CD40 signaling in human dendritic cells is initiated within membrane rafts

Abstract

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.

Fig. 1. Lyn tyrosine phosphorylation following CD40 engagement occurs in membrane rafts. (A) Monocyte-derived DCs were stimulated for 10 min with either an irrelevant mouse IgG1 (–) or with anti-CD40 antibody (+), and analyzed as indicated: IP, immunoprecipitation; blot, immunoblotting. Molecular mass markers (kDa) are shown on the left. (B) Membrane rafts of DCs were separated on a bottom-loaded sucrose step gradient. The distribution of CD55, CD46, Lyn and Syk proteins within fractions (fraction 1 represents the top of the gradient) was analyzed by immunoblotting (equal volume loaded) as indicated. (C) Membrane rafts from unstimulated (IgG) or CD40-stimulated (α-CD40) DCs were fractionated as above and pTyr events were detected within each fraction by western blotting. Molecular mass markers (kDa) are shown on the right. (D) Pooled raft or soluble fractions from DCs, stimulated with either IgG control (–) or anti-CD40 antibody (+), were immunoprecipitated with anti-pTyr antibody and eluates were immunoblotted with either anti-Lyn (p-Lyn) or anti-Syk (p-Syk) antibodies. Total pooled samples were also immunoblotted with Lyn and Syk to control their respective distributions.

Fig. 2. CD40 associates with membrane rafts. (A) DCs were stimulated with either IgG control (–) or anti-CD40 antibody (+) prior to membrane raft separation, and the protein distribution was assayed by immunoblotting as indicated. Numbers 1 and 2 indicate two independent experiments. (B) Membrane rafts were revealed in DCs by GM1 labeling with FITC-conjugated CT-B. Membrane raft patching was induced with anti-CT-B antibody (patched GM1). CD40 and CD46 (red) were detected on fixed cells with specific PE-conjugated immunostaining. Single confocal sections show fluorescence in FITC (GM1) and PE (CD40 or CD46) channels. Bar: 5 µm.

Fig. 3. CD40-induced pTyr events are initiated in membrane rafts and are dependent on Src family kinase activation. (A) Cellular lysates from DCs stimulated with CD40 for the indicated times were immunoprecipitated with anti-p-Tyr antibody. Eluates were immunoblotted with either anti-Lyn (p-Lyn) or anti-Syk (p-Syk) antibodies. (B) DCs were pre-treated or not with the Src-specific inhibitor PP1, or with the cholesterol-binding agents nystatin (Ny) or methyl-β-cyclodextrin (mβC), before CD40 stimulation. Cellular lysates were analyzed by immunoblotting as indicated. Syk served as a loading control.

Fig. 4. Membrane rafts provide a platform for TRAF2 and 3 recruitment to the CD40 cytoplasmic tail. (A) Total pooled raft fractions (R) or soluble fractions (S) from IgG-treated (–) or CD40-stimulated (+) DCs were analyzed for CD40, TRAF3 and TRAF2 content by immunoblotting with the corresponding antibodies as indicated. The asterisk indicates a longer exposure of the TRAF2 immunoblot. (B) CD40 was immunoprecipitated (IP) from raft or soluble fractions and eluates were immunoblotted with either anti-CD40, anti-TRAF2 or anti-TRAF3 antibodies as indicated. (C) DCs were pre-treated or not with methyl-β-cyclodextrin (mβC), before CD40 stimulation. Then CD40 was immunoprecipitated (IP) from raft or soluble fractions and eluates were immunoblotted with either CD40 or anti-TRAF3 antibodies as indicated. The open arrow indicates a non-specific band observed with the anti-TRAF3 antibody.

Fig. 5. Effects of CD40-induced pTyr events on cytokine mRNA expression in DCs. (A) DCs were stimulated with anti-CD40 antibody for the indicated times in the presence or abscence of the Src-specific inhibitor PP1. Total cell lysates were analyzed by immunoblotting for the phos phorylated active form of ERK (p-ERK) or p38 MAPK (p-p38) enzymes. Syk served as a loading control. (B and C) Cells were pre-treated or not with MEK1/2 or p38 MAPK inhibitors, PD98059 or SB203580, respectively (B), or Src-specific inhibitor PP1 (C) before stimulation with either an irrelevant mouse IgG1 or with anti-CD40 antibody as indicated. After 14 h of culture, RNAs were extracted and used for RNase protection analysis. L32 and GAPDH correspond to internal control probes.

Fig. 6. Model of CD40-mediated signaling in human DCs.