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Assembly and regulation of the CD40 receptor complex in human B cells.

Kuhné MR, Robbins M, Hambor JE, Mackey MF, Kosaka Y, Nishimura T, Gigley JP, Noelle RJ, Calderhead DM - J. Exp. Med. (1997)

Bottom Line: CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily.Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor- associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface.In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.

ABSTRACT
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor- associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface. Combined signaling through the interluekin 4 receptor and CD40 induces an increased expression of Fas with a commensurate increase in the level of TRAF2, but not TRAF3, that is recruited to the receptor complex. In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex. These studies show that the TRAF composition of the CD40 receptor complex can be altered by signals that influence B cell differentiation.

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Anti-μ cross-linking reduces TRAF2 association and Fas upregulation induced by CD40 signaling. DND39 cells (2 × 107 cells/lane)  were incubated in media or with 10 μg/ml goat anti–human IgM for 24 h  and both groups were either left unstimulated (minus) or stimulated with  sCD154 (4 nM) (plus) for 15 min. Lysates were immunoprecipitated for  CD40 and immunoblotted for TRAF2 (A) or TRAF3 (B). (C and D)  Immunoblot analysis of cytosolic TRAF content after GST–CD40cyt immunoprecipitations. The lysates used for the anti-CD40 precipitations described in A and B were further precipitated with GST–CD40cyt. 5 ×  106 cells/lane were loaded for the TRAF2 blot, while the TRAF3 blot  received 2 × 107 cell equivalents per lane. (E) Fluorescence-activated cell  sorting analysis of DND39 cells after treatment with anti-IgM and  sCD154. DND39 cells were incubated for 24 h with media (minus), 10  μg/ml anti-IgM, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody (open profile) or a control  mouse IgG1 monoclonal (closed profile). The mean fluorescent intensity of  the cells is indicated in the upper righthand corner. This experiment is  representative of four experiments.
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Figure 3: Anti-μ cross-linking reduces TRAF2 association and Fas upregulation induced by CD40 signaling. DND39 cells (2 × 107 cells/lane) were incubated in media or with 10 μg/ml goat anti–human IgM for 24 h and both groups were either left unstimulated (minus) or stimulated with sCD154 (4 nM) (plus) for 15 min. Lysates were immunoprecipitated for CD40 and immunoblotted for TRAF2 (A) or TRAF3 (B). (C and D) Immunoblot analysis of cytosolic TRAF content after GST–CD40cyt immunoprecipitations. The lysates used for the anti-CD40 precipitations described in A and B were further precipitated with GST–CD40cyt. 5 × 106 cells/lane were loaded for the TRAF2 blot, while the TRAF3 blot received 2 × 107 cell equivalents per lane. (E) Fluorescence-activated cell sorting analysis of DND39 cells after treatment with anti-IgM and sCD154. DND39 cells were incubated for 24 h with media (minus), 10 μg/ml anti-IgM, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody (open profile) or a control mouse IgG1 monoclonal (closed profile). The mean fluorescent intensity of the cells is indicated in the upper righthand corner. This experiment is representative of four experiments.

Mentions: DND39 is a CD40-responsive, human Burkitt B cell lymphoma that has been shown to increase sterile transcripts from the IgE promoter (10) and be rescued from growth inhibition by cross-linking of CD40 (12). Within 15 min of addition of a soluble, multimeric form of CD154 (sCD154) both TRAF2 and TRAF3 could be coimmunoprecipitated with CD40. Immunoprecipitation of CD40 from nonactivated DND39s did not reveal constitutively associated TRAF2 or TRAF3 (Fig. 1). Association of TRAFs was maximal at a 4 nM concentration of ligand and found to peak after 15 min of ligand addition (data not shown). The association of the TRAF molecules with CD40 was mirrored by a decrease in the cytosolic pool of TRAF2 and TRAF3, which could be precipitated with a fusion protein consisting of GST and the CD40 cytoplasmic domain (GST–CD40cyt; reference 6) (see Fig. 3, C and D). However, the reduction in the cytosolic TRAF2 and TRAF3 could only be partially accounted for by recruitment of these molecules to the receptor complex. When total cellular TRAF2 or TRAF3 was immunoprecipitated with anti-TRAF2 or TRAF3 antibodies, a reduction in TRAF content was observed following engagement of CD40 (see Fig. 4). Therefore, in addition to the recruitment of TRAF2 and TRAF3 to CD40, a significant amount of the cellular TRAF2 and TRAF3 is lost from the detergent-soluble fraction. Whether this loss is due to movement of the TRAF molecules to another subcellular location or to degradation of the TRAFs is currently being studied.


Assembly and regulation of the CD40 receptor complex in human B cells.

