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Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes.

Punnonen J, Cocks BG, Carballido JM, Bennett B, Peterson D, Aversa G, de Vries JE - J. Exp. Med. (1997)

Bottom Line: Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro.In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by anti-CD40 mAbs.SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304, USA.

ABSTRACT
In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-mu mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by anti-CD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.

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Effect of B cell activation on mSLAM expression, and expression of SLAM isoforms in activated B cells. (A) B cells negatively selected by  magnetic beads were cultured in the presence of medium only, IL-4 (100 U/ml), anti-CD40 mAbs (10 μg/ml), SAC (0.005%), or anti-μ mAbs (10 μg/ ml) for 6, 20, 48, or 72 h as indicated. Thereafter, the cells were harvested, washed and stained with mAb CD20-FITC and PE-conjugated SLAM-specific mAb A12 or PE-conjugated mouse IgG1 Ab with irrelevant specificity. Open and stippled histograms represent stainings with mAb A12 and control  Ab, respectively. PI was always added to exclude dead cells. mSLAM expression on CD20+ cells was analyzed using FACScan® flow cytometer and  CellQuest software. A representative of three experiments is shown. (B) B cells purified by cell sorting were cultured in the presence of anti-CD40 mAbs  and IL-4 for 24 h, whereas PBMC were cultured in the presence of PMA (1 ng/ml) and Ca2+ ionophore A23187 (500 ng/ml) for 4 h. The cells were  harvested and washed, and expression of different isoforms of SLAM was analyzed by RT-PCR using primers specific for each isoform as described in experimental procedures (m, membrane; s, soluble; c, cytoplasmic; vm, variant membrane). HPRT mRNA was amplified as a positive control.
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Figure 2: Effect of B cell activation on mSLAM expression, and expression of SLAM isoforms in activated B cells. (A) B cells negatively selected by magnetic beads were cultured in the presence of medium only, IL-4 (100 U/ml), anti-CD40 mAbs (10 μg/ml), SAC (0.005%), or anti-μ mAbs (10 μg/ ml) for 6, 20, 48, or 72 h as indicated. Thereafter, the cells were harvested, washed and stained with mAb CD20-FITC and PE-conjugated SLAM-specific mAb A12 or PE-conjugated mouse IgG1 Ab with irrelevant specificity. Open and stippled histograms represent stainings with mAb A12 and control Ab, respectively. PI was always added to exclude dead cells. mSLAM expression on CD20+ cells was analyzed using FACScan® flow cytometer and CellQuest software. A representative of three experiments is shown. (B) B cells purified by cell sorting were cultured in the presence of anti-CD40 mAbs and IL-4 for 24 h, whereas PBMC were cultured in the presence of PMA (1 ng/ml) and Ca2+ ionophore A23187 (500 ng/ml) for 4 h. The cells were harvested and washed, and expression of different isoforms of SLAM was analyzed by RT-PCR using primers specific for each isoform as described in experimental procedures (m, membrane; s, soluble; c, cytoplasmic; vm, variant membrane). HPRT mRNA was amplified as a positive control.

Mentions: We next studied the effect of activation on SLAM expression on B cells. As shown in Fig. 2 A, mSLAM expression was moderately upregulated on B cells cultured in the presence of medium alone. The expression level of mSLAM was not significantly modulated by IL-2, IL-4, or IL-10 (100 U/ml each), when compared to B cells cultured in the presence of medium alone for up to 72 h (Fig. 2 A and data not shown). In contrast, SAC, anti-μ mAbs and anti-CD40 mAbs induced high levels of mSLAM expression on >85% of the B cells during a culture period of 20 h and the high expression levels were sustained for up to 3 d. The most rapid effect was observed in response to anti-μ mAbs, which induced high levels of mSLAM expression on B cells already during a culture period of 6 h, whereas optimal enhancement of mSLAM expression by SAC and anti-CD40 mAbs was observed after 20 h. These data indicate that mSLAM is strongly upregulated on B cells after activation, and suggest that mSLAM is rapidly induced after recognition of antigen by surface Ig.


Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes.

