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The purpose of this study is to assess the role of nitric oxide (NO) in the intestinal lesions of passive anaphylaxis, since this experimental model resembles necrotizing enterocolitis. Sprague-Dawley rats were sensitized with IgE anti-dinitrophenol monoclonal antibody. Extravasation of protein-rich plasma and haemorrhagia were measured in the small intestine. Plasma histamine was measured to assess mast cell activation. The effect of exogenous NO on the lesions was assessed by using two structurally unrelated NO-donors: sodium nitroprusside and S-nitroso-Nacetyl-penicillamine (SNAP). An increased basal production of NO was observed in cells taken after anaphylaxis, associated with a reduced response to platelet-activating factor, interleukin 1beta, and IgE/DNP-bovine serum albumin complexes. The response to bacterial lipopolysaccharide and dibutyryl cyclic adenosine monophosphate (AMP) was enhanced 24 h after challenge, but at earlier times was not significantly different from that observed in controls. Treatment with either sodium nitroprusside or SNAP produced a significant reduction of the haemorrhagic lesions, which are a hallmark of rat anaphylaxis. The extravasation of protein-rich plasma was not influenced by NO-donors. The increase of plasma histamine elicited by the anaphylactic challenge was not influenced by SNAP treatment. NO-donors protect intestinal haemorrhagic lesions of rat anaphylaxis by a mechanism apparently independent of mast cell histamine release.

Objetive: The properties of the materials that constitute the bone tissue are decisive in its mechanical strength but the factors that influence it are partially unknown at present. Material and methods: In this paper, we gauge bone hardness by means of ultra-microindentation tests with a Berkovich tip and a 150 mN load in femurs of Sprague-Dawley rats subjected to a transverse fracture or a subtraction osteotomy. The results are compared in different bone locations and experimental groups. The study includes the following four experimental groups, each consisting of four rats: a) standard diaphyseal fracture; b) fracture plus osteotomy of 2 mm; c) osteotomy treated with human parathyroid hormone, PTH (1-84); d) osteotomy treated with strontium ranelate. Results:We found the hardness of the material was consistently greater in cortical bone than in trabecular bone. It was also consistently higher in the upper femoral epiphyses than in the lower epiphyses (difference of 1.2 standard deviations). The surgery reduced hardness in the operated femur (difference of 0.3 standard deviations, p=5.5 x10-2). PTH treatment induced a slight but consistent increase in hardness at all sites (p=1.8x10-5) while the effect of strontium ranelate was inconsistent. Conclusions: These data show that tissue micro-hardness is influenced by a variety of factors, including anatomy, type of bone tissue, skeletal injury and drug therapy. Therefore, future studies on tissue quality should be carefully designed with these factors in mind. [ABSTRACT FROM AUTHOR]

Background: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function., Results: In this study, we identify in T lymphocytes several adaptor proteins as potential substrates targeted by LYP, including FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2. We also show that LYP co-localizes with SLP76 in microclusters, upon TCR engagement., Conclusions: These data indicate that LYP may modulate T cell activation by dephosphorylating several adaptor proteins, such as FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2 upon TCR engagement., (© 2024. The Author(s).)

Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains β-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U- 13 C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing 13 C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of 13 C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the TCA cycle. Zymosan exhausted NAD + and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD + and poly(ADP-ribose)., Competing Interests: Declaration of competing interest The authors declare that there are not conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) was discovered when the mechanisms involved in the deposition of immune complex in tissues were being scrutinized in the experimental model of rabbit serum sickness. The initial adscription of PAF to IgE-dependent anaphylaxis was soon extended after disclosing its release from phagocytes stimulated by calcium mobilizing agents, formylated peptides, and phagocytosable particles. This explains why ongoing research in the field turned to the analysis of immune cell types and stimuli involved in PAF production with the purpose of establishing its role in pathology. This was spurred by the identification of the chemical structure of PAF and the enzymic mechanisms involved in its biosynthesis and degradation, which showed commonalities with those involved in eicosanoid production and the Lands' cycle of phospholipid fatty acid remodeling. The reassignment of PAF function in immunopathology is explained by the finding that the most robust mechanisms leading to PAF production are associated with opsonic and non-opsonic phagocytosis, depending on the cell type. While polymorphonuclear leukocytes exhibit opsonic phagocytosis, monocyte-derived dendritic cells show a marked preference for non-opsonic phagocytosis associated with C-type lectin receptors. This is particularly relevant to the defense against fungal invasion and explains why PAF exerts an autocrine feed-forwarding mechanism required for the selective expression of some cytokines., (© 2022 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis., (© 2022. The Author(s).)

