Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H 2 O 2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H 2 O 2 -induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with STATs (signal transducers and activators of transcription) are a class of transcription factors bearing SH2 domains that become activated upon tyrosine phosphorylation. STATs are often activated by members of the JAK family of protein-tyrosine kinases (PTKs) in response to cytokine stimulation. This activation mechanism involves the SH2 domain-dependent recruitment of the STATs to tyrosine-phosphorylated cytokine receptors. The STATs then become phosphorylated by receptorassociated JAKs, which induces their dimerization via reciprocal SH2-phosphotyrosine interaction. STAT dimers then enter the nucleus and bind to specific DNA elements, thereby activating the transcription of a number of genes. The JAK-STAT pathway has been the subject of many recent comprehensive reviews (17-21). STAT3, a well characterized 92-kDa protein, has been shown to become activated by both epidermal growth factor and interleukin-6 in human A-431 cells (22). Because the ROI generated in response to various external stimuli can play a role both as regulators of transcription factors, including nuclear factors B (2, 23) and AT (24), and as inhibitors of protein-tyrosine phosphatases (PTPases) (25-27), we have investigated whether H 2 O 2 and other oxidizing agents could modulate STAT3 function in human lymphocytes. Enhanced phosphotyrosine accumulation could then result from the combined effects of increased phosphorylation and decreased dephosphorylation. Moreover, the DNA binding activity of STATs is known to depend primarily on tyrosine phosphorylation (19, 28 -31), although serine phosphorylation is also important in modulating the binding affinity of STAT3 (32-34). Here we show for the first time that STAT3 is phosphorylated on tyrosine residue(s), translocated to the nucleus, and elicited to bind to specific DNA elements upon lymphocyte treatment with H 2 O 2 . An additive effect between H 2 O 2 and vanadate was also evidenced suggesting that inhibition of tyrosine phosphatase(s)

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with 35S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcεRI and galectin-3 (FcεRI>galectin-3), but not against FcεRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

SummaryIt has been demonstrated that neutrophils from healthy donors or from patients with inflammatory disorders can bind immunoglobulin (Ig) E proteins through binding to Mac-2/ebp. Functional responses to allergens were assessed by measuring the respiratory burst and intracellular Ca 2+ levels, and binding of allergens to neutrophils was assessed by flow cytometry analysis and fluorescence microscopy. In this article, we demonstrate that neutrophils sensitized to specific allergens (from allergic patients), but not from healthy donors, are sensitive to allergens of the same type as those that produce clinical allergic symptoms. The activation of neutrophils was analyzed by the induction of a respiratory burst that was detected with luminol-dependent chemiluminescence. Intracellular Ca 2 § levels increased parallel to those of the inducing allergens. In addition, the specific binding of allergens on the cell surface was revealed by flow cytometry and aUergen-FITC-labeled staining analyses. The present data suggest a restricted recognition of allergen by sensitive neutrophils, probably associated with the specific binding of the allergen to its corresponding IgE molecule, which is bound to the Mac-2/~bp structure. These findings demonstrate a functional role of allergen-associated neutrophils during the allergic state.

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca 2؉ , inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H 2 O 2 ), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H 2 O 2 . A similar inhibitory effect of H 2 O 2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-B in human neutrophils is inhibited by cell pretreatment with H 2 O 2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharideinduced NF-B/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H 2 O 2 -dependent NF-B inactivation in neutrophils occurs in concert with inhibition of calcineurin.