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Neutrophils are the most abundant immune cell in the human body and are critically important in clearing infections and in wound healing. They rapidly migrate from circulation to sites of injury or infection by detecting chemicals that are released by the body or that emanate from degrading cells and bacteria. Upon reaching these sites, neutrophils engage pathogens with direct killing processes including pathogen engulfment by phagocytosis and by releasing pre-stored anti-microbial products from specialized vesicles called granules in a process called degranulation. While these destructive processes are effective at neutralizing pathogens, dysregulated activity is implicated in inflammatory diseases. Cleary defining the molecular mechanisms that regulate immune cell functions would present opportunities for new drug targets and therapeutics. However, achieving the comprehensive understanding necessary to derive novel therapies has been hampered by the complex interactions between molecules that regulate neutrophil functions. This dissertation is compilation of my contributions toward untangling these molecular pathways. Chapter 1 provides an introduction to neutrophils, their functions, and an overview of outstanding related questions. Chapter 2 describes discoveries about how neutrophils prioritize competing signals as they migrate, which helps them home in on the correct target. The work described in chapter 2 was a collaboration project with Briana Rocha-Gregg. Chapter 3 focuses on neutrophil function once they reach sites of infection. Specifically, it describes my findings about the molecular pathways that control degranulation, including the identification of components that may distinguish migration and degranulation behaviors. In Chapter 4 closing remarks and comments on future directions are made.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated m

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated m

Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical condition characterized by sustained but ineffective immune system activation, leading to severe and systemic hyperinflammation. It may occur as a genetic or sporadic condition, often triggered by an infection. The multifaceted pathogenesis results in a wide range of non-specific signs and symptoms, hampering early recognition. Despite a great improvement in terms of survival in the last decades, a considerable proportion of patients with HLH still die from progressive disease. Thus, prompt diagnosis and treatment are crucial for survival. Faced with the complexity and the heterogeneity of syndrome, expert consultation is recommended to correctly interpret clinical, functional and genetic findings and address therapeutic decisions. Cytofluorimetric and genetic analysis should be performed in reference laboratories. Genetic analysis is mandatory to confirm familial hemophagocytic lymphohistiocytosis (FHL) and Next Generation Sequencing is increasingly adopted to extend the spectrum of genetic predisposition to HLH, though its results should be critically discussed with specialists. In this review, we critically revise the reported laboratory tools for the diagnosis of HLH, in order to outline a comprehensive and widely available workup that allows to reduce the time between the clinical suspicion of HLH and its final diagnosis.

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli.

BackgroundSpinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury.MethodsContusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array.ResultsSyk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils.ConclusionsSyk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter spar

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by the inability to properly terminate an immune response. Familial HLH (FHLH) and related immune dysregulation syndromes are associated with mutations in the genes PRF1, UNC13D, STX11, STXBP2, LYST, AP3B1, and RAB27A, all of which are required for the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. Loss-of-function mutations in these genes render the cytotoxicity pathway ineffective, thereby failing to eradicate immune stimuli, such as infectious pathogens or malignant cells. The resulting persistent immune system stimulation drives hypercytokinemia, ultimately leading to severe tissue inflammation and end-organ damage. Traditionally, a diagnosis of FHLH requires the identification of biallelic loss-of-function mutations in one of these degranulation pathway genes. However, this narrow definition fails to encompass patients with other genetic mechanisms underlying degranulation pathway dysfunction. In particular, mounting clinical evidence supports a potential digenic mode of inheritance of FHLH in which single loss-of-function mutations in two different degranulation pathway genes cooperate to impair pathway activity. Here, we review the functions of the FHLH-associated genes within the degranulation pathway and summarize clinical evidence supporting a model in which cumulative defects along this mechanistic pathway may underlie HLH.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by the inability to properly terminate an immune response. Familial HLH (FHLH) and related immune dysregulation syndromes are associated with mutations in the genes PRF1, UNC13D, STX11, STXBP2, LYST, AP3B1, and RAB27A, all of which are required for the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. Loss-of-function mutations in these genes render the cytotoxicity pathway ineffective, thereby failing to eradicate immune stimuli, such as infectious pathogens or malignant cells. The resulting persistent immune system stimulation drives hypercytokinemia, ultimately leading to severe tissue inflammation and end-organ damage. Traditionally, a diagnosis of FHLH requires the identification of biallelic loss-of-function mutations in one of these degranulation pathway genes. However, this narrow definition fails to encompass patients with other genetic mechanisms underlying degranulation pathway dysfunction. In particular, mounting clinical evidence supports a potential digenic mode of inheritance of FHLH in which single loss-of-function mutations in two different degranulation pathway genes cooperate to impair pathway activity. Here, we review the functions of the FHLH-associated genes within the degranulation pathway and summarize clinical evidence supporting a model in which cumulative defects along this mechanistic pathway may underlie HLH.

Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor–ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function., QC 20210408

BackgroundSpinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury.MethodsContusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array.ResultsSyk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils.ConclusionsSyk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter spar

Background: Neutrophil extracellular trap (NETs) are implicated in the pathogenesis of myeloperoxidase anti neutrophil cytoplasmic antibody vasculitis (MPO-AAV). Neutrophil elastase (NE) is implicated in initiating NET formation through proteolytic degradation of the nuclear envelope, histone degradation and chromatin decondensation in concert with other neutrophil enzymes to facilitate DNA release. NETs are decorated with multiple proinflammatory enzymes including MPO (the autoantigen in this disease) and NE. NE released through degranulation is degraded by endogenous NE inhibitors, however NE bound to NET derived DNA is protected providing a reservoir of proteolytic NE enabling tissue destruction, microvascular injury and further recruitment of neutrophils. This study investigates the pathogenic contribution of NE release through NET formation in experimental MPO-AAV using animals deficient in neutrophil elastase. Method(s): A 20 day experimental model of anti-MPO GN was induced in B6.129X1-Elanetm1Sds/J mice (neutrophil elastase knock out mice) and wild-type (WT) litter mate controls by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin (anti GBM Ig), [n=10 (WT) and n=10 (Elane-/-)]. Result(s): Elane-/-animals were protected from excessive NET production and glomerular injury with significantly reduced numbers of glomerular neutrophils, extracellular MPO deposition, CD4 T cells and macrophages influx (all, P <0.05 compared to WT). Histological assessment of kidneys demonstrated a reduction in segmental necrosis in the Elane-/-group compared to the WT group (P<0.05). Functional injury measured by creatinine/albuminuria ratio was significantly reduced in the Elane -/-group compared to the WT group (P=0.04). Systemic autoimmunity to MPO was significantly reduced in Elane-/-mice with reduced frequency of MPO specific CD4 effector T cells from the draining lymph nodes and a significant reduct

Background: Neutrophil extracellular trap (NETs) are implicated in the pathogenesis of myeloperoxidase anti neutrophil cytoplasmic antibody vasculitis (MPO-AAV). Neutrophil elastase (NE) is implicated in initiating NET formation through proteolytic degradation of the nuclear envelope, histone degradation and chromatin decondensation in concert with other neutrophil enzymes to facilitate DNA release. NETs are decorated with multiple proinflammatory enzymes including MPO (the autoantigen in this disease) and NE. NE released through degranulation is degraded by endogenous NE inhibitors, however NE bound to NET derived DNA is protected providing a reservoir of proteolytic NE enabling tissue destruction, microvascular injury and further recruitment of neutrophils. This study investigates the pathogenic contribution of NE release through NET formation in experimental MPO-AAV using animals deficient in neutrophil elastase. Method(s): A 20 day experimental model of anti-MPO GN was induced in B6.129X1-Elanetm1Sds/J mice (neutrophil elastase knock out mice) and wild-type (WT) litter mate controls by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin (anti GBM Ig), [n=10 (WT) and n=10 (Elane-/-)]. Result(s): Elane-/-animals were protected from excessive NET production and glomerular injury with significantly reduced numbers of glomerular neutrophils, extracellular MPO deposition, CD4 T cells and macrophages influx (all, P <0.05 compared to WT). Histological assessment of kidneys demonstrated a reduction in segmental necrosis in the Elane-/-group compared to the WT group (P<0.05). Functional injury measured by creatinine/albuminuria ratio was significantly reduced in the Elane -/-group compared to the WT group (P=0.04). Systemic autoimmunity to MPO was significantly reduced in Elane-/-mice with reduced frequency of MPO specific CD4 effector T cells from the draining lymph nodes and a significant reduct

FcgammaRIIa is an activating FcgammaR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcgammaRIIa1. A unique splice variant, we designated FcgammaRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcgammaRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcgammaRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcgammaRIIa3 was identified in macaque and human PBMC. The FcgammaRIIa3 is distinguished from the canonical FcgammaRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcgammaRIIa forms responded distinctly to antibody ligation. Whereas FcgammaRIIa1 was rapidly internalized, FcgammaRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcgammaRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcgammaRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

FcgammaRIIa is an activating FcgammaR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcgammaRIIa1. A unique splice variant, we designated FcgammaRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcgammaRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcgammaRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcgammaRIIa3 was identified in macaque and human PBMC. The FcgammaRIIa3 is distinguished from the canonical FcgammaRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcgammaRIIa forms responded distinctly to antibody ligation. Whereas FcgammaRIIa1 was rapidly internalized, FcgammaRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcgammaRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcgammaRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

Neutrophils are the most abundant leukocytes in blood and disruption in their functions often results in an increased risk of serious infections and inflammatory autoimmune diseases. Following recent discoveries in their influence over disease progression, a resurgence of interest for neutrophil biology has taken place. The multitude of signaling pathways activated by the engagement of numerous types of receptors, with which neutrophils are endowed, reflects the functional complexity of these cells. It is therefore not surprising that there remains a huge lack in the understanding of molecular mechanisms underlining neutrophil functions. Moreover, studies on neutrophils are undoubtedly limited by the difficulty to efficiently edit the cell's genome. Over the past 30 years, compelling evidence has clearly highlighted that Ca(2+) -signaling is governing the key processes associated with neutrophil functions. The confirmation of the role of an elevation of intracellular Ca(2+) concentration has come from studies on NADPH oxidase activation and phagocytosis. In this review, we give an overview and update of our current knowledge on the role of Ca(2+) mobilization in the regulation of pro-inflammatory functions of neutrophils. In particular, we stress the importance of Ca(2+) in the formation of NETs and cytokine secretion in the light of newest findings. This will allow us to embrace how much further we have to go to understand the complex dynamics of Ca(2+) -dependent mechanisms in order to gain more insights into the role of neutrophils in the pathogenesis of inflammatory diseases. The potential for therapeutics to regulate the neutrophil functions, such as Ca(2+) influx inhibitors to prevent autoimmune and chronic inflammatory diseases, has been discussed in the last part of the review.