Punnonen J, Cocks BG, Carballido JM, Bennett B, Peterson D, Aversa G, de Vries JE - J. Exp. Med. (1997)

Effect of B cell activation on mSLAM expression, and expression of SLAM isoforms in activated B cells. (A) B cells negatively selected by  magnetic beads were cultured in the presence of medium only, IL-4 (100 U/ml), anti-CD40 mAbs (10 μg/ml), SAC (0.005%), or anti-μ mAbs (10 μg/ ml) for 6, 20, 48, or 72 h as indicated. Thereafter, the cells were harvested, washed and stained with mAb CD20-FITC and PE-conjugated SLAM-specific mAb A12 or PE-conjugated mouse IgG1 Ab with irrelevant specificity. Open and stippled histograms represent stainings with mAb A12 and control  Ab, respectively. PI was always added to exclude dead cells. mSLAM expression on CD20+ cells was analyzed using FACScan® flow cytometer and  CellQuest software. A representative of three experiments is shown. (B) B cells purified by cell sorting were cultured in the presence of anti-CD40 mAbs  and IL-4 for 24 h, whereas PBMC were cultured in the presence of PMA (1 ng/ml) and Ca2+ ionophore A23187 (500 ng/ml) for 4 h. The cells were  harvested and washed, and expression of different isoforms of SLAM was analyzed by RT-PCR using primers specific for each isoform as described in experimental procedures (m, membrane; s, soluble; c, cytoplasmic; vm, variant membrane). HPRT mRNA was amplified as a positive control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196230&req=5

Figure 2: Effect of B cell activation on mSLAM expression, and expression of SLAM isoforms in activated B cells. (A) B cells negatively selected by magnetic beads were cultured in the presence of medium only, IL-4 (100 U/ml), anti-CD40 mAbs (10 μg/ml), SAC (0.005%), or anti-μ mAbs (10 μg/ ml) for 6, 20, 48, or 72 h as indicated. Thereafter, the cells were harvested, washed and stained with mAb CD20-FITC and PE-conjugated SLAM-specific mAb A12 or PE-conjugated mouse IgG1 Ab with irrelevant specificity. Open and stippled histograms represent stainings with mAb A12 and control Ab, respectively. PI was always added to exclude dead cells. mSLAM expression on CD20+ cells was analyzed using FACScan® flow cytometer and CellQuest software. A representative of three experiments is shown. (B) B cells purified by cell sorting were cultured in the presence of anti-CD40 mAbs and IL-4 for 24 h, whereas PBMC were cultured in the presence of PMA (1 ng/ml) and Ca2+ ionophore A23187 (500 ng/ml) for 4 h. The cells were harvested and washed, and expression of different isoforms of SLAM was analyzed by RT-PCR using primers specific for each isoform as described in experimental procedures (m, membrane; s, soluble; c, cytoplasmic; vm, variant membrane). HPRT mRNA was amplified as a positive control.
Mentions: We next studied the effect of activation on SLAM expression on B cells. As shown in Fig. 2 A, mSLAM expression was moderately upregulated on B cells cultured in the presence of medium alone. The expression level of mSLAM was not significantly modulated by IL-2, IL-4, or IL-10 (100 U/ml each), when compared to B cells cultured in the presence of medium alone for up to 72 h (Fig. 2 A and data not shown). In contrast, SAC, anti-μ mAbs and anti-CD40 mAbs induced high levels of mSLAM expression on >85% of the B cells during a culture period of 20 h and the high expression levels were sustained for up to 3 d. The most rapid effect was observed in response to anti-μ mAbs, which induced high levels of mSLAM expression on B cells already during a culture period of 6 h, whereas optimal enhancement of mSLAM expression by SAC and anti-CD40 mAbs was observed after 20 h. These data indicate that mSLAM is strongly upregulated on B cells after activation, and suggest that mSLAM is rapidly induced after recognition of antigen by surface Ig.

Bottom Line: Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro.In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by anti-CD40 mAbs.SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304, USA.

ABSTRACT
In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-mu mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by anti-CD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.

Show MeSH
Related in: MedlinePlus