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-β secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E 2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Increased glycolysis parallels immune cell activation, but the role of pyruvate remains largely unexplored. We found that stimulation of dendritic cells with the fungal surrogate zymosan causes decreases of pyruvate, citrate, itaconate, and α-ketoglutarate, while increasing oxaloacetate, succinate, lactate, oxygen consumption, and pyruvate dehydrogenase activity. Expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on pyruvate dehydrogenase activity. Mechanistically, pyruvate reinforced histone H3 acetylation, and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because it is a substrate of the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator platelet-activating factor (PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) showed reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and its ensuing autocrine function. Acetyl-CoA therefore intertwines fatty acid remodeling of glycerophospholipids and energetic metabolism during cytokine induction., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

High-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with high-risk hematological malignancies. Polymorphisms for the pentraxin-3 gene ( PTX3 ) showed a significant association with the risk of fungal infection by Candida spp. and, especially, by Aspergillus spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi. CLEC7A polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The PTX3 mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed Candida albicans , thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome. PTX3 mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients., (Copyright © 2018 American Society for Microbiology.)

Objective- Calcific aortic valve disease is the most prevalent valvulopathy in Western countries. An unanticipated pathogenetic clue involving IFN (interferon) was disclosed by the finding of constitutive type I IFN activity associated with aortic valve calcification in children with the atypical Singleton-Merten syndrome. On this basis, the role of type I IFN on inflammation and calcification in human aortic valve interstitial cells (AVIC) was examined. Approach and Results- IFN-α was weakly proinflammatory but potentiated lipopolysaccharide-mediated activation of NF (nuclear factor)-κB and the ensuing induction of proinflammatory molecules in human AVIC. Stimulation with IFN-α and in combination with lipopolysaccharide promoted osteoblast-like differentiation characterized by increased osteoblastic gene expression, BMP (bone morphogenetic protein)-2 secretion, and ectopic phosphatase activity. Sex differences were observed. Likewise, IFN-α treatment of human AVICs in osteogenic medium resulted in increased formation of calcific nodules. Strikingly, IFN-α-mediated calcification was significantly higher in AVICs from males, and was blocked by tofacitinib, a JAK (Janus kinase) inhibitor, and by a BMP antagonist. A female-specific protective mechanism involving the activation of PI3K-Akt (protein kinase B) pathways and cell survival was disclosed. Females exhibited higher levels of BCL2 in valve cells and tissues and lower annexin V staining on cell stimulation. Conclusions- IFN-α acts as a proinflammatory and pro-osteogenic cytokine in AVICs, its effects being potentiated by lipopolysaccharide. Results also uncovered sex differences with lower responses in female AVICs and sex-specific mechanisms involving apoptosis. Data point to JAK/STAT (signal transducer and activator of transcription) system as a potential therapeutic target for calcific aortic valve disease.

Inflammation, the primary response of innate immunity, is essential to initiate the calcification process underlying calcific aortic valve disease (CAVD), the most prevalent valvulopathy in Western countries. The pathogenesis of CAVD is multifactorial and includes inflammation, hemodynamic factors, fibrosis, and active calcification. In the development of CAVD, both innate and adaptive immune responses are activated, and accumulating evidences show the central role of inflammation in the initiation and propagation phases of the disease, being the function of Toll-like receptors (TLR) particularly relevant. These receptors act as sentinels of the innate immune system by recognizing pattern molecules from both pathogens and host-derived molecules released after tissue damage. TLR mediate inflammation via NF-κB routes within and beyond the immune system, and play a crucial role in the control of infection and the maintenance of tissue homeostasis. This review outlines the current notions about the association between TLR signaling and the ensuing development of inflammation and fibrocalcific remodeling in the pathogenesis of CAVD. Recent data provide new insights into the inflammatory and osteogenic responses underlying the disease and further support the hypothesis that inflammation plays a mechanistic role in the initiation and progression of CAVD. These findings make TLR signaling a potential target for therapeutic intervention in CAVD.