Neutrophils are the most abundant leukocytes in blood and disruption in their functions often results in an increased risk of serious infections and inflammatory autoimmune diseases. Following recent discoveries in their influence over disease progression, a resurgence of interest for neutrophil biology has taken place. The multitude of signaling pathways activated by the engagement of numerous types of receptors, with which neutrophils are endowed, reflects the functional complexity of these cells. It is therefore not surprising that there remains a huge lack in the understanding of molecular mechanisms underlining neutrophil functions. Moreover, studies on neutrophils are undoubtedly limited by the difficulty to efficiently edit the cell's genome. Over the past 30 years, compelling evidence has clearly highlighted that Ca(2+) -signaling is governing the key processes associated with neutrophil functions. The confirmation of the role of an elevation of intracellular Ca(2+) concentration has come from studies on NADPH oxidase activation and phagocytosis. In this review, we give an overview and update of our current knowledge on the role of Ca(2+) mobilization in the regulation of pro-inflammatory functions of neutrophils. In particular, we stress the importance of Ca(2+) in the formation of NETs and cytokine secretion in the light of newest findings. This will allow us to embrace how much further we have to go to understand the complex dynamics of Ca(2+) -dependent mechanisms in order to gain more insights into the role of neutrophils in the pathogenesis of inflammatory diseases. The potential for therapeutics to regulate the neutrophil functions, such as Ca(2+) influx inhibitors to prevent autoimmune and chronic inflammatory diseases, has been discussed in the last part of the review.

Copyright © 2019 by the American Thoracic Society. Rationale: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown. Objectives: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF. Methods: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects. Measurements and Main Results: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes. Conclusions: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Natural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion., QC 20240906

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Mast cells are sentinel cells with a tissue-specific localization in the interface between the host and the external environment. Their quick and selective response upon encountering pathogens is part of the innate host response and typically initiates the following adaptive immune response. Among several pattern recognition receptors (PRRs) involved in the recognition of pathogens by mast cells, the C-type lectin receptor Dectin-1 has been associated with the recognition of fungi. Our previous studies have shown that mast cells are the predominant cell type expressing Dectin-1 in human skin, and they also recognize and respond to Malassezia sympodialis by producing cytokines connected to the innate host response and upregulating the expression of Dectin-1. In the present study, we investigated mast cell responses to Curdlan, a β-glucan that acts as an agonist for the fungi receptor Dectin-1, and found a unique response pattern with induced degranulation, but surprisingly without synthesis of Leukotriene C4, IL-6 or CCL2. Since mast cells are the predominant Dectin-1 expressing cell in the human skin, this study suggests that mast cell degranulation in response to fungi is an important part of the first line of defense against these pathogens.

Endometriosis is a chronic estrogen-dependent inflammatory disease of unclear etiology that affects 15-20% of women of reproductive age. Efforts are now focusing on understanding new mechanisms involved in its physiopathology, like novel target pathways and different molecules. There is evidence that mast cells (MCs) play a role in this disease. This article summarizes recent achievements in preclinical studies and clinical activities investigating the role of MCs in endometriosis. Targeting MCs might offer new alternatives to treat this disease. Areas covered: A systematic literature search was performed (PubMed, Cochrane Library and ClinicalTrials.gov) using the keywords 'endometriosis and mast cells'. All relevant articles (34) found in PubMed were examined and their reference lists reviewed in order to pinpoint further studies for potential inclusion. Expert opinion: Since endometriosis is a multifactorial disease, and considering that numbers of MCs and activated MCs were clearly increased in endometriotic lesions in both animals and humans, use of MC stabilizers and inhibitors may prove to be effective to treat endometriosis and its associated pain. However, more data from preclinical studies and clinical trials will help to better define the status of MCs in the treatment of this pathology.

Neutrophils are essential components of immunity and are rapidly recruited to infected or injured tissue. Upon their activation, neutrophils release granules to the cell's exterior, through a process called degranulation. These granules contain proteins with antimicrobial properties that help combat infection. The enteropathogenic bacterium Yersinia pseudotuberculosis successfully persists as an extracellular bacterium during infection by virtue of its translocation of virulence effectors (Yersinia outer proteins [Yops]) that act in the cytosol of host immune cells to subvert phagocytosis and proinflammatory responses. Here, we investigated the effect of Y. pseudotuberculosis on neutrophil degranulation upon cell contact. We found that virulent Y. pseudotuberculosis was able to prevent secondary granule release. The blocking effect was general, as the release of primary and tertiary granules was also reduced. Degranulation of secondary granules was also blocked in primed neutrophils, suggesting that this mechanism could be an important element of immune evasion. Further, wild-type bacteria conferred a transient block on neutrophils that prevented their degranulation upon contact with plasmid-cured, avirulent Y. pseudotuberculosis and Escherichia coli Detailed analyses showed that the block was strictly dependent on the cooperative actions of the two antiphagocytic effectors, YopE and YopH, suggesting that the neutrophil target structures constituting signaling molecules needed to initiate both phagocytosis and general degranulation. Thus, via these virulence effectors, Yersinia can impair several mechanisms of the neutrophil's antimicrobial arsenal, which underscores the power of its virulence effector machinery.

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the

Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de nova synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer.

