Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation.

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.

Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency.

Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. unc13d-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including Atg9b. Downregulation of TFEB decreased Atg9b levels and decreased macroautophagy in unc13d-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders. Abbreviations ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type.

The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration.

Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A.

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

Rab27a regulates GM-CSF-dependent priming of neutrophil exocytosis.

Neutrophil secretory proteins are mediators of systemic inflammation in infection, trauma, and cancer. In response to specific inflammatory mediators, neutrophil granules are mobilized and cargo proteins released to modulate the microenvironment of inflammatory sites and tumors. In particular, GM-CSF, a cytokine secreted by several immune, nonimmune, and tumor cells, regulates neutrophil priming and exocytosis. Whereas a comprehensive understanding of this process is necessary to design appropriate anti-inflammatory therapies, the molecular effectors regulating GM-CSF-dependent priming of neutrophil exocytosis are currently unknown. With the use of neutrophils deficient in the small GTPase Rab27a or its effector Munc13-4, we show that although both of these secretory factors control matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) exocytosis in response to GM-CSF, their involvement in exocytosis after GM-CSF priming is very different. Whereas GM-CSF priming-induced exocytosis is abolished in the absence of Rab27a for all secondary stimuli tested, including TLR7, TLR9, and formyl peptide receptor 1 (Fpr1) ligands, cells lacking Munc13-4 showed a significant exocytic response to GM-CSF priming. The mobilization of CD11b was independent of both Rab27a and Munc13-4 in GM-CSF-primed cells unless the cells were stimulated with nucleic acid-sensing TLR ligand, thus highlighting a role for both Rab27a and Munc13-4 in endocytic TLR maturation. Finally, the observation that the absence of Rab27a expression impairs the exocytosis of MMP-9 and MPO under both primed and unprimed conditions suggests that Rab27a is a possible target for intervention in inflammatory processes in which GM-CSF-dependent neutrophil priming is involved.

Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis.

Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils compared to wild type was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, azurophilic granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1ß secretion by neutrophils was undetectable under these experimental conditions. The increased azurophilic granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an azurophilic granule-selective dysregulation of neutrophil exocytosis in CAPS.

MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation: DRUGGABILITY OF THE SMALL GTPase Rab27a.

Neutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the up-regulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion.

Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy.

A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via surface expressed fibrinogen or other membrane molecules. The identification of the sites for these viral ligands should therefore provide additional targets for the design of peptide-based or antigen-based therapeutic strategies.

Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses.

The molecular mechanisms that regulate late endosomal maturation and function are not completely elucidated, and direct evidence of a calcium sensor is lacking. Here we identify a novel mechanism of late endosomal maturation that involves a new molecular interaction between the tethering factor Munc13-4, syntaxin 7, and VAMP8. Munc13-4 binding to syntaxin 7 was significantly increased by calcium. Colocalization of Munc13-4 and syntaxin 7 at late endosomes was demonstrated by high-resolution and live-cell microscopy. Munc13-4-deficient cells show increased numbers of significantly enlarged late endosomes, a phenotype that was mimicked by the fusion inhibitor chloroquine in wild-type cells and rescued by expression of Munc13-4 but not by a syntaxin 7-binding-deficient mutant. Late endosomes from Munc13-4-KO neutrophils show decreased degradative capacity. Munc13-4-knockout neutrophils show impaired endosomal-initiated, TLR9-dependent signaling and deficient TLR9-specific CD11b up-regulation. Thus we present a novel mechanism of late endosomal maturation and propose that Munc13-4 regulates the late endocytic machinery and late endosomal-associated innate immune cellular functions.

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: possible implications in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-ß, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-ß in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-ß and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF.

Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood.

Complement C3 is a key fluid-phase protein of the immune system that covalently tags pathogenic cells and molecules for subsequent clearance. Previously, we reported that complement activation results in the formation of multiple C3b:plasma protein complexes in serum. However, it is not known if C3b attaches to any plasma protein in close proximity or preferentially binds damaged proteins. The objective of this study was to determine if C3b couples to plasma proteins in a non-native state and if this could be a potential mechanism to detect and clear damaged proteins from the blood. Using a purified in vitro system with alternative pathway proteins C3, factors B and D it was observed that guanidinium-HCl denaturation of three purified plasma proteins (albumin, alpha-1 proteinase inhibitor, vitamin D binding protein) greatly increased their capacity to form covalent complexes with C3b. However, native vitamin D binding protein, covalently attached to C3b, still retained the ability to bind its natural ligand G-actin, indicating that C3b links to plasma proteins in their native configuration but denaturation substantially increases this interaction. Serum complement activation generated a large number of C3b:plasma protein complexes that bound red blood cell membranes, suggesting a CR1-mediated clearance mechanism. Thermally denatured (60°C) serum activated the alternative pathway when added to fresh serum as evidenced by factor B cleavage and iC3b generation, but this heat-treated serum could not generate the pro-inflammatory peptide C5a. These results show that C3 recognizes and tags damaged plasma proteins for subsequent removal from the blood without triggering proinflammatory functions.