Cyclic AMP regulatory element binding protein and signal transducer and activator of transcription 3 (STAT3) may control inflammation by several mechanisms, one of the best characterized is the induction of the expression of the anti-inflammatory cytokine interleukin-10 (IL-10). STAT3 also down-regulates the production of pro-inflammatory cytokines induced by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, a mechanism termed cross-inhibition. Because signalling via ITAM-dependent mechanisms is a hallmark of fungal pattern receptors, STAT3 activation might be involved in the cross-inhibition associated with invasive fungal infections. The fungal surrogate zymosan produced the phosphorylation of Y705-STAT3 and the expression of Ifnb1 and Socs3, but did not induce the interferon (IFN)-signature cytokines Cxcl9 and Cxcl10 in bone marrow-derived dendritic cells. Unlike lipopolysaccharide (LPS), zymosan induced IL-10 and phosphorylated Y705-STAT3 to a similar extent in Irf3 and Ifnar1 knockout and wild-type mice. Human dendritic cells showed similar results, although the induction of IFNB1 was less prominent. These results indicate that LPS and zymosan activate STAT3 through different routes. Whereas type I IFN is the main effector of LPS effect, the mechanism involved in Y705-STAT3 phosphorylation by zymosan is more complex, cannot be associated with type I IFN, IL-6 or granulocyte-macrophage colony-stimulating factor, and seems dependent on several factors given that it was partially inhibited by the platelet-activating factor antagonist WEB2086 and high concentrations of COX inhibitors, p38 mitogen-activate protein kinase inhibitors, and blockade of tumour necrosis factor-α function. Altogether, these results indicate that fungal pattern receptors share with other ITAM-coupled receptors the capacity to produce cross-inhibition through a mechanism involving STAT3 and induction of SOCS3 and IL-10, but that cannot be explained through type I IFN signalling., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: This study aimed to set up an experimental model of long bone atrophic nonunion and to explore the potential role of PTH-1-84 (PTH 1-84) and strontium ranelate (SrR). A model of atrophic nonunion was created in Sprague-Dawley rats at the femoral midshaft level. The animals were randomised into four groups. Group A1: control rodents, fracture without bone gap; Group A2: rodents with subtraction osteotomy (non-union model control) treated with saline; Group B: rodents with subtraction osteotomy treated with human-PTH (PTH 1-84); and Group C: rodents with subtraction osteotomy treated with strontium ranelate (SrR). The groups were followed for 12 weeks. X-rays were be obtained at weeks 1, 6 and 12. After sacrificing the animals, we proceeded to the biomechanical study and four point bending tests to evaluate the resistance of the callus and histological study. In second phase, the expression of genes related to osteoblast function was analysed by reverse transcription-quantitative PCR in rats subjected to substraction osteotomy and treated for 2 weeks. The animals were randomised into three groups: Group A2: rodents treated with saline; Group B: rodents treated with PTH 1-84 and Group C: rodents treated with SrR., Results: No significant histological differences were found between animals subjected to subtraction osteotomy and treated with either saline or PTH (p=0.628), but significant difference existed between animals receiving saline or SrR (p=0.005). There were no significant differences in X-ray score between the saline and PTH groups at either 6 or 12 weeks (p=0.33 and 0.36, respectively). On the other hand, better X-ray scores were found in the SrR group (p=0.047 and 0.006 in comparison with saline, at 6 and 12 weeks, respectively). In line with this, biomechanical tests revealed improved results in the SrR group. Gene expression analysis revealed a slightly decreased levels of DKK1, a Wnt pathway inhibitor, in rats treated with SrR., Conclusions: SrR increases has a beneficial effect in this atrophic non-union model in rats. This suggests that it might have a role may have important implications for the potential clinical role in the treatment of fracture nonunion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) is an adaptor protein associated with the cytoskeleton that is mainly expressed in hematopoietic cells. Mutations in PSTPIP1 cause the rare autoinflammatory disease called pyogenic arthritis, pyoderma gangrenosum, and acne. We carried out this study to further our knowledge on PSTPIP1 function in T cells, particularly in relation to the phosphatase lymphoid phosphatase (LYP), which is involved in several autoimmune diseases. LYP-PSTPIP1 binding occurs through the C-terminal homology domain of LYP and the F-BAR domain of PSTPIP1. PSTPIP1 inhibits T-cell activation upon T-cell receptor (TCR) and CD28 engagement, regardless of CD2 costimulation. This function of PSTPIP1 depends on the presence of an intact SH3 domain rather than on the F-BAR domain, indicating that ligands of the F-BAR domain, such as the PEST phosphatases LYP and PTP-PEST, are not critical for its negative regulatory role in TCR signaling. Additionally, PSTPIP1 mutations that cause the pyogenic arthritis, pyoderma gangrenosum and acne syndrome do not affect PSTPIP1 function in T-cell activation through the TCR., (© 2014 FEBS.)

Eicosanoids tailor the innate immune response by supporting local inflammation and exhibiting immunomodulatory properties. Prostaglandin (PG) E2 is the most abundant eicosanoid in the inflammatory milieu due to the robust production elicited by pathogen-associated molecular patterns on cells of the innate immune system. The different functions and cell distribution of E prostanoid receptors explain the difficulty encountered thus far to delineate the actual role of PGE2 in the immune response. The biosynthesis of eicosanoids includes as the first step the Ca(2+)- and kinase-dependent activation of the cytosolic phospholipase A2, which releases arachidonic acid from membrane phospholipids, and later events depending on the transcriptional regulation of the enzymes of the cyclooxygenase routes, where PGE2 is the most relevant product. Acting in an autocrine/paracrine manner in macrophages, PGE2 induces a regulatory phenotype including the expression of interleukin (IL)-10, sphingosine kinase 1, and the tumor necrosis factor family molecule LIGHT. PGE2 also stabilizes the suppressive function of myeloid-derived suppressor cells, inhibits the release of IL-12 p70 by macrophages and dendritic cells, and may enhance the production of IL-23. PGE2 is a central component of the inflammasome-dependent induction of the eicosanoid storm that leads to massive loss of intravascular fluid, increases the mortality rate associated with coinfection by Candida ssp. and bacteria, and inhibits fungal phagocytosis. These effects have important consequences for the outcome of infections and the polarization of the immune response into the T helper cell types 2 and 17 and can be a clue to develop pharmacological tools to address infectious, autoimmune, and autoinflammatory diseases.

