Fcgamma Receptor-mediated Phagocytosis Research Articles

Identification of hub genes, signaling pathways and immune infiltration of recurrent spontaneous abortion based on bioinformatics analysis with clinical verification

ObjectiveThe present study was aimed to identify hub genes associated with recurrent spontaneous abortion (RSA) via both bioinformatics analysis and clinical verification, also to evaluate the related pathways and immune infiltration situation of RSA, for exploring its underlying mechanism. Materials and methodsWe screened candidate hub genes associated with RSA via bioinformatic analysis in the microarray datasets GSE22490 downloaded from the Gene Expression Omnibus (GEO) database. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to validate these hub genes. Several kinds of enrichment analysis were carried out to find out the pathways related to RSA. Additionally, CIBERSORT was used for evaluation of local immune Infiltration status of RSA. ResultsThere were 536 differentially expressed genes (DEGs) including 301 upregulated and 235 downregulated genes in RSA group compared with healthy control group. Four hub genes (STAT3, TLR2, TLR4 and CD86) were finally screened out according to the protein–protein interaction (PPI) network analysis, RT-qPCR and Western blotting. Enrichment analysis showed that Toll-like receptor signaling pathway, neutrophil chemotaxis, chemokine signaling pathway and Fc gamma receptor-mediated phagocytosis were strongly associated with RSA. And in immune infiltration analysis, RSA tissue was found containing a higher proportion of monocytes and eosinophils. ConclusionThis study screened out four hub genes and several important pathways changed in the trophoblastic tissue of RSA patients. We also found that monocytes and eosinophils may be involved in RSA. These findings provide theoretical basis for further studies on the mechanisms of RSA.

Identification of Shared Genes and Pathways in Periodontitis and Type 2 Diabetes by Bioinformatics Analysis.

IntroductionIt is well known that the presence of diabetes significantly affects the progression of periodontitis and that periodontitis has negative effects on diabetes and diabetes-related complications. Although this two-way relationship between type 2 diabetes and periodontitis could be understood through experimental and clinical studies, information on common genetic factors would be more useful for the understanding of both diseases and the development of treatment strategies.Materials and MethodsGene expression data for periodontitis and type 2 diabetes were obtained from the Gene Expression Omnibus database. After preprocessing of data to reduce heterogeneity, differentially expressed genes (DEGs) between disease and normal tissue were identified using a linear regression model package. Gene ontology and Kyoto encyclopedia of genes and genome pathway enrichment analyses were conducted using R package ‘vsn’. A protein-protein interaction network was constructed using the search tool for the retrieval of the interacting genes database. We used molecular complex detection for optimal module selection. CytoHubba was used to identify the highest linkage hub gene in the network.ResultsWe identified 152 commonly DEGs, including 125 upregulated and 27 downregulated genes. Through common DEGs, we constructed a protein-protein interaction and identified highly connected hub genes. The hub genes were up-regulated in both diseases and were most significantly enriched in the Fc gamma R-mediated phagocytosis pathway.DiscussionWe have identified three up-regulated genes involved in Fc gamma receptor-mediated phagocytosis, and these genes could be potential therapeutic targets in patients with periodontitis and type 2 diabetes.

Genome assembly and transcriptome analysis provide insights into the antischistosome mechanism of Microtus fortis

Microtus fortis is the only mammalian host that exhibits intrinsic resistance against Schistosoma japonicum infection. However, the underlying molecular mechanisms of this resistance are not yet known. Here, we perform the first de novo genome assembly of M. fortis, comprehensive gene annotation analysis, and evolution analysis. Furthermore, we compare the recovery rate of schistosomes, pathological changes, and liver transcriptomes between M. fortis and mice at different time points after infection. We observe that the time and type of immune response in M. fortis are different from those in mice. M. fortis activates immune and inflammatory responses on the 10th day post infection, such as leukocyte extravasation, antibody activation, Fc-gamma receptor-mediated phagocytosis, and the interferon signaling cascade, which play important roles in preventing the development of schistosomes. In contrast, an intense immune response occurrs in mice at the late stages of infection and could not eliminate schistosomes. Infected mice suffer severe pathological injury and continuous decreases in cell cycle, lipid metabolism, and other functions. Our findings offer new insights into the intrinsic resistance mechanism of M. fortis against schistosome infection. The genome sequence also provides the basis for future studies of other important traits in M. fortis.