Cell migration to CXCL12 requires simultaneous IKKα and IKKß-dependent NF-κB signaling.

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKß and IKKα-dependent NF-κB activation. IKKß-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKß and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKß, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.

Generation of multiple fluid-phase C3b:plasma protein complexes during complement activation: possible implications in C3 glomerulopathies.

The complement system is tightly regulated to safeguard against tissue damage that results from unwanted activation. The key step of C3 cleavage to C3b is regulated by multiple mechanisms that control the initiation and extent of activation. This study demonstrated that C3b:plasma protein complexes form in the fluid-phase during complement activation. Several different plasma proteins displayed a discrete high molecular SDS-resistant band when any of the three complement activating pathways were triggered in normal human serum or plasma. Serum depleted of individual complement proteins revealed that C3 and factors B and D were essential for complex formation. Inactivation of the thioester bond in C3 also prevented complex formation. In vitro, complexes could be generated using four purified proteins-C3, factor B, factor D, and target protein-and Mg(2+) to allow C3 convertase formation. These studies showed that the complexes consisted of a plasma protein covalently bound to C3b in a 1:1 molar ratio; the C3b portion was rapidly degraded by factors H and I. Analysis of plasma samples from patients with dense deposit disease and C3 glomerulonephritis demonstrated that C3b:protein complexes form spontaneously in the blood of patients with dense deposit disease and, to a lesser extent, in C3 glomerulonephritis patients, but not in healthy controls. This finding supports the underlying hypothesis that these C3 glomerulopathies are diseases of fluid-phase complement dysregulation. These complexes could normally function as a passive mechanism to intercept C3b from depositing on host cells. However, excessive generation and/or defective clearance of fluid-phase C3b:protein complexes may have pathological consequences.

Soluble gC1qR is an autocrine signal that induces B1R expression on endothelial cells.

Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein-coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1ß. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174-180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37 °C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174-180 and 154-162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R.

Neutrophil recruitment to the lung in both C5a- and CXCL1-induced alveolitis is impaired in vitamin D-binding protein-deficient mice.

Knowledge of how neutrophils respond to chemotactic signals in a complex inflammatory environment is not completely understood. Moreover, even less is known about factors in physiological fluids that regulate the activity of chemoattractants. The vitamin D-binding protein (DBP) has been shown to significantly enhance chemotaxis to complement activation peptide C5a using purified proteins in vitro, and by ex vivo depletion of DBP in physiological fluids, but this function has not been determined in vivo. DBP null ((-/-)) mice were used to investigate how a systemic absence of this plasma protein affects leukocyte recruitment in alveolitis models of lung inflammation. DBP(-/-) mice had significantly reduced (~50%) neutrophil recruitment to the lungs compared with their wild-type DBP(+/+) counterparts in three different alveolitis models, two acute and one chronic. The histology of DBP(-/-) mouse lungs also showed significantly less injury than wild-type animals. The chemotactic cofactor function of DBP appears to be selective for neutrophil recruitment, but, in contrast to previous in vitro results, in vivo DBP can enhance the activity of other chemoattractants, including CXCL1. The reduced neutrophil response in DBP(-/-) mice could be rescued to wild-type levels by administering exogenous DBP. Finally, in inflammatory fluids, DBP binds to G-actin released from damaged cells, and this complex may be the active chemotactic cofactor. To our knowledge, results show for the first time that DBP is a significant chemotactic cofactor in vivo and not specific for C5a, suggesting that this ubiquitous plasma protein may have a more significant role in neutrophil recruitment than previously recognized.

Cofactor regulation of C5a chemotactic activity in physiological fluids. Requirement for the vitamin D binding protein, thrombospondin-1 and its receptors.

Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937-C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought.