Regulation of K-Cl cotransport by Syk and Src protein tyrosine kinases in deoxygenated sickle cells

Protein tyrosine kinases (PTK) of the Src family are thought to suppress K-Cl cotransport (KCC) activity via negative regulation of protein phosphatases. However, some PTK inhibitors reduce KCC activity, suggesting opposite regulation by different PTK families. We have reported previously that deoxygenation of sickle cells stimulates KCC and activates Syk (a Syk family PTK), but not Lyn (an Src family PTK). In this study the same results were obtained when PTK activities were measured under the conditions used to measure KCC activity and which prevent any change in intracellular [Mg(2+)]. Methyl-2,5-dihydroxycinnamate (DHC), a PTK inhibitor, was more selective for Syk than Lyn, while staurosporine (ST), a broad-specificity protein kinase inhibitor, inhibited Lyn more than Syk. Deoxygenation or 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d] pyrimidine (pp2, a specific Src inhibitor) stimulated KCC independently. These effects were not additive and were inhibited by DHC. In contrast, ST-induced KCC activation was resistant to DHC, suggesting a different pathway of activation. Overall, these data indicate that Syk activity is required for KCC activation, either induced by deoxygenation of sickle cells, or mediated by Src inhibition in oxygenated cells, and that Syk and Src PTKs exert opposing and interconnected regulatory effects on the activity of the transporter.     

Rethinking the role of Src family protein tyrosine kinases in the allergic response

Antigen-induced cross-linking of immunoglobulin E (IgE) antibodies bound to the high-affinity IgE receptor (FcepsilonRI), on mast cells results in the release of mediators that initiate an inflammatory response. This normal immune response has been abducted by immunological adaptation, through the production of IgE antibodies to normally innocuous substances, to cause allergic disease. Therefore, understanding the molecular requirements in IgE-dependent mast-cell activation holds promise for therapeutic intervention in disease. Recent investigation on the functional coupling of FcepsilonRI to the intracellular signaling apparatus has provided paradigm-altering insights on the importance and function of Src family protein tyrosine kinases (Src PTK) in mast-cell activation. In this synopsis, we review the current knowledge on the role of the Src PTKs, Fyn and Lyn, in mast-cell activation and discuss the implications of our findings on allergic disease.     

Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury

Mer signaling participates in a novel inhibitory pathway in TLR activation. The purpose of the present study was to examine the role of Mer signaling in the down-regulation of TLR4 activation-driven immune responses in mice, i.t.-treated with LPS, using the specific Mer-blocking antibody. At 4 h and 24 h after LPS treatment, expression of Mer protein in alveolar macrophages and lung tissue decreased, sMer in BALF increased significantly, and Mer activation increased. Pretreatment with anti-Mer antibody did not influence the protein levels of Mer and sMer levels. Anti-Mer antibody significantly reduced LPS-induced Mer activation, phosphorylation of Akt and FAK, STAT1 activation, and expression of SOCS1 and -3. Anti-Mer antibody enhanced LPS-induced inflammatory responses, including activation of the NF-κB pathway; the production of TNF-α, IL-1β, and MIP-2 and MMP-9 activity; and accumulation of inflammatory cells and the total protein levels in BALF. These results indicate that Mer plays as an intrinsic feedback inhibitor of the TLR4- and inflammatory mediator-driven immune responses during acute lung injury.     

Intracellular sensing of microbes and danger signals by the inflammasomes

The cells of the innate immune system mobilize a coordinated immune response towards invading microbes and after disturbances in tissue homeostasis. These immune responses typically lead to infection control and tissue repair. Exaggerated or uncontrolled immune responses, however, can also induce acute of chronic inflammatory pathologies that are characteristic for many common diseases such as sepsis, arthritis, atherosclerosis, or Alzheimer's disease. In recent years, the concerted efforts of many scientists have uncovered numerous mechanisms by which immune cells detect foreign or changed self-substances that appear in infections or during tissue damage. These substances stimulate signaling receptors, which leads to cellular activation and the induction of effector mechanisms. Here, we review the role of inflammasomes, a family of signaling molecules that form multi-molecular signaling platforms and activate inflammatory caspases and interleukin-1β cytokines.     