Network pharmacology analysis of the therapeutic mechanisms of the traditional Chinese herbal formula Lian Hua Qing Wen in Corona virus disease 2019 (COVID-19), gives fundamental support to the clinical use of LHQW

BackgroundThe traditional Chinese Medicine (TCM) herbal formula Lian Hua Qing Wen (LHQW) improves the results of COVID-19 treatment. Three very recent studies analyzed with network pharmacology some working mechanisms of LHQW. However, we used more techniques and also included Angiotensin converting enzyme 2 (ACE2) (a SARS-CoV receptor, possibly the viral entry point in alveolar lung cells) and the immune system, as cytokine storm is essential in the late phase. PurposeExtensive detailed Network Pharmacology analysis of the LHQW- treatment mechanism in COVID-19. MethodsTCM-herb-meridian and protein interaction network (PIN) of LHQW, based on LHQW herbs meridian information and the protein-protein interaction (PPI) information of the LHQW-component targets. Hub and topological property analyses to obtain crucial targets and construct the crucial LHQW-PIN. Functional modules determination using MCODE, GO and KEGG pathway analysis of biological processes and pathway enrichment. Intersection calculations between the LHQW-proteins and ACE2 co-expression-proteins. ResultsLHQW herbs have relationships to Stomach-, Heart-, Liver- and Spleen-systems, but most (10 of the 13 herbs) to the Lung system, indicating specific effects in lung diseases. The crucial LHQW PIN has the scale-free property, contains 2,480 targets, 160,266 PPIs and thirty functional modules. Six modules are enriched in leukocyte-mediated immunity, the interferon-gamma-mediated signaling pathway, immune response regulating signaling pathway, interleukin 23 mediated signaling pathway and Fc gamma receptor-mediated phagocytosis (GO analysis). These 6 are also enriched in cancer, immune system-, and viral infection diseases (KEGG). LHQW shared 189 proteins with ACE2 co-expression proteins. ConclusionsDetailed network analysis shows, that LHQW herbal TCM treatment modulates the inflammatory process, exerts antiviral effects and repairs lung injury. Moreover, it also relieves the “cytokine storm” and improves ACE2-expression-disorder-caused symptoms. These innovative findings give a rational pharmacological basis and support for treating COVID-19 and possibly other diseases with LHQW.

Alleviation of Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Rats by Yiqi Huayu Jiedu Decoction: A Tandem Mass Tag-Based Proteomics Study.

BackgroundTo study the effect of Yiqi Huayu Jiedu Decoction (YQHYJD) on protein expression in the lung tissue of acute respiratory distress syndrome (ARDS) rats and to explore the underlying molecular therapeutic mechanism of YQHYJD.MethodsSprague Dawley rats were administered with YQHYJD by oral gavage for 1 week. The rats were injected with lipopolysaccharide (LPS) to induce ARDS. The lung injury was assessed pathologically. Differentially expressed proteins (DEPs) were screened by quantitative proteomics and analyzed using bioinformatic tools, such as Metascape and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper. DEPs were verified by parallel reaction monitoring (PRM).ResultsYQHYJD alleviated the LPS-induced pathological damage of lung tissue in rats. There were 134 DEPs among the YQHYJD treatment and model groups. The Genomes pathway analyses revealed that the DEPs were closely related to immune system pathway. The mass spectrometry analysis revealed that YQHYJD exhibits a protective effect on lung tissue by significantly upregulating hematopoietic cell kinase (Hck), phospholipid phosphatase 3 (Plpp3), myristoylated-alanine rich C-kinase substrate (Marcks), and Actin-related protein 2/3 complex subunit 2 (Arpc2), which are related to Fc gamma receptor-mediated phagocytosis pathway.ConclusionYQHYJD can alleviate the lung injury of ARDS rats by regulating the Fc gamma receptor-mediated phagocytosis pathway, which is related to immune system.