Background and Aims: Natural killer (NK) cells have been shown to attenuate hepatic fibrosis in transgenic HBV murine models. There is a paucity of data regarding the role of human NK and NKT cells in hepatitis B virus associated fibrogenesis. The aim of this study was to investigate the potential anti-fibrotic effects of human NK and or NKT cells from immune active HBV patients on HSC. Method(s): Immune active (IA) HBV was defined as ALT >=5 ULN and Immune control (IC) was defined as normal ALT. Human NK (90-96% purity) and NKT cells (75-85% purity) were magnetically isolated from peripheral blood mononuclear cells and cultured with LX2 human hepatic stellate cells (HSC). NK and NKT cells were further phenotyped by Tim-3, NKG2A, NKG2D, NKp46, FasL and TRAIL expression. Induction of LX2 apoptosis was analysed by caspase-3 activation. Intracellular cytokine production (IFN-alpha and TNF-alpha) and CD107a degranulation were used to determine NK and NKT cell functional activity. HSC collagen production was determined by the Sircol assay. Result(s): 15 patients were recruited: 6 IA HBV, 4 IC HBV and 5 healthy controls (HC) [serum ALT (IU/ml): 260.83+/-232 vs 24.25+/-8.93 and 24+/-5.42 respectively, p = 0.007]. Median HBVDNA viral load was higher in IA compared to IC (6.29 log10 IU/ml, IQR: 4.8-7.49 vs 2.26 log10 IU/ml, IQR: 1.73-2.88; p = 0.019). NK and NKT cells from IA HBV were significantly more effective in HSC apoptosis induction than similar cells from IC HBV or HC (16.15% +/- 13.8 vs 5.17% +/- 2.31 and 2.67% +/- 0.85, respectively; p < 0.0001). HSC caspase-3 expression correlated with serum ALT [Spearman's rho (rho) = 0.66, p < 0.0001], reduced proline incorporation (rho = -0.62, p < 0.0001) and increased NKp46 expression on NKBright cells (rho = 0.56, p = 0.04) and NKG2D expression on NKT cells (rho = 0.54, p = 0.03). There was no significant difference in cytokine production, TRAIL or FasL expression. Conclusion(s): NK and NKT cells from immune active HBeAg neg

Background and Aims: Natural killer (NK) cells have been shown to attenuate hepatic fibrosis in transgenic HBV murine models. There is a paucity of data regarding the role of human NK and NKT cells in hepatitis B virus associated fibrogenesis. The aim of this study was to investigate the potential anti-fibrotic effects of human NK and or NKT cells from immune active HBV patients on HSC. Method(s): Immune active (IA) HBV was defined as ALT >=5 ULN and Immune control (IC) was defined as normal ALT. Human NK (90-96% purity) and NKT cells (75-85% purity) were magnetically isolated from peripheral blood mononuclear cells and cultured with LX2 human hepatic stellate cells (HSC). NK and NKT cells were further phenotyped by Tim-3, NKG2A, NKG2D, NKp46, FasL and TRAIL expression. Induction of LX2 apoptosis was analysed by caspase-3 activation. Intracellular cytokine production (IFN-alpha and TNF-alpha) and CD107a degranulation were used to determine NK and NKT cell functional activity. HSC collagen production was determined by the Sircol assay. Result(s): 15 patients were recruited: 6 IA HBV, 4 IC HBV and 5 healthy controls (HC) [serum ALT (IU/ml): 260.83+/-232 vs 24.25+/-8.93 and 24+/-5.42 respectively, p = 0.007]. Median HBVDNA viral load was higher in IA compared to IC (6.29 log10 IU/ml, IQR: 4.8-7.49 vs 2.26 log10 IU/ml, IQR: 1.73-2.88; p = 0.019). NK and NKT cells from IA HBV were significantly more effective in HSC apoptosis induction than similar cells from IC HBV or HC (16.15% +/- 13.8 vs 5.17% +/- 2.31 and 2.67% +/- 0.85, respectively; p < 0.0001). HSC caspase-3 expression correlated with serum ALT [Spearman's rho (rho) = 0.66, p < 0.0001], reduced proline incorporation (rho = -0.62, p < 0.0001) and increased NKp46 expression on NKBright cells (rho = 0.56, p = 0.04) and NKG2D expression on NKT cells (rho = 0.54, p = 0.03). There was no significant difference in cytokine production, TRAIL or FasL expression. Conclusion(s): NK and NKT cells from immune active HBeAg neg

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes.

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes.

Objective: The main objective of this preliminary study is to confirm the synergistic anticarcinogenic potential of Vitex trifolia and Triticum aestivum ethanolic extracts. Materials and Methods: Rat hepatic microsomal degranulation is a short - term technique that has been used for the detection of potential chemical carcinogens, in vitro. The present study has been carried out to study the inhibition of ribosome- membrane disruption against 3, 8-Diamino-5-ethyl-6-pheylphenanthridinium bromide (EB), as the degranulating agent, by measuring the RNA/protein ratios of microsomal membranes in the presence or absence of V.trifolia and T. aestivum extracts. These two extracts were further evaluated for cytotoxic effect in HCT 116 and A549 cell lines. Results: V. trifolia and T. aestivum protects hepatic microsomes against the degranulatory attack by the carcinogen EB showed a significant reduction in the proliferation of the HCT 116 and A549 cancer cell lines. Conclusion: The ethanolic extracts of the plants, V. trifolia and T. aestivum individually possessed anti-degranulatory potential. Importantly they act synergistically, possess appreciable anticarcinogenic properties, based on their ability to inhibit EB induced liver microsomal degranulation. Further these extracts inhibit cell proliferation of cancer cell lines.