Src kinase-mediated signaling in leukocytes

A concert of antigens, antibodies, cytokines, adhesion molecules, lipid factors, and their different receptors mediate leukocyte development and inflammatory responses. Regardless of the stimulus and receptor type, members of the Src family of protein tyrosine kinases (PTKs) play a critical role in initiating the numerous intracellular signaling pathways. Recruited and activated by the receptor, these Src PTKs amplify and diversify the signal. Multiple pathways arise, which affect cell migration, adhesion, phagocytosis, cell cycle, and cell survival. Essential nonredundant properties of Src PTKs have been identified through the use of gene targeting in mice or in the somatic cell line DT40. Because of their role in mediating leukocyte proliferation and activation, Src PTKs serve as excellent drug targets. Inhibitors of Src family members and dependent pathways may be useful in the treatment of human diseases similar to drugs known to inhibit other signal transduction pathways.     

Signaling through HLA-DR induces PKC beta-dependent B cell death outside rafts

Signals through HLA-DR molecules contribute to optimal activation of antigen-presenting cells (APC) during T cell/APC interactions participating in the generation of productive interactions, and to the induction of APC death, which has been postulated to play a role in the termination of the immune response. To understand how these molecules accommodate both cellular responses, we studied the not yet well-defined signaling events and the biochemical requirements for HLA-DR-mediated death. We demonstrate that in B cells the HLA-DR-activated protein kinase C (PKC) beta is required for HLA-DR-mediated death whereas the HLA-DR-activated Src family of PTK is redundant. In contrast to HLA-DR-mediated activation of Src kinase Lyn, the aggregation of HLA-DR molecules in lipid rafts is not required for HLA-DR-mediated PKC beta activation nor for the induction of cell death. Indeed, the bulk of HLA-DR-activated PKC beta reside outside rafts. This is the first report showing that HLA-DR-induced PKC beta activation is essential for the induction of B cell death via HLA-DR, and that these HLA-DR-mediated events do not require the integrity of rafts.     

The context of HLA-DR/CD18 complex in the plasma membrane governs HLA-DR-derived signals in activated monocytes

HLA-DR-derived signals in activated monocytes mediate both pro-inflammatory cytokine production and caspase-independent death, and have been postulated to play a role in inflammation and in its resolution, respectively. Herein, using the monocytic/macrophagic human cell line THP-1 primed with IFNgamma (IFNgamma-primed THP-1), we investigated how HLA-DR may integrate both signals. Our inhibition studies demonstrated that if cell death is dependent on PKCbeta activation, the induction of TNFalpha gene expression relies on PTK activation, in particular the Src family of kinases, but both cell responses implicate the beta2-integrin CD18. Accordingly, sequential immunoprecipitation experiments demonstrated that following engagement of HLA-DR on IFNgamma-primed THP-1 cells, the HLA-DR/CD18 complex physically associates with PKCbeta and with PTK. Pharmacological disruption of lipid rafts microdomains abolished the assembly of HLA-DR/CD18/PTK signaling complex, HLA-DR-mediated tyrosine activation, and the PTK-dependent TNFalpha expression in IFNgamma-primed THP-1 cells. In contrast, HLA-DR/CD18/PKCbeta complex was still formed and able to mediate cell death after cholesterol depletion of these cells. These results indicate that while the integrity of lipid rafts is necessary for the transduction of cytokine gene expression through the HLA-DR/CD18 complex, it is not necessary for the induction of the HLA-DR/CD18-dependent cell death. Thus, our study provides experimental evidence indicating the compartmentalization of HLA-DR/CD18 complex within or outside lipid rafts as a mechanism through which HLA-DR can integrate both PTK and PKCbeta signals leading to activation and death, respectively, of activated monocytes. This might provide new insights into how MHC class II signaling may regulate inflammatory response.     