Ginsenoside Rg3 promotes Fc gamma receptor-mediated phagocytosis of bacteria by macrophages via an extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent mechanism

Ginsenoside Rg3 is a steroidal saponin abundant in Korean red ginseng that has high anti-inflammatory activity. Rg3 exerts an immunomodulatory effect in acute inflammatory conditions such as bacterial infections. In this study, we determined the effect of Rg3 on bacterial uptake by macrophages and the related intracellular signaling pathways. Rg3 increased macrophage phagocytosis of IgG-opsonized Escherichia coli and IgG-opsonized beads (IgGbeads), but not of non-opsonized beads. Rg3 also enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38 MAPK), but not that of Akt. The inclusion of IgGbeads in macrophage cultures also increased the phosphorylation of ERK1/2 and p38, but co-culture of macrophages with non-opsonized beads did not affect the phosphorylation of ERK1/2 and p38. The Rg3-induced promotion of phagocytosis was inhibited by PD98059, an ERK1/2 inhibitor, and SB203580, a p38 inhibitor. PD98059 inhibited Rg3-induced p38 MAPK phosphorylation, but SB203580 did not suppress ERK1/2 phosphorylation. Culture of macrophages with Rg3 increased actin polymerization, and this effect was inhibited by SB203580 and PD98059. The Rg3-induced increase in phagocytosis was also inhibited by NSC23766, a Rac1 inhibitor and CASIN, a Cdc42 inhibitor. Intraperitoneal injection of Rg3 increased the phosphorylation of ERK1/2 and p38 as well as the phagocytosis of bacteria by lung cells. These results demonstrate that ginsenoside Rg3 enhances macrophage phagocytosis of bacteria by activating the ERK1/2 and p38 MAPK pathways.

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Splice variants as novel targets in pancreatic ductal adenocarcinoma

Despite a wealth of genomic information, a comprehensive alternative splicing (AS) analysis of pancreatic ductal adenocarcinoma (PDAC) has not been performed yet. In the present study, we assessed whole exome-based transcriptome and AS profiles of 43 pancreas tissues using Affymetrix exon array. The AS analysis of PDAC indicated on average two AS probe-sets (ranging from 1–28) in 1,354 significantly identified protein-coding genes, with skipped exon and alternative first exon being the most frequently utilised. In addition to overrepresented extracellular matrix (ECM)-receptor interaction and focal adhesion that were also seen in transcriptome differential expression (DE) analysis, Fc gamma receptor-mediated phagocytosis and axon guidance AS genes were also highly represented. Of note, the highest numbers of AS probe-sets were found in collagen genes, which encode the characteristically abundant stroma seen in PDAC. We also describe a set of 37 ‘hypersensitive’ genes which were frequently targeted by somatic mutations, copy number alterations, DE and AS, indicating their propensity for multidimensional regulation. We provide the most comprehensive overview of the AS landscape in PDAC with underlying changes in the spliceosomal machinery. We also collate a set of AS and DE genes encoding cell surface proteins, which present promising diagnostic and therapeutic targets in PDAC.

Investigating Actin Mechanics during Phagocytic Uptake and Transport

Phagocytosis, the internalization and consecutive digestion of biological material by macrophages is a major part of the innate mammalian immune response. Fc gamma receptor-mediated uptake is driven by actin recruitment to the internalization site and consecutive formation of membrane protrusions around the target object. Molecular motors and cytoskeletal elements contribute to the subsequent transport of phagosomes inside the cells. Although a large number of molecules that are involved in the phagocytic uptake and the phagosomal transport are identified by now, the mechanics of these processes are largely unknown. We therefore investigated the mechanics of phagocytic uptake and phagosomal transport by using live cell microscopy in combination with optical and magnetic tweezers with a focus on the role of actin filaments. We hypothesize that the actin recruitment during phagocytic uptake leads to a transient and localized increase of the stiffness of the uptake region.To test this hypothesis we induced Fc gamma receptor-mediated phagocytosis in J774A.1 mouse macrophages by offering the cells target microbeads coated with immunoglobulin-G. During the uptake we measured local cell stiffness changes with a ‘blinking optical traps’ technique executed via periodic intensity modulation of the optical force. Preliminary data indicates that that the stiffness of the uptake region increases temporarily during the uptake. In addition, we investigated the influence of actin filaments on the phagosomal transport by magnetic tweezers-based bead displacement assays and cytochalasin treatment of the cells.Our measurements are expected to identify characteristic length- and time-scales for the variation of cell mechanical properties during phagocytosis and to contribute to a more comprehensive understanding of this medically relevant process.