The protein tyrosine phosphatase LYP, a key regulator of TCR signaling, presents a single nucleotide polymorphism, C1858T, associated with several autoimmune diseases such as type I diabetes, rheumatoid arthritis, and lupus. This polymorphism changes an R by a W in the P1 Pro rich motif of LYP, which binds to CSK SH3 domain, another negative regulator of TCR signaling. Based on the analysis of the mouse homologue, Pep, it was proposed that LYP and CSK bind constitutively to inhibit LCK and subsequently TCR signaling. The detailed study of LYP/CSK interaction, here presented, showed that LYP/CSK interaction was inducible upon TCR stimulation, and involved LYP P1 and P2 motifs, and CSK SH3 and SH2 domains. Abrogating LYP/CSK interaction did not preclude the regulation of TCR signaling by these proteins.

Contact of apoptotic cells (AC) with phagocytes tilts the balance of pro-inflammatory and anti-inflammatory cytokines. To address the cell- and stimulus-dependency of this mechanism, human monocyte-derived dendritic cells were treated with Jurkat AC in the presence and absence of different stimuli. AC reduced the production of IL-23 and enhanced the production of IL-10 elicited by zymosan, but they did not influence IL-12 p70 production nor did they modify the effect of LPS. Since formation of lipid bodies (LB) and PGE(2) production have been associated with IL-10 induction, the effect of PGE(2), the formation of LB, and the role of PPAR-γ were assessed. Exogenous PGE(2) enhanced IL-10 expression, but no evidence of PGE(2) production elicited by AC was obtained. Inhibition of PPAR-γ activity reduced the production of IL-10 both in the presence and in the absence of AC, but formation of LB in response to zymosan and AC was not observed. Notably, AC induced a transient nuclear translocation of both the CREB coactivator CRTC2/TORC2 and the homeodomain protein PBX1, which are involved in the CREB/HOX/PBX/MEIS transcription complex. These data show a selective effect of AC on the production of cytokines elicited by the fungal surrogate zymosan through the enhancement of CREB-dependent transcription., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, sex ratio, spawning season, fork length (L(F)) at maturity (L(F50)), batch fecundity and spawning frequency were characterized for the continental population of Ocyurus chrysurus from the Campeche Bank, in the southern Gulf of Mexico. A total of 1657 specimens were collected from February 2008 to January 2009. The overall sex ratio (male:female) and sex ratios by size-class showed no significant differences from an expected 1:1 ratio. The Campeche Bank population did not conform to the reproductive seasonality pattern characteristic of a continental population. A protracted spawning season that extended from January to September with peaks occurring mainly between April and May and additionally in September was observed. The population conformed, however, to the sexual maturity pattern observed for populations and species associated with a continental margin. Fish of both sexes reached the onset of sexual maturity at a similar and small L(F) of c. 14 cm, and L(F50) (L(F) at which 50% of females and males become mature) was 21·3 and 19·4 cm. Asynchronous-type ovarian development was observed for this species and batch fecundity estimates ranged from 14,102 to 164,756 oocytes (mean ±S.D. = 43,852 ± 32,684 oocytes). The overall spawning frequency estimate was once every 8·3 days or 26 times during the 9 month spawning season., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-γ. These results indicate that the selective induction of IL-23 by β-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.

Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated kappaB-binding activity, but inhibition of NF-kappaB was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE(2), and inhibitors of PKA, COX, and glycogen-synthase kinase-3beta showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE(2) and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3beta, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.

Cyclooxygenase (COX)-2 is a central enzyme of arachidonic acid metabolism, and its modulation by statins may explain some of the myocardial protective effects of these drugs. Dendritic cells (DCs) play a central role in microbial defense and in atherogenesis, and COX-2 expression in DCs is important for their migration to lymph nodes and antibody response, thus explaining why prostaglandin E(2) is a main component of the cocktails used to prepare DCs for clinical applications. On this basis, we addressed the effect of atorvastatin (ATV) on the release of arachidonic acid and on the expression of COX-2 in human monocyte-derived DCs. Although ATV on its own lacked any effect on COX-2 protein induction expression, it enhanced the release of arachidonic acid, the expression of COX-2 protein, and the production of prostaglandin E(2) induced by the fungal wall extract zymosan, and to a lower extent the effect of peptidoglycan. The effect on COX-2 protein was observed mainly 24 h after stimulation by zymosan and was not reverted by mevalonate, thus pointing to an effect unrelated to cholesterol metabolism. It is noteworthy that COX-2 protein showed a great stability, with a t((1/2)) of approximately 12 h, which was enhanced in the presence of ATV. In view of the important role played by COX-2 on DC function, these data indicate that ATV, by enhancing COX-2 stability, may increase DC function after infectious bouts and also counteract some of the risks associated with sustained inhibition of COX-2.

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

Francisella tularensis is a virulent Gram-negative intracellular pathogen. To address the signaling routes involved in the response of host cells to LPS from F. tularensis live vaccine strain (LVS), experiments were performed in transiently transfected 293 cells. Induction of kappaB-driven transcriptional activity by 2.5 mug ml(-1) F. tularensis LPS isolated by phenol-water and ether-water extraction, was observed in cells transfected with Toll-like receptor (TLR) 4 and MD-2, although CD14 was required for optimal induction. Conversely, TLR2, TLR2/TLR1 or TLR2/TLR6 transfected cells did not show kappaB-driven transcriptional activity in the presence of F. tularensis LPS. In human monocytic cells, F. tularensis LPS activated extracellular signal-regulated kinases and the production of pro-inflammatory proteins. Concentrations of 5-10 mug ml(-1) F. tularensis LPS elicited a similar pattern of mRNA and protein induction than 0.1 mug ml(-1) E. coli LPS, including the expression of CXC chemokines (IL-8, Gro and IFN-gamma-inducible protein-10); CC chemokines (monocyte chemoattractant protein-1 and -2, macrophage-derived chemoattractant, macrophage inflammatory protein-1alpha and -1beta and RANTES (regulated upon activation, normal T cell expressed and secreted) and pro-inflammatory cytokines (IL-6 and tumor necrosis factor alpha). Altogether, these data indicate that LPS from F. tularensis LVS signals via TLR4 at higher concentrations than those required for E. coli LPS, which may explain the inflammatory reaction and the low endotoxic response associated to vaccination with LVS in humans.

Antigen-antibody complexes can damage tissues by triggering inflammation. Recent studies have enabled the description of a sequence of steps, which depend on the intra- or perivascular location of complex formation. Acute lethal toxicity and circulatory shock as a result of the acute release of inflammatory mediators can occur after intravascular complex formation. The lesions associated with perivascular complexes are characterized by plasma leakage and the recruitment of polymorphonuclear leukocytes. These lesions are modulated by mediators released from endothelial cells, namely nitric oxide, endothelins and lipid mediators, and provide an appropriate basis for the activation of both arms of hemostasis: coagulation and fibrinolysis. The balance between both activation systems can explain the late occurrence of both tissue fibrosis and organ remodeling.

Antigen-antibody complexes can be formed both intravascularly and perivascularly and damage tissues by inducing inflammatory mechanisms. Recent studies have characterized a definite sequence of steps involved in these inflammatory mechanisms, and identified the predominance of particular chemical mediator(s) in each step. The lesions associated with this type of inflammation are characterized by the early development of plasma leakage, followed by the recruitment of polymorphonuclear leukocytes mediated by chemokines generated by FcgammaR-dependent mechanisms. The development of these lesions is modulated by endothelial cell-derived paracrine mediators, and activation of the coagulation system can ensue. The activation of platelets and coagulation, if not properly counterbalanced by fibrinolysis, might be a major factor for the late development of fibrotic changes and organ remodeling.

Group IIA secreted phospholipase A(2) (sPLA(2)-IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A(2), which shows properties different from those displayed by the cytosolic phospholipase A(2) involved in the release of arachidonic acid. sPLA(2)-IIA behaves as a ligand for a group of receptors inside the C-type multilectin mannose receptor family and also interacts with heparan sulfate proteoglycans such as glypican, the dermatan/chondroitin sulfate-rich decorin, and the chondroitin sulfate-rich versican, thus being able to internalize to specific compartments within the cell and producing biological responses. This review provides a short summary of the biological actions of sPLA(2)-IIA on intracellular signaling pathways.

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcgammaRI, FcgammaRIIA, and FcgammaRIII, LPS, TNF-alpha, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcgammaR with mAb reacting with FcgammaRI and FcgammaRIIA, but not with a mAb reacting with FcgammaRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcgammaRI and FcgammaRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcgammaR cross-linking, although expression of IFN-gamma-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1alpha, MIP-1beta, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1alpha using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein beta sites and ruled out the functional significance of both NF-AT and AP-1 sites.

THP-1 monocytic cells were stimulated with IgG-ovalbumin equivalence immune complexes (IC) and mAb reacting with both FcgammaRI and FcgammaRIIA. All of these stimuli were capable of activating the cells; however, different patterns of response were observed as regards activation of the p42-MAP/ERK kinase, triggering of the NF-kappaB/Rel system, production of chemotactic cytokines, and induction of the expression of cyclooxygenase-2 (COX-2). Activation of p42-MAP/ERK kinase was a constant finding, which occurred regardless of the type of stimulus applied, for instance, homotypic stimulation of a single type of receptor by cross-linking with specific mAb or heterotypic stimulation with both types of antibodies and IC. However, the activation of the MAP/ERK kinase cascade was not connected to the triggering of cytosolic phospholipase A(2) (cPLA(2)) and arachidonic acid (AA) release. The heterotypic stimulation of FcgammaR induced the expression of COX-2 in a time and dose-dependent manner and activated the NF-kappaB system as judged from the degradation of IkappaB-alpha protein. In summary, the present data indicate that activation of the p42-MAP/ERK pathway occurs after cross-linking FcgammaRI and FcgammaRIIA receptors in monocytic cells; however, this is not coupled to the cPLA(2) route, which leads to the release of AA. Noteworthy,heterotypic activation involving combined cross-linking of both FcgammaRI and FcgammaRIIA has a robust effect on the oxidative metabolism of AA by a mechanism involving kappaB-dependent trans-activation of COX-2.

