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Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6′-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

No MeSH data available.


Mincle-mediated degranulation in RBL-2H3 cells.RBL-2H3 cells and cells expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 30 min (DNP). The amount of β-hexosaminidase released from these cells was determined. Data are representative of more than three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). Similar results were obtained from the other cloned cell lines.
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f6: Mincle-mediated degranulation in RBL-2H3 cells.RBL-2H3 cells and cells expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 30 min (DNP). The amount of β-hexosaminidase released from these cells was determined. Data are representative of more than three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). Similar results were obtained from the other cloned cell lines.

Mentions: Because a previous study demonstrated that exposure of M. tuberculosis to rat peritoneal mast cells causes the release of histamine and β-hexosaminidase31, we examined whether Mincle stimulation induces degranulation of RBL-2H3 cells. As expected, expression of WT Mincle and the R42I mutant had no considerable effect on β-hexosaminidase release induced by engagement of FcεRI (Fig. 6). In contrast, engagement of Mincle by the anti-myc mAb caused the significant release of β-hexosaminidase from cells expressing WT Mincle (P = 6.97 × 10−6), but not the R42I mutant, at a comparable level to stimulation of FcεRI. Therefore, these results demonstrate that Mincle-mediated signaling efficiently induces degranulation as much as FcεRI signaling in RBL-2H3 cells.


Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk
Mincle-mediated degranulation in RBL-2H3 cells.RBL-2H3 cells and cells expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 30 min (DNP). The amount of β-hexosaminidase released from these cells was determined. Data are representative of more than three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). Similar results were obtained from the other cloned cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385489&req=5

f6: Mincle-mediated degranulation in RBL-2H3 cells.RBL-2H3 cells and cells expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 30 min (DNP). The amount of β-hexosaminidase released from these cells was determined. Data are representative of more than three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). Similar results were obtained from the other cloned cell lines.
Mentions: Because a previous study demonstrated that exposure of M. tuberculosis to rat peritoneal mast cells causes the release of histamine and β-hexosaminidase31, we examined whether Mincle stimulation induces degranulation of RBL-2H3 cells. As expected, expression of WT Mincle and the R42I mutant had no considerable effect on β-hexosaminidase release induced by engagement of FcεRI (Fig. 6). In contrast, engagement of Mincle by the anti-myc mAb caused the significant release of β-hexosaminidase from cells expressing WT Mincle (P = 6.97 × 10−6), but not the R42I mutant, at a comparable level to stimulation of FcεRI. Therefore, these results demonstrate that Mincle-mediated signaling efficiently induces degranulation as much as FcεRI signaling in RBL-2H3 cells.

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the &gamma;-subunit of high-affinity IgE receptor (Fc&epsilon;RI&gamma;) and activates Syk by recognizing its specific ligand, trehalose-6,6&prime;-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only Fc&epsilon;RI&gamma; but also Fc&epsilon;RI&beta; in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of Fc&epsilon;RI&beta;&gamma; subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase C&gamma;2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the Fc&epsilon;RI&beta;&gamma; complex.

No MeSH data available.