Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation.
Bottom Line: Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction.Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs.The transferred material is ultimately processed and presented by DCs and can activate T cells.
Affiliation: Department of Pathology, The University of New Mexico School of Medicine, Albuquerque, NM 87131.Show MeSH
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Mentions: We disrupted integrin activity by preincubation of MCs with the small molecule inhibitors LDV (VLA-4) or Zyn (LFA-1; Chigaev et al., 2011a,b). LDV was derived from the LDV sequence within the VLA-4–binding region of fibrinogen and has been shown to block interaction with VCAM-1 (Chigaev et al., 2001), whereas Zyn is an α/β I–like allosteric β2 integrin antagonist compound (Shimaoka and Springer, 2003; Chigaev et al., 2015). Although inhibitor treatment was not sufficient to completely abrogate cell contacts, MC polarization was dramatically reduced from 58% to 6.7% with LFA-1 inhibition and to 16% with VLA-4 inhibition (Fig. 6 A). Consistent with this, the small molecule inhibitors also reduced the number of DCs containing transferred material, from 80% of DCs showing material transfer in untreated samples to 46% and 33% showing transfer with Zyn or LDV treatment, respectively (Fig. 6 A). The requirement for integrin engagement for synapse stability was further demonstrated by single-cell force spectroscopy measurements. In the presence of either the small molecule inhibitors or an antibody cocktail that blocks VLA-4 and LFA-1 binding, adhesion between the cells was decreased to levels similar for unactivated MCs and imDCs (Fig. 6 B). We also treated cells with the Src Family kinase inhibitor PP2. PP2 prevents FcεRI phosphorylation and, therefore, any downstream signaling that would up-regulate integrin affinity, but it does not interfere with receptor cross-linking or internalization (Andrews et al., 2008). Pretreatment with PP2 completely blocked the increase in cell–cell adhesion when MCs were activated (Fig. 6 B). These results demonstrate that cell adhesion mediated by integrins is important for the efficient formation of the MC–DC synapse. To test whether integrins simply act to mediate cell–cell contact, we subjected MC–DCs to centrifugation to force cell–cell proximity/interaction. In this assay, MC activation also lead to an increase in material transfer (27.4 ± 5.7% with actMCs compared with 10.4 ± 4.3% with unactivated MCs). We note the increased background of material transfer when compared with the experiments with DCs plated on glass in which transfer was not observed for unactivated MCs. Inhibition of VLA-4 by LDV treatment still blocked material transfer (6.9 ± 3.4%) even when MCs and DCs were forcibly conjugated through centrifugation, demonstrating that signaling through integrins is required for efficient material transfer between MCs and DCs.
Affiliation: Department of Pathology, The University of New Mexico School of Medicine, Albuquerque, NM 87131.