Human brucellosis is characterized by the presence of both acute inflammatory episodes and chronic inflammation with granuloma formation. On this basis, the proinflammatory effects of smooth lipopolysaccharide of Brucella (S-LPS) were addressed and compared to those of LPS from Escherichia coli. For this purpose, the induction of cyclooxygenase-2 (COX-2), the production of the chemokine monocyte chemoattractant protein-1 (MCP-1), and the activation of the nuclear factor kappa B (NF-kappa B) were studied. S-LPS was found to induce both COX-2 expression and MCP-1 production; however, the potency of E. coli LPS exceeded that of Brucella S-LPS by some orders of magnitude. However, at concentrations above 1 microg/ml, all of the LPS produced comparable effects, including their ability to activate the NF-kappa B system. These observations help explain the inflammatory events associated with Brucella infection and the ability of Brucella to produce monocyte recruitment and granuloma formation.

Astrocytes constitute the most abundant cell type in the nervous system. Under physiological conditions, they respond to the stimuli to which neurons are also responsive. The use of astrocytoma cell lines with well-defined morphological and functional markers has been helpful for addressing the mechanisms of signal transduction that operate in the nervous system. On the basis of the effects produced by agonists of different types of receptor (muscarinic ACh receptors, thrombin receptors, phospholipases A2 receptors and tumor necrosis factor alpha receptors), several different transcriptional programs that involve the MAP kinase-cytosolic phospholipase A2 system and the transcription factor NF-kappaB have been described.

Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucella in humans.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that elicits cell responses by activating the mitogen-activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor-kappaB (NF-kappaB). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2), the effect of TNF-alpha on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF-alpha produced a phosphorylation of cPLA2, which was preceded by an activation of both c-Jun N-terminal kinase (JNK) and p38-MAP kinase, and this was associated with the release of [3H]AA. In contrast, TNF-alpha did not activate the extracellular signal-regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic response. Analysis of [3H]AA metabolites by reverse-phase HPLC showed that all of the [3H]AA released during the first hour after TNF-alpha addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF-alpha, 25% of the [3H]AA had been converted into COX products as compared with only 9% in control cells. In keeping with these findings, TNF-alpha produced an increase of COX-2 expression, as judged from both RT-PCR studies and immunoblot of COX-2 protein, and a long-lasting activation of NF-kappaB. These data show that TNF-alpha produces in astrocytoma cells an early activation of both p38-MAP kinase and JNK, which is followed by the phosphorylation of cPLA2 and the release of AA. On the other hand, the activation of NF-kappaB may explain the induction of the expression of COX-2 and the delayed generation of prostanoids.

The effect of secretory phospholipase A2 (sPLA2) on intracellular Ca2+ signaling in human astrocytoma cells was studied. sPLA2 increased cytosolic [Ca2+] ([Ca2+]c) in both Ca2+-containing and Ca2+-free medium, thus suggesting Ca2+ release from intracellular stores. The activation by sPLA2 of arachidonate release via cytosolic PLA2 (cPLA2) was also independent of extracellular Ca2+. As sPLA2 requires Ca2+ for activity, these results indicate that both Ca2+ mobilization and cPLA2 activation induced by sPLA2 are unrelated to phospholipase activity but dependent on signaling mechanisms. The sPLA2-induced [Ca2+]c peak was sensitive to Bordetella pertussis toxin and inhibited by caffeine, suggesting its mediation by inositol 1,4,5-trisphosphate (IP3). sPLA2 induced tyrosine phosphorylation and membrane targeting of phospholipase Cgamma-1 (PLCgamma-1). Moreover, the Ca2+ peak was sensitive to protein tyrosine kinase inhibitors. sPLA2 activates two signaling pathways: one leading to the activation of the MAP kinase/cPLA2 cascade and another leading to PLCgamma activation and Ca2+ release., (Copyright 1999 Academic Press.)

1. The effect of two derivatives of salicylate, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal), on the activation of NF-kappaB elicited by tumour necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVEC) was tested. 2. The expression of the mRNA of vascular cell adhesion molecule-1 (VCAM-1) was studied as an example of a gene the expression of which is regulated by NF-kappaB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. 3. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF-kappaB. The calculation of the IC50 values showed approximately 2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. 4. Comparison of the potency of these compounds on VCAM-1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36-43% inhibition at the same concentration. 5. Inhibition of NF-kappaB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose-dependent inhibition of nitrite production by the L-arginine pathway via iNOS. IC50 values for this effect were 1.13+/-0.12 mM (triflusal), 1.84+/-0.34 (HTB), 6.08+/-1.53 mM (aspirin) and 9.16+/-1.9 mM (salicylate). 6. These data indicate that the incorporation of a 4-trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF-kappaB activation, VCAM-1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo-oxygenase.

