Ryanodine Receptor Activation by Cav1.2 Is Involved in Dendritic Cell Major Histocompatibility Complex Class II Surface Expression

Mirko Vukcevic,Giulio C Spagnoli,Giandomenica Iezzi,Francesco Zorzato,Susan Treves

doi:10.1074/jbc.m804472200

Mirko Vukcevic, Giulio C Spagnoli + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m804472200

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Abstract

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.

Highlights

Ryanodine receptors belong to a family of intracellular Ca2ϩ release channels composed of at least three different isoforms that have been characterized extensively biochemically, functionally, and at the molecular level [5,6,7]
Both B-lymphocytes and dendritic cells (DCs) can act as antigen presenting cells and initiate T cell-driven immune responses [20], DCs play a central role in the immune system; they are strategically located in peripheral tissues where they continuously sample their environment for the presence of antigens
Upstream Events Leading to RyR1 Activation—In an earlier report [17] we showed that (i) immature DCs (iDCs) only express the type 1 ryanodine receptors (RyRs) isoform, (ii) the addition of caffeine, 4-chloro-m-cresol, or KCl to iDC causes a RyR1-dependent increase in the intracellular Ca2ϩ concentration ([Ca2ϩ]i), and (iii) the signaling cascade activated by the Ca2ϩ increase acts synergistically with signals generated via Toll-like receptors, stimulating DC maturation [17]

Summary

EXPERIMENTAL PROCEDURES

Isolation and Generation of Dendritic Cells—Human iDCs were generated from peripheral blood mononuclear leukocytes as previously described [17]. To monitor surface expression of MHC class I and II molecules, stimulated cells were washed with ice-cold PBS, labeled, and analyzed by flow cytometry using FITC-labeled anti-human HLA DR and HLA A, B, C or, in the case of mouse CD11cϩ cells, with FITC-labeled anti mouse I-Ab monoclonal antibody (BD Pharmingen). For MHC II induction untreated T-lymphocytes or T-lymphocytes preincubated with 100 nM charybdotoxin (Alexa Biochemicals) for 45 min and washed to remove excess toxin were used Both batches of T cells were placed in 1.5-ml Eppendorf tubes together with the CD11c positive B6 or B6.C-H-2bm DCs. Cells were spun down for 30 s using a Tomy PMC-060 capsulefuge and incubated at 37 °C for an additional 5 min.

RESULTS

DISCUSSION

Although the expression of the