Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and ␤-hexosaminidase and produced tumor necrosis factor ␣ (TNF-␣), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF-␣ production. Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-␣ production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.

Close physical proximity between mast cells and T cells has been demonstrated in several T cell-mediated inflammatory processes. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We previously identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells and showed that this pathway is associated with degranulation and cytokine release. In this study, we provide evidence that mast cells may also be activated by microparticles released from activated T cells that are considered miniature versions of a cell. Microparticles were isolated from supernatants of activated T cells by Centricon filtration or by high-speed centrifugation and identified by electron microscopy, flow cytometry (Annexin stain), and expression of the integrin LFA-1. Stimulated T cells were found to generate microparticles that induce degranulation and cytokine (IL-8 and oncostatin M) release from human mast cells. Mast cell activation by T cell microparticles involved the MAPK signaling pathway. The results were similar when mast cells were stimulated by activated fixed T cells or by whole membranes of the latter. This suggests that microparticles carry mast cell-activating factors similar to cells from which they originate. By releasing microparticles, T cells might convey surface molecules similar to those involved in the activation of mast cells by cellular contact. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site.

SUMMARYThere has been substantial evidence that suggests that heparin may modulate various aspects of immune function and inflammation in addition to its well known anticoagulant activity. In this regard heparin was found to suppress cell-mediated immune responses or asthmatic reactions to allergen challenge. In the present study we analyse the effects of low molecular weight heparin (LMWH) on mast cell degranulation and cytokine production in vitro and on the elicitation of IgE-mediated mast celldependent late cutaneous allergic inflammation in vivo. We have established that LMWH preferentially inhibited tumour necrosis factor-alpha (TNF-a) and IL-4 production without having any significant effect on mast cell degranulation. These effects have been observed in mast cells derived from three different origins that were activated by either immunological or non-immunological stimuli. We have shown that there is inhibition of TNF-a production (and not neutralization of activity), as elimination of the drug after a short preincubation and addition of LMWH to rTNF-a had no effect on TNF-a-mediated cytotoxic activity. These results were also confirmed by ELISA. In vivo, s.c. injection of the LMWH inhibited the leucocyte infiltration associated with the late cutaneous response which followed passive cutaneous anaphylaxis (PCA) reaction, without affecting mast cell numbers or degranulation. These data suggest that LMWH may have an inhibitory role in mast cell-mediated allergic inflammation, and thus might be considered as a possible therapeutic modality.

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