Deposition of immune complexes in tissues is the pathogenic mechanism underlying tissue injury in a number of diverse clinical conditions affecting the skin, joints, blood vessels and renal glomeruli. Initial approaches to the understanding of these conditions have stressed the roles of both the activation of the complement system and the accumulation of polymorphonuclear leukocytes as the main molecular and cellular mechanisms explaining the sequence of events leading to tissue damage. Recent findings on (i) the molecular biology of the leukocyte chemoattractants, (ii) the chemical structure and function of receptors for the Fc portion of the antibody molecule and (iii) the signaling events coupled to the engagement of these receptors have led to an understanding of the biochemical events involved in immune-complex injury and have provided a promising avenue for the development of therapeutic approaches. This review will focus on our current understanding of signal transduction events in the effector phase of immune-complex-mediated tissue injury.

Objective and Design: Since IgE-dependent reactions induce the inducible isoform of NO synthase, we postulated the involvement of the transcription factor NF-kappaB., Materials: 72 Wistar rats were divided into 6 groups and used to study the hemorrhagic necrosis of the small intestine elicited by anaphylaxis., Treatment: Passive anaphylaxis was produced by i.p. sensitization with IgE anti-dinitrophenyl monoclonal antibody and i.v. challenge with the cognate antigen., Methods: Competitive PCR was used to assay the expression of p50 subunit of NF-kappaB. kappaB-binding activity was assayed by electrophoretic mobility shift assay., Results: The PCR assay showed a time-dependent increase of mRNA coding for the p50 subunit of NF-kappaB, which was maximal 1 h after challenge (40+/-3, versus 230+/-32 fM, mean +/- SEM) and decreased to prechallenge level at 4h. kappaB-binding activity was also increased., Conclusions: IgE-mediated reactions trigger a pathway for nuclear signaling that seems to be related to the intermediate/late response of the allergic reaction.

The production of nitric oxide (NO) by rat adherent peritoneal cells stimulated with preformed IgE/Dinitrophenyl-BSA (DNP-BSA) complexes and its dependence on the activation of the transcription factor NF-kappa B were studied. Stimulation with IgE/DNP-BSA complexes at equivalence induced both the production of NO and an increased expression of the inducible isoform of NO synthase (iNOS) protein. Both events were also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23, thus suggesting Fc epsilon RII/CD23 antigen as the IgE-binding structure involved in the triggering of the response and ruling out an interaction of the antibody via its Fc portion. Inhibition of redox-sensitive signaling mechanisms by the antioxidant pyrrolidine dithiocarbamate (PDTC) blocked NO production, iNOS expression, and NF-kappa B activation elicited by both IgE/DNP-BSA complexes and anti-CD23 F(ab')2, thus suggesting the involvement of NF-kappa B in the signaling pathway leading to the transcriptional activation of iNOS. These results show the existence in rat peritoneal macrophages of a signaling pathway triggered by CD23 engagement that promotes nuclear translocation of NF-kappa B and transcriptional activation of the inducible isoform of NO synthase.

The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways.

The induction of nitric oxide (NO) production and the expression of cytokine-induced neutrophil chemoattractant (CINC-1) were studied in rat peritoneal adherent cells stimulated with insoluble immune complexes containing rabbit IgG Ab and OVA as the cognate Ag (IC). Incubation with IC at concentrations as low as 10 microg/ml induced NO production and the expression of inducible NO synthase (iNOS) protein. This was accompanied by the expression of CINC-1 mRNA and the activation of nuclear factor-kappaB (NF-kappaB). However, the expression of iNOS and CINC-1 mRNA induced by IC showed a different temporal pattern and a different sensitivity to both the antioxidant agent pyrrolidine dithiocarbamate (PDTC) and modulation by NO itself. Whereas iNOS mRNA and protein expression were blunted by PDTC and NO-generating compounds, CINC-1 mRNA expression was either enhanced or not affected by PDTC and NO donors. The time course of NF-kappaB activation was parallel to that of iNOS induction and was influenced in the same sense as iNOS induction by antioxidants, NO donors, the protease inhibitor N-tosyl phenylalanine chloromethyl ketone, and inhibitors of protein tyrosine phosphorylation reactions. These data indicate the existence in rat macrophages of a signaling mechanism triggered by Fc gammaR occupancy that leads to nuclear signaling, is initiated by protein tyrosine phosphorylation reactions, and shows specific sensitivities to antioxidants and NO. Whereas trans-activation of the iNOS gene can be fully explained by the stimulation of NF-kappaB, induction of CINC-1 mRNA expression seems influenced by additional regulatory elements.

The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP-specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes.