IgG4-related sclerosing cholangitis: all we need to know

Our knowledge and experience of IgG4-related sclerosing cholangitis (ISC) have expanded in the last decade. ISC is one of the common organ manifestations of IgG4-related disease (IgG4-RD); approximately 60 % of patients with this systemic condition have ISC in the proximal and/or distal bile ducts. ISC needs to be discriminated from primary sclerosing cholangitis, cholangiocarcinoma, and other rare forms of lymphoplasmacytic cholangiopathy (e.g., follicular cholangitis and sclerosing cholangitis with granulocytic epithelial lesions). Its diagnosis requires a multidisciplinary approach, in which serology, histology, and imaging play crucial roles. Treatments with high-dose corticosteroids typically lead to the rapid and consistent induction of disease remission. Another promising therapeutic approach is B-cell depletion with rituximab. Although disease relapse is relatively common, provided that appropriate treatments are administered, ISC is considered a "benign" disease with a low risk of liver failure and biliary malignancy. Its molecular pathology is characterized by Th2-dominant immune reactions, regulatory T-cell activation, and CCL1-CCR8 interactions. Particular subsets of B cells such as plasmablasts and regulatory B cells also expand. A recent global proteomic study demonstrated that three significantly activated immunological cascades in ISC were all B-cell- or immunoglobulin-related (Fc-gamma receptor-mediated phagocytosis, B-cell receptor signaling pathway, and Fc-epsilon receptor I signaling pathway), suggesting the crucial roles of B cells in the underlying immune reactions. Despite the expansion of our knowledge of the pathophysiology of ISC, the exact role of IgG4 remains unclear. A better understanding of its immunopathology will offer some potential drug targets for this emerging biliary disease.

Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions

BackgroundParasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate.ResultsWe performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure.ConclusionsThe gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2237-2) contains supplementary material, which is available to authorized users.

Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder.

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of postmortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q ≤ 0.01, 53 genes; q ≤ 0.05, 213 genes; q ≤ 0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (p < 10−7 ) and BPD (p = 0.029). To our knowledge, this is the first time that a substantially large number of genes shows concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor mediated phagocytosis, regulation of actin skeleton pathways, along with several cancer pathways. Functional analyses of these genes revealed an interconnected pathway network centered on lysosomal function and the regulation of actin cytoskeleton. These pathways and their interacting network were principally confirmed by an independent transcriptome sequencing dataset of hippocampus. Dysregulation of lysosomal function and cytoskeleton remodeling has direct impacts on endocytosis, phagocytosis, exocytosis, vesicle trafficking, neuronal maturation and migration, neurite outgrowth, and synaptic density and plasticity, and different aspects of these processes have been implicated in SCZ and BPD.

Cdc42 and the RhoGEF Intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation

Vaccinia virus enhances its cell-to-cell spread by inducing Arp2/3-dependent actin polymerisation. This process is initiated by Src- and Abl-mediated phosphorylation of the viral transmembrane protein A36, leading to recruitment of a signalling network consisting of Grb2, Nck, WIP and N-WASP. Nck is a potent activator of N-WASP-Arp2/3-dependent actin polymerisation. However, recent observations demonstrate that an interaction between Nck and N-WASP is not required for vaccinia actin tail formation. We found that Cdc42 cooperates with Nck to promote actin tail formation by stabilising N-WASP beneath the virus. Cdc42 activation is mediated by the Rho guanine-nucleotide-exchange factor (GEF) intersectin-1 (ITSN1), which is recruited to the virus prior to its actin-based motility. Moreover, Cdc42, ITSN1 and N-WASP function collaboratively in a feed-forward loop to promote vaccinia-induced actin polymerisation. Outside the context of infection, we demonstrate that ITSN1 also functions together with Cdc42, Nck and N-WASP during phagocytosis mediated by the Fc gamma receptor. Our observations suggest that ITSN1 is an important general regulator of Cdc42-, Nck- and N-WASP-dependent actin polymerisation.

Rab20 Regulates Phagosome Maturation in RAW264 Macrophages during Fc Gamma Receptor-Mediated Phagocytosis

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.