Aim: To define the presence of intrarenal degranulated mast cells (MCs) in humans with MPO-ANCA glomerulonephritis and determine whether a MC stabiliser, disodium chromoglycate (DSCG) limits experimental murine anti- MPO glomerulonephritis. Background(s): MCs are tuneable cells that potentially have pro- or antiinflammatory roles. However, MC degranulation is a proinflammatory event. Method(s): Human renal biopsies MPO-ANCA glomerulonephritis (n = 24), controls (thin basement membrane or minimal change disease n = 10) were examined for the presence of MCs and their degranulation. Experimental anti-MPO glomerulonephritis was induced using a standard protocol, by MPO immunisation and glomerulonephritis triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. Daily administration of DSCG (or saline control) to prevent MC degranulation commenced either 2 days prior to MPO immunisation or 10 days after establishment of MPO autoimmunity. Result(s): MPO-ANCA glomerulonephritis patients had significant interstitial MC infiltration (MPO-ANCA: 0.8 +/- 0.1 vs ctrl: 0.2 +/- 0.1 cells per high power field, P = 0.16) and increased MC numbers correlated with low presenting eGFR (r = -0.49, P = 0.15). A high proportion of MCs were degranulated (MPOANCA: 58 +/- 8 vs ctrl: 1 +/- 1%, P = 0.001) and were associated with severe tubulointerstitial injury (TI) (mild TI injury score <30%: 0.2+/-.06 vs severe TI injury score >30%TI; 0.9 +/- 0.2, P = 0.17). In experimental anti-MPO glomerulonephritis, DSCG treatment prior to initial MPO immunisation resulted in diminished anti-MPO autoimmunity (IFN-gamma; Ctrl: 13.5 +/- 3.7 vs DSCG: 3.6 +/- 1.3 ng/mL, P < 0.05), enhanced IL-10 (Ctrl: 124 +/- 10 vs DSCG: 198 +/- 16pg/mL, P = 0.01) and attenuated glomerulonephritis (segmental glomerular necrosis; Ctrl: 62 +/- 6 vs DSCG: 34 +/- 4%, P = 0.01). When treatment commenced in established MPO autoimmunity, DSCG limited the development of glomerulonephritis (segmental glom

Aim: To define the presence of intrarenal degranulated mast cells (MCs) in humans with MPO-ANCA glomerulonephritis and determine whether a MC stabiliser, disodium chromoglycate (DSCG) limits experimental murine anti- MPO glomerulonephritis. Background(s): MCs are tuneable cells that potentially have pro- or antiinflammatory roles. However, MC degranulation is a proinflammatory event. Method(s): Human renal biopsies MPO-ANCA glomerulonephritis (n = 24), controls (thin basement membrane or minimal change disease n = 10) were examined for the presence of MCs and their degranulation. Experimental anti-MPO glomerulonephritis was induced using a standard protocol, by MPO immunisation and glomerulonephritis triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. Daily administration of DSCG (or saline control) to prevent MC degranulation commenced either 2 days prior to MPO immunisation or 10 days after establishment of MPO autoimmunity. Result(s): MPO-ANCA glomerulonephritis patients had significant interstitial MC infiltration (MPO-ANCA: 0.8 +/- 0.1 vs ctrl: 0.2 +/- 0.1 cells per high power field, P = 0.16) and increased MC numbers correlated with low presenting eGFR (r = -0.49, P = 0.15). A high proportion of MCs were degranulated (MPOANCA: 58 +/- 8 vs ctrl: 1 +/- 1%, P = 0.001) and were associated with severe tubulointerstitial injury (TI) (mild TI injury score <30%: 0.2+/-.06 vs severe TI injury score >30%TI; 0.9 +/- 0.2, P = 0.17). In experimental anti-MPO glomerulonephritis, DSCG treatment prior to initial MPO immunisation resulted in diminished anti-MPO autoimmunity (IFN-gamma; Ctrl: 13.5 +/- 3.7 vs DSCG: 3.6 +/- 1.3 ng/mL, P < 0.05), enhanced IL-10 (Ctrl: 124 +/- 10 vs DSCG: 198 +/- 16pg/mL, P = 0.01) and attenuated glomerulonephritis (segmental glomerular necrosis; Ctrl: 62 +/- 6 vs DSCG: 34 +/- 4%, P = 0.01). When treatment commenced in established MPO autoimmunity, DSCG limited the development of glomerulonephritis (segmental glom

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed alpha, beta, and gamma subunits of high-affinity immunoglobulin E (IgE) receptor (FcERI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of 1L-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-alpha, and cyclooxygenase 2, and production of prostaglandin D2 and leukotriene C4 in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-alpha expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcERI- and TLR-mediated effector functions of mast cells.