Serum-circulating His-tRNA synthetase inhibits organ-targeted immune responses

His-tRNA synthetase (HARS) is targeted by autoantibodies in chronic and acute inflammatory anti-Jo-1-positive antisynthetase syndrome. The extensive activation and migration of immune cells into lung and muscle are associated with interstitial lung disease, myositis, and morbidity. It is unknown whether the sequestration of HARS is an epiphenomenon or plays a causal role in the disease. Here, we show that HARS circulates in healthy individuals, but it is largely undetectable in the serum of anti-Jo-1-positive antisynthetase syndrome patients. In cultured primary human skeletal muscle myoblasts (HSkMC), HARS is released in increasing amounts during their differentiation into myotubes. We further show that HARS regulates immune cell engagement and inhibits CD4⁺ and CD8⁺ T-cell activation. In mouse and rodent models of acute inflammatory diseases, HARS administration downregulates immune activation. In contrast, neutralization of extracellular HARS by high-titer antibody responses during tissue injury increases susceptibility to immune attack, similar to what is seen in humans with anti-Jo-1-positive disease. Collectively, these data suggest that extracellular HARS is homeostatic in normal subjects, and its sequestration contributes to the morbidity of the anti-Jo-1-positive antisynthetase syndrome.     

IL-17 Producing γδ T Cells are Required for a Controlled Inflammatory Response after Bleomycin-induced Lung Injury

BACKGROUND: gammadelta T cells play a key role in the regulation of inflammatory responses in epithelial tissue, and in adaptive immunity, as gammadelta T cell deficient mice have a severely impaired capacity to clear lung pathogens. gammadelta T cells regulate the initial inflammatory response to microbial invasion and thereby protect against tissue injury. Here we examined the response of gammadelta T cells to lung injury induced by bleomycin, in an effort to study the inflammatory response in the absence of any adaptive immune response to a pathogen. RESULTS: After lung injury by bleomycin, we localized the gammadelta T cells to the lung lesions. gammadelta T cells were the predominant source of IL-17 (as detected by flow cytometry and real-time PCR). Moreover, gammadelta T cell knockout mice showed a significant reduction in cellular infiltration into the airways, reduced expression of IL-6 in the lung, and a significant delay in epithelial repair. CONCLUSION: Mouse gammadelta T cells produce IL-17 in response to lung injury and are required for an organized inflammatory response and epithelial repair. The lack of gammadelta T cells correlates with increased inflammation and fibrosis.     

Japanese encephalitis virus infection stimulates Src tyrosine kinase in neuron/glia

Japanese encephalitis virus (JEV) is a neurotropic virus. The clinically manifestation of JEV-induced encephalitis is characterized by the brain inflammation and neuronal dysfunction and/or destruction. Currently, the cellular signaling molecules that underlie JEV-induced cerebral inflammation and cellular alterations are not well understood. Protein tyrosine phosphorylation events are key regulators of cellular signaling processes, including inflammation. We investigated whether Src protein tyrosine kinase (PTK) function in JEV-induced cellular changes in neuron/glia cultures. JEV infection modulated tyrosine phosphorylation events. Src PTK was hyperphosphorylated at the early stage of infection. Biochemical studies demonstrated that both inhibitors of the Src family PTK and Ras attenuated JEV-induced extracellular signal-regulated kinase (ERK) activation. Our results further revealed that PTK, Ras, and ERK inhibitors effectively suppressed JEV-induced pro-inflammatory cytokine expression and neurotoxicity. Pharmacological studies suggested that microglia secreted pro-inflammatory cytokine via Src/Ras/ERK pathway in responding to JEV infection. Another interesting observation was that nonstructural protein 3 (NS3) was able to interact with Src and showed tyrosine phosphorylation. However, the biological consequences of their interaction and exact control of NS3 tyrosine phosphorylation required further investigation. Our results suggest that the Src/Ras/ERK signaling cascade is involved in JEV-induced pro-inflammatory cytokine expression and neurotoxicity.     

呼吸机相关性肺损伤的炎症反应机制

机械通气是治疗急性呼吸窘迫综合征的主要手段，但呼吸机应用不当也可能加重原有的肺损伤，引起呼吸机相关性肺损伤。呼吸机相关性肺损伤本质上是生物伤，异常的机械力作用于细胞，激活细胞内信号转导通路，活化炎性细胞，产生大量炎症介质，加重炎症反应。探讨呼吸机相关性肺损伤的炎症反应机制，阻断机械力作用向细胞内的传导和细胞内信号转导途径的激活，减少炎症细胞的激活和炎症介质基因的表达，对于预防呼吸机相关性肺损伤发挥重要作用。     

Regulatory effects of interleukin-11 during acute lung inflammatory injury.