Kuhné MR, Robbins M, Hambor JE, Mackey MF, Kosaka Y, Nishimura T, Gigley JP, Noelle RJ, Calderhead DM - J. Exp. Med. (1997)

Anti-μ cross-linking reduces TRAF2 association and Fas upregulation induced by CD40 signaling. DND39 cells (2 × 107 cells/lane)  were incubated in media or with 10 μg/ml goat anti–human IgM for 24 h  and both groups were either left unstimulated (minus) or stimulated with  sCD154 (4 nM) (plus) for 15 min. Lysates were immunoprecipitated for  CD40 and immunoblotted for TRAF2 (A) or TRAF3 (B). (C and D)  Immunoblot analysis of cytosolic TRAF content after GST–CD40cyt immunoprecipitations. The lysates used for the anti-CD40 precipitations described in A and B were further precipitated with GST–CD40cyt. 5 ×  106 cells/lane were loaded for the TRAF2 blot, while the TRAF3 blot  received 2 × 107 cell equivalents per lane. (E) Fluorescence-activated cell  sorting analysis of DND39 cells after treatment with anti-IgM and  sCD154. DND39 cells were incubated for 24 h with media (minus), 10  μg/ml anti-IgM, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody (open profile) or a control  mouse IgG1 monoclonal (closed profile). The mean fluorescent intensity of  the cells is indicated in the upper righthand corner. This experiment is  representative of four experiments.
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Related In: Results  -  Collection

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Figure 3: Anti-μ cross-linking reduces TRAF2 association and Fas upregulation induced by CD40 signaling. DND39 cells (2 × 107 cells/lane) were incubated in media or with 10 μg/ml goat anti–human IgM for 24 h and both groups were either left unstimulated (minus) or stimulated with sCD154 (4 nM) (plus) for 15 min. Lysates were immunoprecipitated for CD40 and immunoblotted for TRAF2 (A) or TRAF3 (B). (C and D) Immunoblot analysis of cytosolic TRAF content after GST–CD40cyt immunoprecipitations. The lysates used for the anti-CD40 precipitations described in A and B were further precipitated with GST–CD40cyt. 5 × 106 cells/lane were loaded for the TRAF2 blot, while the TRAF3 blot received 2 × 107 cell equivalents per lane. (E) Fluorescence-activated cell sorting analysis of DND39 cells after treatment with anti-IgM and sCD154. DND39 cells were incubated for 24 h with media (minus), 10 μg/ml anti-IgM, sCD154, or both (as indicated). Fas expression was detected with anti-CD95 monoclonal antibody (open profile) or a control mouse IgG1 monoclonal (closed profile). The mean fluorescent intensity of the cells is indicated in the upper righthand corner. This experiment is representative of four experiments.
Mentions: DND39 is a CD40-responsive, human Burkitt B cell lymphoma that has been shown to increase sterile transcripts from the IgE promoter (10) and be rescued from growth inhibition by cross-linking of CD40 (12). Within 15 min of addition of a soluble, multimeric form of CD154 (sCD154) both TRAF2 and TRAF3 could be coimmunoprecipitated with CD40. Immunoprecipitation of CD40 from nonactivated DND39s did not reveal constitutively associated TRAF2 or TRAF3 (Fig. 1). Association of TRAFs was maximal at a 4 nM concentration of ligand and found to peak after 15 min of ligand addition (data not shown). The association of the TRAF molecules with CD40 was mirrored by a decrease in the cytosolic pool of TRAF2 and TRAF3, which could be precipitated with a fusion protein consisting of GST and the CD40 cytoplasmic domain (GST–CD40cyt; reference 6) (see Fig. 3, C and D). However, the reduction in the cytosolic TRAF2 and TRAF3 could only be partially accounted for by recruitment of these molecules to the receptor complex. When total cellular TRAF2 or TRAF3 was immunoprecipitated with anti-TRAF2 or TRAF3 antibodies, a reduction in TRAF content was observed following engagement of CD40 (see Fig. 4). Therefore, in addition to the recruitment of TRAF2 and TRAF3 to CD40, a significant amount of the cellular TRAF2 and TRAF3 is lost from the detergent-soluble fraction. Whether this loss is due to movement of the TRAF molecules to another subcellular location or to degradation of the TRAFs is currently being studied.

Bottom Line: CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily.Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor- associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface.In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.

ABSTRACT
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor- associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface. Combined signaling through the interluekin 4 receptor and CD40 induces an increased expression of Fas with a commensurate increase in the level of TRAF2, but not TRAF3, that is recruited to the receptor complex. In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex. These studies show that the TRAF composition of the CD40 receptor complex can be altered by signals that influence B cell differentiation.

Show MeSH