Background Hepatitis C (HCV) recurrence post liver transplantation is universal, with a subgroup developing rapid hepatic fibrosis. Natural killer (NK) cells are critical to anti-viral immunity and the role of NK cell function in HCV rapid fibrosis post transplant is unknown. Aims This study determines NK cell marker expression in HCV patients post liver transplant and the relationship to HCV viral load and graft fibrosis progression. Methods Peripheral blood mononuclear cells (PBMCs) from 35 HCV patients post transplant, 10 non-HCV patients post transplant (matched for sex, age and time post transplant) and 8 healthy controls were labelled for CD3 and CD56, activation markers CD69 and NKG2D, degranulation marker CD107a, inhibitory KIR 2DL2/2DL3 (CD158b) and baseline intracellular TNFalpha and IFNgamma and analysed by flow cytometry. Rate of fibrosis progression was calculated using post transplant liver biopsies graded by Metavir scoring (F0-4; R = fibrosis stage/year post transplant). Rapid fibrosis was defined as > 0.4 units/yr, dichotomized about the median observed rate (0.4). Results Both HCV and non-HCV patients post transplant had reduced NK cell CD158b (NK cells p = 0.037, CD56bright NK cells p = 0.0081) and NKG2D expression (NK cells p = 0.030, CD56dim NK cells p = 0.003) compared with healthy controls. NK cell NKG2D expression inversely correlated with HCV viral load (p = 0.012, r = -0.57). HCV NK CD56dim cells had greater CD69 expression compared with post transplant controls (p = 0.043) and CD69 expression correlated with both TNF and IFNgamma production (TNF p = 0.043, r = 0.43; IFNgamma p = 0.004, r = 0.58). HCV NK cells produced greater TNFalpha (NK CD56 dim p = 0.005, NK CD56bright p = 0.044) and IFNgamma (NK CD56dim p = 0.008, NK CD56bright p = 0.016) at baseline compared with non-HCV post transplant patients. There was no relationship between NK cell marker expression and HCV fibrosis progression post transplant. Conclusion Our data suggest that i

Background Hepatitis C (HCV) recurrence post liver transplantation is universal, with a subgroup developing rapid hepatic fibrosis. Natural killer (NK) cells are critical to anti-viral immunity and the role of NK cell function in HCV rapid fibrosis post transplant is unknown. Aims This study determines NK cell marker expression in HCV patients post liver transplant and the relationship to HCV viral load and graft fibrosis progression. Methods Peripheral blood mononuclear cells (PBMCs) from 35 HCV patients post transplant, 10 non-HCV patients post transplant (matched for sex, age and time post transplant) and 8 healthy controls were labelled for CD3 and CD56, activation markers CD69 and NKG2D, degranulation marker CD107a, inhibitory KIR 2DL2/2DL3 (CD158b) and baseline intracellular TNFalpha and IFNgamma and analysed by flow cytometry. Rate of fibrosis progression was calculated using post transplant liver biopsies graded by Metavir scoring (F0-4; R = fibrosis stage/year post transplant). Rapid fibrosis was defined as > 0.4 units/yr, dichotomized about the median observed rate (0.4). Results Both HCV and non-HCV patients post transplant had reduced NK cell CD158b (NK cells p = 0.037, CD56bright NK cells p = 0.0081) and NKG2D expression (NK cells p = 0.030, CD56dim NK cells p = 0.003) compared with healthy controls. NK cell NKG2D expression inversely correlated with HCV viral load (p = 0.012, r = -0.57). HCV NK CD56dim cells had greater CD69 expression compared with post transplant controls (p = 0.043) and CD69 expression correlated with both TNF and IFNgamma production (TNF p = 0.043, r = 0.43; IFNgamma p = 0.004, r = 0.58). HCV NK cells produced greater TNFalpha (NK CD56 dim p = 0.005, NK CD56bright p = 0.044) and IFNgamma (NK CD56dim p = 0.008, NK CD56bright p = 0.016) at baseline compared with non-HCV post transplant patients. There was no relationship between NK cell marker expression and HCV fibrosis progression post transplant. Conclusion Our data suggest that i

In this work, the effect of aqueous and methanolic extracts of the plants species Critoniella acuminata, Salvia rubescens, Phenax rugosus (Poir.) Wedd and Tabebuia chrysanta G. on the enzymes elastase and myeloperoxidase, involved in degranulation leukocyte process, was evaluated, identifying the potential direct inhibitory effect on the enzyme and/or inhibition of the desgranulation of polymorphonuclear neutrophils. Extracts of Critoniella acuminata andSalvia rubescens presented effects on the degranulation process and the inhibition of the enzyme elastase and myeloperoxidase; the extracts of Phenax rugosus do not showed significant effect. Tabebuia chrysanta methanolic extract only showed effect on inhibition of elastase activity., Se evaluó el efecto de extractos metanólicos y acuosos de las especies vegetales Critoniella acuminata, Salvia rubescens, Phenax rugosus (Poir.) Wedd y Tabebuia chrysanta G. Nicholson sobre las enzimas elastasa y mieloperoxidasa, relacionadas con el proceso de desgranulación leucocitaria, y se determinó el potencial efecto inhibitorio directo sobre la enzima o la inhibición de la desgranulación de los neutrófilos polimorfonucleares. Los extractos de Critoniella acuminata y Salvia rubescens presentaron efectos sobre el proceso de desgranulación y la actividad de las enzimas mieloperoxidasa y elastasa; en el caso de los extractos de Phenax rugosus, estos no mostraron un efecto significativo sobre ninguna de las enzimas. De la especieTabebuia chrysanta solamente el extracto metanólico mostró efecto sobre la inhibición de la actividad elastasa.

Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived-neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose-dependent inhibition of all three granule proteins. LY294002 (10(-6) M) also inhibited the release of all proteins. Genistein (10(-6) M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration-dependent patterns of release of all three proteins. Staurosporine (10(-7) M), GÖ6976 (10(-6) M) and PD98059 (10(-5) M) caused an increased release of the three proteins. PI3-kinases play an important role in the C3b-induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b-induced degranulation.