Adaptor Protein SLAT Modulates Fcγ Receptor-mediated Phagocytosis in Murine Macrophages

SLAT (SWAP-70-like adaptor protein of T cells) is an adaptor protein expressed in cells of the hematopoietic system. SLAT interacts with and alters the function of small GTPase Rac1 in fibroblasts. In these nonhematopoietic models, the SLAT-Rac interaction leads to changes in F-actin and causes cytoskeletal reorganization. In T cells, SLAT expression regulates the development of T helper cells through Cdc42- and Rac1-mediated activation of the NF-AT transcription factor. Here we show that SLAT is expressed in macrophages. Overexpression of SLAT in a macrophage cell line inhibits the IgG Fcgamma receptor-mediated phagocytic ability of THP1 cells. In bone marrow-derived macrophages, SLAT protein is recruited to the early phagosomes formed via Fcgamma receptor engagement. SLAT recruitment to the phagosome was most efficient when the macrophages express at least one isoform of Rac (Rac1 or Rac2), because SLAT recruitment was reduced in macrophages of Rac-deficient mice. Macrophages derived from animals lacking SLAT show an elevation in the rate of Fcgamma receptor-mediated phagocytosis. The absence of SLAT is associated with an increase in the amount of F-actin formed around these phagosomes as well as an increase in the amount of Rac1 protein recruited to the phagosome. Our results suggest that SLAT acts as a gatekeeper for the amount of Rac recruited to the phagosomes formed by Fcgamma receptor engagement and thus is able to regulate F-actin re-organization and consequently phagocytosis.

Agonists of proteinase‐activated receptor‐2 affect transendothelial migration and apoptosis of human neutrophils

Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase-activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR(2) agonists up-regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR(2) agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR(2) agonist also enhanced protective interferon (IFN)gamma-induced FcgammaRI expression at neutrophil cell surface. Of note, IFNgamma is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR(2) expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR(2) may be involved in the pathophysiology of neutrophil-endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcgamma receptor-mediated phagocytosis.

Gene expression analysis illuminates the transcriptional programs underlying the functional activity of ex vivo-expanded granulocytes

Global gene expression analysis established the temporal expression patterns and programs underlying the development of functional activity of ex vivo-expanded (EXE) human granulocytes, as well as differences compared with peripheral blood (PB) granulocytes. CD34(+) progenitor cells were cultured for 3 wk to induce rapid expansion and granulocytic differentiation, with 40% CD15(+) cells by day 3 and 90% by day 12. Phagocytic and respiratory burst activity increased with the fraction of CD15(++)CD11b(+) cells (myelocytes to segmented) and peaked by day 17. However, only 25% of CD15(++)CD11b(+) cells were phagocytic, and respiratory burst activity was one-third that of PB granulocytes. EXE granulocytes from later days and PB granulocytes showed similar expression of Fc gamma receptors (-1A, -2A, -2C, -3A) and complement receptors (-1, -3, -4). Later downregulation of CD36 (expressed by macrophages) suggests lineage plasticity early in granulocytic differentiation. Expression in mature EXE and PB granulocytes was similar for most Fc gamma receptor-mediated phagocytosis signaling proteins, including high-level expression of Hck, Fgr, and the actin-related protein 2/3 complex. Lower expression of Lyn, Cdc42, pleckstrin, and PKC beta(I) by EXE granulocytes may explain decreased phagocytosis. PB and mature EXE granulocytes expressed similar levels of NADPH oxidase complex genes and receptors for fMLP-mediated respiratory burst. Lower burst activity by EXE granulocytes may result from lower expression of Raf1 and PKC zeta. Elevated expression of toll-like receptor (TLR)2, TLR1, and CD14 in mature EXE and PB granulocytes supports a role for the TLR2 and CD14 pathway in zymosan-mediated respiratory burst activity. Lower activity in EXE granulocytes may be due to greater expression of IRAK3, which inhibits TLR-mediated signaling.

A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis.

Fcγ receptor (FcγR)–mediated phagocytosis of IgG-coated particles is regulated by 3′-phosphoinositides (3′PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcγR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcγR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome.

Complement‐mediated phagocytosis ‐ the role of Syk

Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serum-treated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis.