Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCtheta inhibitors.

Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).

Mast cell-dependent contraction of human airway smooth muscle cell-containing collagen gels: influence of cytokines, matrix metalloproteases, and serine proteases.

In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13Ralpha2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.

Altered regulation of aquaporin gene expression in allergen and IL-13-induced mouse models of asthma.

IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis revealed differential regulation of AQPs in these two models. AQP1 and AQP4 mRNA expression was downregulated in the OVA/OVA model, but not in the IL-13 model. AQP5 mRNA was reduced in both models, whereas AQP3 was upregulated only in the IL-13 model. Western analysis showed that diminished expression of an apically localized aquaporin, (AQP5), and concomitant upregulation of a basolateral aquaporin (AQP3 or AQP4) are characteristic features of both inducible asthma models. These results demonstrate that aquaporins are common targets of gene expression in both allergen and IL-13 induced mouse models of asthma.

CAT-2 amplifies the agonist-evoked force of airway smooth muscle by enhancing spermine-mediated phosphatidylinositol-(4)-phosphate-5-kinase-gamma activity.

We investigated the effect the loss of the CAT-2 gene (CAT-2-/-) has on lung resistance (R(L)) and tracheal isometric tension. The R(L) of CAT-2-/- mice at a maximal dose of acetylcholine (ACh) was decreased by 33.66% (P = 0.05, n = 8) compared with that of C57BL/6 (B6) mice. The isometric tension of tracheal rings from CAT-2-/- mice showed a significant decrease in carbachol (CCh)-induced force generation (33.01%, P < 0.05, n = 8) compared with controls. The isoproterenol- or the sodium nitroprusside-induced relaxation was not affected in tracheal rings from CAT-2-/- mice. The activity of iNOS and arginase in lung tissue lysates of CAT-2-/- mice was indistinguishable from that of B6 mice. Furthermore, the expression of phospholipase-Cbeta (PLC-beta) and phosphatidylinositol-(4)-phosphate-5-kinase-gamma (PIP-5K-gamma) was examined in the lung tissue of CAT-2-/- and B6 mice. The expression of PIP-5K-gamma but not PLC-beta was significantly reduced in CAT-2-/- compared with B6 mice. The reduced airway smooth muscle (ASM) contractility to CCh seen in the CAT-2-/- tracheal rings was completely reversed by pretreating the rings with 100 muM spermine. This increase in the CAT-2-/- tracheal ring contraction upon spermine pretreatment correlated with a recovery of the expression of PIP-5K-gamma. Our data indicates that CAT-2 exerts control over ASM force development through a spermine-dependent pathway that directly correlates with the expression level of PIP-5K-gamma in the lung.

Temporal associations between interleukin 22 and the extracellular domains of IL-22R and IL-10R2.

Interleukin 22 (IL-22) is a cytokine induced during both innate and adaptive immune responses. It can effect an acute phase response, implicating a role for IL-22 in mechanisms of inflammation. IL-22 requires the presence of the IL-22 receptor (IL-22R) and IL-10 receptor 2 (IL-10R2) chains, two members of the class II cytokine receptor family (CRF2), to effect signal transduction within a cell. We studied the interaction between human IL-22 and the extracellular domains (ECD) of its receptor chains in an enzyme-linked immunoabsorbant assay (ELISA)-based format, using biotinylated IL-22 (bio-IL-22) and receptor-fusions containing the ECD of a receptor fused to the Fc of hIgG1 (IL-22R-Fc and IL-10R2-Fc). IL-22 has measurable affinity for IL-22R-Fc homodimer and undetectable affinity for IL-10R2. IL-22 has substantially greater affinity for IL-22R/IL-10R2-Fc heterodimers. Further analyses involving sequential additions of receptor homodimers and cytokine indicates that the IL-10R2(ECD) binds to a surface created by the interaction between IL-22 and the IL-22R(ECD), and thereby further stabilizes the association of IL-22 within this cytokine-receptor-Fc complex. Both a neutralizing rat monoclonal antibody, specific for human IL-22, and human IL-22BP-Fc, an Fc-fusion of the secreted IL-22 binding-protein and proposed natural antagonist for IL-22, bind to similar cytokine epitopes that may overlap the binding site for IL-22R(ECD). Another rat monoclonal antibody, specific for IL-22, binds to an epitope that may overlap a separate binding site for IL-10R2(ECD). We propose, based on this data, a temporal model for the development of a functional IL-22 cytokine-receptor complex.

IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity.

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.