Aspirin and salicylates inhibit the IL-4- and IL-13-induced activation of STAT6.

Allergic diseases, including asthma, represent a major threat to human health. Over the three last decades, their incidence has risen in western countries. Aspirin treatment has been shown to improve allergic diseases, especially asthma, and the decreased use of aspirin has been hypothesized to contribute to the increase in childhood asthma. Because salicylate compounds suppress a number of enzymatic activities, and signaling through IL-4R participates in the development of allergic responses, we tested the effect of salicylates on IL-4 signal transduction. We found that treatment of cell lines and primary cells with aspirin and salicylates, but not acetaminophen, inhibited the activation of STAT6 by IL-4 and IL-13. This effect correlated with the inhibition of IL-4-induced CD23 expression. Although salicylates inhibited the in vivo activation of Janus kinases, their kinase activity was not affected in vitro by salicylates, suggesting that other kinases were involved in IL-4-induced STAT6 activation. Furthermore, we found that an Src kinase was involved in STAT6 activation because 1) Src kinase activity was induced by IL-4, 2) Src kinase activity, but not Janus kinase, was inhibited by salicylates in vitro, 3) cells expressing viral Src had constitutive STAT6 phosphorylation, and 4) cells lacking Src showed low STAT6 phosphorylation in response to IL-4. Because STAT6 activation by IL-4 and IL-13 participates in the development of allergic diseases, our results provide a mechanism to explain the beneficial effects of aspirin and salicylate treatment of these diseases.     

Extracellular adenine nucleotides inhibit the activation of human CD4+ T lymphocytes

ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE(2), inhibit human CD4(+) T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4(+) T cells. The rank order of potency of the various nucleotides was: adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) approximately 2'- and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP > dATP, 2-propylthio-beta,gamma-dichloromethylene-D-ATP, UDP, UTP. This effect did not involve the activation of A(2)Rs by adenosine or the synthesis of prostaglandins. ATPgammaS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPgammaS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-gamma; expression of CD25; and proliferation after activation of CD4(+) T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4(+) T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.     

Interleukin-4 fails to inhibit interferon-gamma-induced activation of human colostral macrophages.

IL-4 abrogates the IFN-gamma-mediated activation of peripheral blood monocytes (M. Lehn, W. Y. Weiser, S. E. Engelhorn, S. Gillis, and H. G. Remold, 1989, J. Immunol. 143, 3020). In contrast, in colostral macrophages IL-4 fails to inhibit IFN-gamma-induced increase of H2O2 production and of antileishmanial activity. Flow cytometric analysis shows that the number of IL-4 receptors (IL-4R) is 2.4 times higher on colostral macrophages than on peripheral blood monocytes and that 23% of the colostral macrophages have detectable IL-4R in contrast to 2% of the blood monocytes. Thus, colostral macrophages are functionally different from peripheral blood monocytes in their response to IL-4 and in the numbers of IL-4R. This difference could reflect specific requirements for their protective performance in the neonatal intestine.     

Stretch-induced activation of ERK in myocytes is p38 and calcineurin-dependent

Activation of specific mitogen-activated protein kinases (MAPKs) has been suggested to be involved in phenotype modulation of cells subjected to mechanical strain, which may be common to different mechano-sensitive cell types. We have submitted C2C12 myocytes to a static stretch and examined its effect upon the activation of ERK. Stretch induced a rapid but transient activation of ERK. This activation was however prevented when cells were pre-treated with inhibitors of p38 and calcineurin. The dependence of strain-induced ERK activation upon p38 suggests a cross-talk between these two pathways when mediating a response to a mechanical stimulus in this cell type. This suggests that cross relationships between these MAP kinases may be of crucial importance for myocyte phenotype modulation and differentiation in response to a mechanical stimulus.     

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.     

A polymorphic dinucleotide repeat in the human IL-10 promoter

The polymorphism data reported in this paper have been submitted to the Human Genome databse and have been assigned the accession number G00-603-930     

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.     

Chromosomal mapping of the mouse IL-4 and human IL-5 genes

We mapped the mouse interleukin (IL)-4 gene on chromosome 11 by restriction fragment length polymorphism using recombinant inbred mouse strains. The human IL-5 gene was mapped on chromosome 5q 23.3-31.1 by in situ hybridization. Because the granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-3 genes were previously mapped on mouse chromosome 11 (within a 230-kb region) and human chromosome 5, the IL-4 and IL-5 genes are likely to cluster on the same chromosomes with the GM-CSF and IL-3 genes in both species.     

Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.     

Glucocorticoids inhibit formation of inositol phosphates in macrophages.

Glucocorticoids inhibited the zymosan-induced formation of inositol phosphates in macrophages. No inhibition was observed with progesterone. Inhibitors of protein (cycloheximide) and RNA (actinomycin D) synthesis exhibited similar inhibitory effects. The activity of phospholipase C in subcellular fractions was not altered by hormone treatment of the cells. However, the incorporation of inositol into membrane lipids was reduced by dexamethasone. These data indicate that glucocorticoids are able to inhibit the formation of inositol phosphates; the effect of the hormone is rather due to an inhibition of the incorporation of inositol in membrane lipids than to an inhibition of phospholipase C. The anti-inflammatory action of glucocorticoids may, therefore, also be attributed to their effect on the polyphosphoinositide cycle and inositol phosphate-mediated processes.     

The effects of IL-4 on human natural killer cells. A potent regulator of IL-2 activation and proliferation

NK cells are directly activated by rIL-2 and subsequently undergo rIL-2-dependent proliferation in vitro. Herein, we report that rIL-4 is a potent regulator of human NK cells. Although rIL-4 had no effect on the cytotoxic activity of resting NK cells, it was capable of inhibiting in a concentration-dependent manner the rIL-2-induced cytolytic activation of NK cells against NK cell-resistant tumor cell targets. rIL-4 acted directly on NK cells and did not require accessory cells. rIL-4-induced inhibition of NK cell activation was specific for rIL-2 in that activation of NK cell cytolysis by IFN-alpha was not affected. These results represent the first direct evidence that rIL-2 and IFN-alpha activate NK cells by different pathways. rIL-4 also effectively blocked the rIL-2-dependent proliferation of NK cells. The results presented in this study clearly demonstrate that rIL-4 is a potent regulator of IL-2-dependent mechanisms of NK cell activation and proliferation and thus may play an important physiologic role in vivo.