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

1. The effector mechanism of intestinal necrosis in rat anaphylaxis was studied following several complementary approaches: (i) the use of monoclonal antibodies (mAb) belonging to different classes (IgG1, IgG2b and IgE anti-DNP), (ii) the assay of mediators, and (iii) the use of pharmacological tools. 2. Lethality and haemorrhagic necrosis of the small intestine were observed in IgE-sensitized rats, whereas IgG mAb produced milder physiological disturbances. 3. Inhibition of leukotriene biosynthesis reduced the drop of systemic blood pressure (BP) and the extent of protein-rich plasma exudation but it did not influence the haemorrhagic component of intestinal necrosis. 4. The antihistamine, pyrilamine, partially diminished the haemorrhagic component of the intestinal necrosis. 5. The involvement of mediators related to platelet-activating factor (PAF) was studied by examining the pharmacological effects of these autacoids and of PAF-receptor antagonists (PCA4248, UR12460 and BB823). PAF induced intestinal lesions similar to those observed in IgE-sensitized rats and PAF-receptor antagonists markedly decreased haemorrhage in IgE-sensitized rats. 6. PAF levels were transiently increased after dinitrophenol (DNP)- bovine serum albumin (BSA) challenge in the small intestine of IgE-sensitized rats. 7. These data stress differences in the outcome of anaphylaxis related to the type of receptors for the Fc portion of immunoglobulins that are involved. IgE is the antibody class that elicits the most severe response due to the activation of mast cells via Fc epsilon RI (surface receptors that bind IgE antibodies with high affinity), and the only one able to produce intestinal haemorrhagic necrosis. 8. The mast-cell-derived mediators PAF/acyl-PAF and histamine, most probably associated with tumour necrosis factor alpha/cachectin (TNF-alpha), seem to play a central role in the production of the vascular changes required for the extravasation of erythrocytes in the small intestine mucosa.

The production of platelet-activating factor (PAF) and the release of [3H]arachidonate were studied in human polymorphonuclear leukocytes (PMN) stimulated with thapsigargin, an inhibitor of endomembrane Ca(2+)-ATPase. Concentrations of thapsigargin as low as 10-25 nM primed PMN for both PAF production and [3H]arachidonate release in response to the chemotactic peptide (fMLP), whereas concentrations in the range 25-200 nM induced a time- and dose-dependent production of PAF, which occurred in the absence of both [3H]arachidonate release and [3H]phosphatidylethanol formation. Studies in fura-2/AM-loaded cells showed that concentrations of thapsigargin that elicited PAF production induced a protracted and long lasting elevation of cytosolic free calcium concentration ([Ca2+]i) between 200 and 700 nM. The lower concentrations primed the cells for a late [Ca2+]i elevation in response to fMLP similar to that elicited by cytochalasin B or ionomycin. PAF production showed a good correlation with the increase of [Ca2+]i (r = 0.91) irrespective of the procedure used to grade [Ca2+]i. In contrast, phorbol 12,13-dibutyrate failed to induce both PAF production and elevation of [Ca2+]i, but it was a very effective stimulator of [3H]arachidonate release and [3H]phosphatidylethanol production. These data indicate that PAF production and [3H]arachidonate release in PMN differ in both biochemical pathway and modulatory mechanisms. Whereas PAF production seems extremely sensitive to changes in [Ca2+]i, which seems to exert its modulatory effect at the lyso-PAF:acetyl-CoA acetyltransferase step, [3H]arachidonate release seems tightly modulated by protein kinase C-dependent mechanisms and is coincidental with activation of phospholipase D.

Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor.

1. The role of platelet-activating factor (PAF) and peptidoleukotrienes as putative mediators of some of the vascular changes triggered by antigen was investigated in rats passively sensitized with monoclonal anti-DNP (2,4-dinitrophenyl) IgE. 2. Lethal anaphylaxis with respiratory distress, systemic hypotension, detachment of the intestinal mucosa, leukopenia and extravasation of protein-rich plasma was observed after antigen challenge of rats sensitized with partially purified monoclonal IgE at concentrations of 15 mg protein kg-1. 3. Analysis of the peritoneal fluid obtained after i.v. challenge with DNP-BSA (bovine serum albumin) showed the presence of significant amounts of PAF (101 +/- 8 pg/rat), whereas this mediator was undetectable in control animals. Leukotriene D4 was the predominant peptidoleukotriene that could be recovered after antigen challenge, and showed an extremely high concentration (92 +2- 15 ng/rat) as compared to PAF levels. 4. Extravasation of protein-rich plasma was observed shortly after challenge and reached a maximum at 30 min. Treatment of animals with i.v. PCA 4248 (1-2 mg kg-1) and WEB 2086 (1 mg kg-1), two chemically unrelated compounds which are antagonists of the PAF-receptor, produced a significant reduction of the extravasation of protein-rich plasma. 5. The same degree of protection could be afforded by MK-886, an inhibitor of leukotriene biosynthesis. Combined treatment with WEB 2086 and MK-886 provided greater inhibition of protein-rich plasma extravasation than either compound alone. PCA 4248 was also found to inhibit in a dose-dependent manner the systemic hypotension observed upon DNP-BSA challenge.6. These data indicate that the lipid mediators PAF and peptidoleukotrienes are major effectors of the vascular disturbances observed in rat passive IgE-mediated anaphylaxis.