The role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1.     

衰变加速因子介导Jurkat细胞的PTK信号传递机制的实验研究

为研究衰变加速因子 (DAF )介导Jurkat细胞信号传递的机制 ,本文应用免疫印迹及化学发光技术 (ECL)发现交联DAF单抗可使CD3+ 与CD3 Jurkat细胞总蛋白酪氨酸磷酸化水平增加 ,但磷酸化水平不同。免疫共沉淀实验检测到DAF与Src家族酪氨酸激酶 (SrcPTK )可发生共沉淀反应。去污剂不溶膜复合物 (DIG )抽提与鉴定检测到DIG内有SrcPTK与DAF的特异性聚集。说明DAF对Jurkat细胞的信号传递有辅助激活效应 ,此效应可能是通过其以与膜微区相关的所联系的SrcPTK实现的     

Strategies that modulate inflammasomes—insights from host–pathogen interactions

The innate immune system is a dynamic and complex network for recognizing and responding to cellular insult or tissue damage after infection or injury. The primary effector mechanism of innate immunity is the generation of acute and chronic inflammatory responses through regulation of the processing and activation of proinflammatory caspases, particularly caspase 1, and cytokines, most notably IL-1β and IL-18. Inflammasomes, cytosolic multi-protein complexes that function as molecular scaffolds for caspase activation, have recently emerged as the pivotal mechanism by which host innate immune and inflammatory responses are regulated. In this review, we investigate the mechanisms by which inflammasomes are modulated, both by endogenous host systems and by microbial pathogens.     

A model for lupus brain disease

Systemic lupus erythematosus is an autoimmune disease characterized by antibodies that bind target autoantigens in multiple organs in the body. In peripheral organs, immune complexes engage the complement cascade, recruiting blood-borne inflammatory cells and initiating tissue inflammation. Immune complex-mediated activation of Fc receptors on infiltrating blood-borne cells and tissue resident cells amplifies an inflammatory cascade with resulting damage to tissue function, ultimately leading to tissue destruction. This pathophysiology appears to explain tissue injury throughout the body, except in the central nervous system. This review addresses a paradigm we have developed for autoantibody-mediated brain damage. This paradigm suggests that antibody-mediated brain disease does not depend on immune complex formation but rather on antibody-mediated alterations in neuronal activation and survival. Moreover, antibodies only access brain tissue when blood-brain barrier integrity is impaired, leading to a lack of concurrence of brain disease and tissue injury in other organs. We discuss the implications of this model for lupus and for identifying other antibodies that may contribute to brain disease.     

Key mechanisms governing resolution of lung inflammation

Innate immunity normally provides excellent defence against invading microorganisms. Acute inflammation is a form of innate immune defence and represents one of the primary responses to injury, infection and irritation, largely mediated by granulocyte effector cells such as neutrophils and eosinophils. Failure to remove an inflammatory stimulus (often resulting in failed resolution of inflammation) can lead to chronic inflammation resulting in tissue injury caused by high numbers of infiltrating activated granulocytes. Successful resolution of inflammation is dependent upon the removal of these cells. Under normal physiological conditions, apoptosis (programmed cell death) precedes phagocytic recognition and clearance of these cells by, for example, macrophages, dendritic and epithelial cells (a process known as efferocytosis). Inflammation contributes to immune defence within the respiratory mucosa (responsible for gas exchange) because lung epithelia are continuously exposed to a multiplicity of airborne pathogens, allergens and foreign particles. Failure to resolve inflammation within the respiratory mucosa is a major contributor of numerous lung diseases. This review will summarise the major mechanisms regulating lung inflammation, including key cellular interplays such as apoptotic cell clearance by alveolar macrophages and macrophage/neutrophil/epithelial cell interactions. The different acute and chronic inflammatory disease states caused by dysregulated/impaired resolution of lung inflammation will be discussed. Furthermore, the resolution of lung inflammation during neutrophil/eosinophil-dominant lung injury or enhanced resolution driven via pharmacological manipulation will also be considered.