1, -6, -8, -10 and -12) and other genes like COX-2 and SOCS.…”