Aim: To define the role of Mast Cells (MCs) in renal fibrosis induced by unilateral ureteric obstruction (UUO). Background(s): MCs are pluripotent innate immune cells, which after degranulation release cytokines, chemokines and pro-fibrotic factors including transforming growth factor beta (TGFbeta). Method(s): To define a role for MCs in renal fibrosis, we performed UUO on C57BL/6 wild type (WT) and mast cell deficient, KitWsh/Wsh mice. Experiments ended 7 days later. We measured collagen deposition, intrarenal mRNA expression of pro-fibrotic factors, TGFbeta and smooth muscle actin (alphaSMA) and interstitial leukocyte recruitment. To confirm these results were exclusively due to MC deficiency we measured renal fibrosis in KitWsh/Wsh mice reconstituted with MCs from WT mice. Finally we assessed renal fibrosis after administration of sodium chromoglycate, a mast cell stabilizer. NEPHROLOGY 2011; 16 (Suppl. 1), 44-69 doi:10.1111/j.1440-1797.2011.01491.x Results: Compared to WT mice, renal fibrosis was decreased in KitWsh/Wsh mice 7 days after UUO. Histological assessment demonstrated decreased collagen deposition (12.7 +/- 1.2 vs. 9.9 +/- 0.9%, P < 0.05). Intrarenal TGFbeta (1.0 +/- 0.1 vs. 0.7 +/- 0.1, P < 0.05) and alphaSMA (1.0 +/- 0.1 vs. 0.7 +/- 0.1, P < 0.05) expression was also decreased in KitWsh/Wsh mice. Fewer interstitial macrophages (score 2.4 +/- 0.1 vs. 1.6 +/- 0.1 P < 0.001) and CD4+T cells (7.1 +/- 0.4 vs. 4.1 +/- 0.6 cells/high power field, P < 0.001) were seen in KitWsh/Wsh mice. Collagen deposition (17.0 +/- 2.5 vs. 9.1 +/- 1.2%, P < 0.05) and intrarenal mRNA expression of TGFbeta and alphaSMA was decreased in (unreconstituted) KitWsh/Wsh mice compared to KitWsh/Wsh mice reconstituted with MCs from WT mice. Pre-emptive administration of sodium chromoglycate to WT mice resulted in decreased collagen deposition (16.3 +/- 1.3 vs. 10.5 +/- 0.7%, P < 0.01) and decreased intrarenal mRNA expression of TGFbeta (1.0 +/- 0.1 vs. 0.7 +/- 0.1, P < 0.01) and a

Aim: To demonstrate a role for mast cells (MC) in immunomodulating autoimmune anti-Myeloperoxidase (MPO) glomerulonephritis (AIMPOGN). Background(s): Upon degranulation, MCs release cytokines involved in effector and regulatory immune responses. Preliminary studies suggest that MCs interact with regulatory T cells (Tregs) modulating autoimmunity. Method(s): We compared autoimmunity and GN between WT, MC deficient KitWsh/Wsh and KitWsh/Wsh mice reconstituted with WT or IL-10-/- MCs. AIMPOGN was induced by immunizing mice with MPO and GN triggered using low dose anti-glomerular basement membrane antibody. Four days later, renal injury and systemic immune responses were assessed. Result(s): Compared with WT mice, KitWsh/Wsh mice developed enhanced autoimmunity (MPO induced lymph node [LN] CD4 IL-17A recall responses (1403 +/- 420.6 vs 520.7 +/- 92.2 pg/ml P < 0.05) and reduced immunoregulatory IL-10 (15.5 +/- 1.2 vs 49.3 +/- 11.9 pg/ml P < 0.05). GN was more severe in KitWsh/Wsh mice with increased proteinuria (7.4 +/- 1.9 vs 2.5 +/- 0.6 mg/24 hr P < 0.05), glomerular CD4 cells (0.6 +/- 0.1 vs 0.2 +/- 0.1 cell/glomerular cross section [c/gcs] P < 0.05), macrophages influx (1.7 +/- 0.2 vs 0.8 +/- 0.2 c/gcs P < 0.05) and histological injury. The percentage of Tregs in LNs draining immunization sites was reduced (17.1 +/- 0.3 vs 15.9 +/- 0.4 P < 0.05). MC reconstituted KitWsh/Wsh mice showed attenuation of increased autoimmunity and GN seen in KitWsh/Wsh mice. However reconstitution with IL-10-/- MCs did not significantly attenuate autoimmunity (IL-17A: 5.5 +/- 1.1 vs 1.9 +/- 0.7 ng/ml P < 0.05) and GN severity (proteinuria: 11.5 +/- 1.8 vs 6.8 +/- 0.4 mg/24 hr P < 0.05). Ex vivo culture of isolated anti-MPO CD4 effectors with combinations of MCs, IL-10-/- MCs and Foxp3+ Tregs demonstrated that MCs modulate Teffector function by IL-10 production to enhanced Treg functional capacities. Conclusion(s): In AIMPOGN MCs play an important role in limiting anti-MPO autoimmunity a

