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Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

Quintana FJ, Solomon A, Cohen IR, Nussbaum G - PLoS ONE (2008)

Bottom Line: We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3.In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10.Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. fquintana@rics.bwh.harvard.edu

ABSTRACT
B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

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TLR4 and BCR interact in the membrane of LPS-activated B-cells.A. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated and bound proteins were separated by PAGE-SDS and analyzed by western blot using an IgM-specific antibody. D. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated with a specific biotinylated antibody and then captured on streptavidin-coated microplates and bound IgM was detected with a specific HRP-conjugated antibody. * P<0.05 and ** P<0.01 vs. control (no LPS).
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pone-0003509-g004: TLR4 and BCR interact in the membrane of LPS-activated B-cells.A. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated and bound proteins were separated by PAGE-SDS and analyzed by western blot using an IgM-specific antibody. D. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated with a specific biotinylated antibody and then captured on streptavidin-coated microplates and bound IgM was detected with a specific HRP-conjugated antibody. * P<0.05 and ** P<0.01 vs. control (no LPS).

Mentions: To confirm these results, we performed immuno-precipitation and pull-down assays. IgM and TLR4 were co-precipitated from extracts prepared from wild-type LPS-treated B cells, but not from TLR4del B-cells (Figure 4A and Figure S4B). No co-precipitation was detected using extracts from LPS-treated TLR4P712H B cells, suggesting that physical association depends on functional signaling through TLR4 (data not shown). To confirm these results, we preincubated cell extracts from LPS activated (or control) B cells with biotinylated antibodies to TLR4, and then captured the biotinylated antibody-antigen complexes on streptavidin coated microtiter plates. The plates where washed and the BCR molecules pulled-down with TLR4 were quantified using an HRP-conjugated anti-mouse IgM detection antibody. TLR4/IgM complexes could be pulled down from TLR4WT B cells, but not from their TLR4del counterparts (Figure 4B). These results suggest that the TLR4 and IgM molecules are associated on the surface of LPS-activated B cells, either directly or through yet uncharacterized interaction partners.


Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

Quintana FJ, Solomon A, Cohen IR, Nussbaum G - PLoS ONE (2008)

TLR4 and BCR interact in the membrane of LPS-activated B-cells.A. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated and bound proteins were separated by PAGE-SDS and analyzed by western blot using an IgM-specific antibody. D. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated with a specific biotinylated antibody and then captured on streptavidin-coated microplates and bound IgM was detected with a specific HRP-conjugated antibody. * P<0.05 and ** P<0.01 vs. control (no LPS).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2566810&req=5

pone-0003509-g004: TLR4 and BCR interact in the membrane of LPS-activated B-cells.A. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated and bound proteins were separated by PAGE-SDS and analyzed by western blot using an IgM-specific antibody. D. Purified B cells from TLR4WT or TLR4del mice were incubated for 1 hr with 0, 0.1 or 1 µg/ml of LPS, TLR4 was immunoprecipitated with a specific biotinylated antibody and then captured on streptavidin-coated microplates and bound IgM was detected with a specific HRP-conjugated antibody. * P<0.05 and ** P<0.01 vs. control (no LPS).
Mentions: To confirm these results, we performed immuno-precipitation and pull-down assays. IgM and TLR4 were co-precipitated from extracts prepared from wild-type LPS-treated B cells, but not from TLR4del B-cells (Figure 4A and Figure S4B). No co-precipitation was detected using extracts from LPS-treated TLR4P712H B cells, suggesting that physical association depends on functional signaling through TLR4 (data not shown). To confirm these results, we preincubated cell extracts from LPS activated (or control) B cells with biotinylated antibodies to TLR4, and then captured the biotinylated antibody-antigen complexes on streptavidin coated microtiter plates. The plates where washed and the BCR molecules pulled-down with TLR4 were quantified using an HRP-conjugated anti-mouse IgM detection antibody. TLR4/IgM complexes could be pulled down from TLR4WT B cells, but not from their TLR4del counterparts (Figure 4B). These results suggest that the TLR4 and IgM molecules are associated on the surface of LPS-activated B cells, either directly or through yet uncharacterized interaction partners.

Bottom Line: We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3.In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10.Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. fquintana@rics.bwh.harvard.edu

ABSTRACT
B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

Show MeSH