Mechanisms of allergy and clinical immunology
Modulation of FcεRI-dependent mast cell response by OX40L via Fyn, PI3K, and RhoA

https://doi.org/10.1016/j.jaci.2012.03.032Get rights and content

Background

The interaction of mast cells (MCs) with regulatory T cells through the OX40 ligand (OX40L):OX40 axis downregulates FcεRI-dependent immediate hypersensitivity responses both in vitro and in vivo. Little is known on OX40L-mediated intracellular signaling or on the mechanism by which OX40L engagement suppresses MC degranulation.

Objective

We explored the role of OX40L engagement on IgE/antigen-triggered MCs both in vitro and in vivo.

Methods

The soluble form of OX40 molecule was used to selectively trigger OX40L on MCs in vitro and was used to dissect OX40L contribution in an in vivo model of systemic anaphylaxis.

Results

OX40L:OX40 interaction led to the recruitment of C-terminal src kinase into lipid rafts, causing a preferential suppression of Fyn kinase activity and subsequent reduction in the phosphorylation of Gab2, the phosphatidylinositol 3-OH kinase regulatory subunit p85, and Akt, without affecting the Lyn pathway. Dampening of Fyn kinase activity also inhibited RhoA activation and microtubule nucleation, key regulators of MC degranulation. The in vivo administration of a blocking antibody to OX40L in wild-type mice caused enhanced immediate hypersensitivity, whereas the administration of soluble OX40 to regulatory T-cell–depleted or OX40-deficient mice reduced MC degranulation.

Conclusions

The engagement of OX40L selectively suppresses Fyn-initiated signals required for MC degranulation and serves to limit immediate hypersensitivity. Our data suggest that soluble OX40 can restore the aberrant or absent regulatory T-cell activity, revealing a previously unappreciated homeostatic role for OX40L in setting the basal threshold of MC response.

Section snippets

Mice and reagents

C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me) and were used in accordance with the animal study proposal A010-04-03 approved by the National Institutes of Health (NIH) and the National Institute of Arthritis, Musculoskeletal and Skin Diseases. C57BL/6 Tnfrsf4-deficient (OX40-deficient) mice were from Nigel Killeen (University of California, San Francisco). DNP-specific IgE was previously described.20 Di-nitro-phenyl (DNP)-human serum albumin (Ag) was purchased from

OX40L engagement impairs FcεRI activation and Gab2/PI3K/Akt, but not Syk/LAT, phosphorylation

The effect of OX40L engagement on FcεRI-mediated signaling early events was investigated in IgE/Ag-activated BM-derived MCs, in the absence or presence of sOX40. Phosphorylation of pY47 in FcεRI-γ subunit was ∼50% reduced 2 and 5 minutes after Ag triggering in the presence of sOX40 (Fig 1, A). This was followed by no significant alterations in Lyn-supported phosphorylation of Syk (Y323) or LAT (Y191) on OX40L engagement (Fig 1, B). In contrast, Fyn-mediated phosphorylation of Gab2 (Y452), PI3K

Discussion

Evidence of the OX40:OX40L axis as a crucial element for the maintenance of immunological tolerance is rapidly accumulating. Interference with this molecular interplay has been connected to the development of a proinflammatory microenvironment characterized by MC-dependent hyperresponsiveness, atopy, and autoimmunity in the host.31, 32

In the present work, we demonstrate that OX40L engagement blunts FcεRI-dependent MC activation in a manner that has not previously been appreciated.

MC

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    Supported by the Italian Ministry of Health, AIRC (Associazione Italiana Ricerca sul Cancro), Ministero dell'Istruzione, Università e Ricerca (PRIN 2009), Fondazione Compagnia di San Paolo, Turin, Italy, and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (Bethesda, Md). S.P. was supported by a My First AIRC Grant (8726).

    Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

    Silvia Piconese is currently with the Dipartimento di Medicina Interna e Specialità Mediche, “Sapienza” Università di Roma, Policlinico Umberto I, Rome, Italy.

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