Aim: To define the role of Mast Cells (MCs) in cisplatin induced Acute Kidney Injury (AKI). Background(s): MCs are pluripotent innate immune cells, which after degranulation release inflammatory molecules, including tissue necrosis factor (TNF). Method(s): To determine the role of MCs in AKI we administered cisplatin to C57BL/6 wild type (WT) and mast cell deficient, KitWsh/Wsh mice. Renal injury and leukocyte recruitment was assessed after 4 days. Subsequently we reconstituted KitWsh/Wsh mice with MCs from WT and TNF-/- mice, and then administered cisplatin. Finally we assessed renal injury after pre-emptive administration of sodium chromoglycate, a mast cell stabilizer. Result(s): Functional and histological kidney injury and leukocyte recruitment, chemokine and TNF expression was decreased in KitWsh/Wsh mice. Blood urea nitrogen (BUN) (109.4 +/- 6.1 vs. 45.3 +/- 11.8 mmol/l, P < 0.01) and histological injury (score 2.7 +/- 0.2 vs. 1.6 +/- 0.1, P < 0.001) was decreased in KitWsh/Wsh mice. Renal neutrophil and CD4+ T cell recruitment were also diminished in KitWsh/ Wsh mice. Serum TNF levels (84.4 +/- 7.4 vs. 60.5 +/- 4.9 pg/ml) were lower in KitWsh/Wsh mice. Intrarenal TNF mRNA expression, as well as mRNA for key T cell (RANTES/CCL5, IP-10/CXCL10), and neutrophil chemokines (KC/CXCL1 and MIP2/CXCL2) were decreased in KitWsh/Wsh mice. To define the mechanism of MC-mediated AKI, KitWsh/Wsh mice, reconstituted with MCs derived from WT or TNF-/- mice, were given cisplatin. Compared to KitWsh/Wsh mice reconstituted with WT MCs, renal injury was attenuated in KitWsh/Wsh mice reconstituted with TNF-/- MCs (BUN 100.3 +/- 16.2 vs. 41.6 +/- 15.9 mmol/l, P < 0.01; histological injury 3.3 +/- 01 vs. 2.2 +/- 0.2, P < 0.001). Giving WT mice sodium chromoglycate before cisplatin protected mice (BUN 20.4 +/- 2.6 vs. 12.5 +/- 1.2, P < 0.05; histological injury 3.1 +/- 0.2 vs. 2.1 +/- 0.1, P < 0.001). Conclusion(s): MCs mediate AKI by producing TNF. Targeting MCs represents a novel

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)- and antigen-induced mast cell degranulation and subsequent gene expression. Bone marrow-derived mast cells (BMMCs) from DBA/2 mice were cultured with heat-killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE- and antigen-induced beta-hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)-4, IL-13, tumor necrosis factor-alpha, and cyclooxygenase-2 expression levels that was induced by 1-2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll-like receptor (TLR)2-neutralizing antibody. In addition, downregulation of cell surface Fc epsilon RI alpha expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE-mediated mast cell degranulation and subsequent late-phase reactions involving mast cells via a TLR2-dependent mechanism with Fc epsilon RI alpha downregulation., The definitive version is available at www.blackwell-synergy.com

Aim: To demonstrate a role for mast cells (MC) in immunomodulating autoimmune anti-Myeloperoxidase (MPO) glomerulonephritis (AIMPOGN). Background(s): Upon degranulation, MCs release cytokines involved in effector and regulatory immune responses. Preliminary studies suggest that MCs interact with regulatory T cells (Tregs) modulating autoimmunity. Method(s): We compared autoimmunity and GN between WT, MC deficient KitWsh/Wsh and KitWsh/Wsh mice reconstituted with WT or IL-10-/- MCs. AIMPOGN was induced by immunizing mice with MPO and GN triggered using low dose anti-glomerular basement membrane antibody. Four days later, renal injury and systemic immune responses were assessed. Result(s): Compared with WT mice, KitWsh/Wsh mice developed enhanced autoimmunity (MPO induced lymph node [LN] CD4 IL-17A recall responses (1403 +/- 420.6 vs 520.7 +/- 92.2 pg/ml P < 0.05) and reduced immunoregulatory IL-10 (15.5 +/- 1.2 vs 49.3 +/- 11.9 pg/ml P < 0.05). GN was more severe in KitWsh/Wsh mice with increased proteinuria (7.4 +/- 1.9 vs 2.5 +/- 0.6 mg/24 hr P < 0.05), glomerular CD4 cells (0.6 +/- 0.1 vs 0.2 +/- 0.1 cell/glomerular cross section [c/gcs] P < 0.05), macrophages influx (1.7 +/- 0.2 vs 0.8 +/- 0.2 c/gcs P < 0.05) and histological injury. The percentage of Tregs in LNs draining immunization sites was reduced (17.1 +/- 0.3 vs 15.9 +/- 0.4 P < 0.05). MC reconstituted KitWsh/Wsh mice showed attenuation of increased autoimmunity and GN seen in KitWsh/Wsh mice. However reconstitution with IL-10-/- MCs did not significantly attenuate autoimmunity (IL-17A: 5.5 +/- 1.1 vs 1.9 +/- 0.7 ng/ml P < 0.05) and GN severity (proteinuria: 11.5 +/- 1.8 vs 6.8 +/- 0.4 mg/24 hr P < 0.05). Ex vivo culture of isolated anti-MPO CD4 effectors with combinations of MCs, IL-10-/- MCs and Foxp3+ Tregs demonstrated that MCs modulate Teffector function by IL-10 production to enhanced Treg functional capacities. Conclusion(s): In AIMPOGN MCs play an important role in limiting anti-MPO autoimmunity a