Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278).

To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y(191)MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85ß regulatory subunits and p110α, p110δ and p110ß catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.

Expression of functional NK1 receptors in human alveolar macrophages: superoxide anion production, cytokine release and involvement of NF-kappaB pathway.

1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory-immune processes. We previously demonstrated that SP activates guinea-pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non-smokers, by evaluating the presence of tachykinin NK(1) receptors (NK-1R) and SP's ability to induce superoxide anion (O(2)(-)) production and cytokine release, as well as activation of the nuclear factor-kappaB (NF-kappaB) pathway. 3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK-1R are present on human AMs, a three-fold enhanced expression being observed in healthy smokers. These NK-1R are functional, as SP and NK(1) agonists dose-dependently induce O(2)(-) production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor-alpha as compared to healthy non-smokers, but has inconsistent effects on IL-10 release. The NK(1) selective antagonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]-1-azabicyclo-octan-3-amine)) competitively antagonized SP-induced effects. 4 SP activates the transcription factor NF-kappaB, a three-fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor-mediated, as it is reproduced by the NK(1) selective agonist [Sar(9)Met(O(2))(11)]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK-1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells.

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4+ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti-CD3 mAb. ICOS strikingly potentiated secretion of IL-2, IFN-gamma, IL-10, and TNF-alpha, but not IL-4, promoted by optimal stimulation of CD3+CD28, and it was the key switching-factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL-2. In these conditions, blockade of IL-2 and IFN-gamma showed that ICOS builds up a positive feedback loop with IFN-gamma, which required IL-2 and was inhibited by IL-4. By contrast, in the absence of CD28 triggering or exogenous IL-2, ICOS-induced costimulation mainly supported expression of TGF-beta1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4+ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL-2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.