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Aggregation of a-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloidderived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1b and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes-based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis. [ABSTRACT FROM AUTHOR]

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment--derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2-/- mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d--CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/lpr model of SLE. [ABSTRACT FROM AUTHOR]

Complement activation generates the core effector protein C5a, a potent immune molecule that is linked to multiple inflammatory diseases. Two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), mediate the biological activities of C5a. Although C5aR1 has broadly acknowledged proinflammatory roles, C5aR2 remains at the center of controversy, with existing findings supporting both immune-activating and immune-dampening functions. Recent progress has been made toward resolving these issues. Instead of being a pure recycler and sequester of C5a, C5aR2 is capable of mediating its own set of signaling events and through these events exerting significant immunomodulatory effects not only toward C5aR1 but also other pattern recognition receptors and innate immune systems, such as NLRP3 inflammasomes. This review highlights the existing knowns and unknowns concerning C5aR2 and provides a timely update on recent breakthroughs which are expected to have a substantial impact on future fundamental and translational C5aR2 research. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis ("Dragot" is a Greek surname meaning "sentinel"), as it represents sharing of a microbe between two sentinel cells of the innate immune system. [ABSTRACT FROM AUTHOR]

Human CD21low B cells present with an activated phenotype and accumulate in distinct disorders connected with chronic immune stimulation. Signaling studies had revealed an increased basal phosphorylation of spleen tyrosine kinase (SYK) and phospholipase Cg2. Additional BCR stimulation of these constitutively active cells, however, led to reduced activation of these signaling molecules and subsequently NF-κB and Ca2+ activation. In this article, we demonstrate that high SYK expression is a common feature of CD21low B cells independent of the underlying disorder, and that this high expression is sufficient to drive constitutive phosphorylation of SYK and its immediate targets Bruton's tyrosine kinase and phospholipase Cg2. Inhibition of SYK activity eliminated features of the constitutive activation in these cells and partly restored BCR signaling. High SYK expression is especially induced by CpG or CD40L in combination with IL-21, but not BCR stimulation, suggesting the importance of the immune-stimulatory context for the induction of this B cell phenotype. In summary, high SYK expression is a common feature of human CD21low B cells and presumably results from chronic activation in inflammatory environments present in a subgroup of patients with heterogeneous disorders like chronic infection, autoimmunity, and immunodeficiency. High SYK expression by itself drives the constitutive activation observed in these B cells, which in turn may contribute to the hyporesponsiveness upon BCR stimulation. Given the high prevalence of autoreactive clones among CD21low B cells in autoimmune disorders, the dominant role of SYK in CD21low B cells may provide a new option for therapeutic interventions in patients with expanded CD21low B cells and humoral autoimmunity. [ABSTRACT FROM AUTHOR]

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial .S', aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR-/- mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR-/- mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CDllb and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincuhation with an anti-CDllb blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18-/- and C3-/- mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR in IgMmediated phagocytosis of S. aureus by microglia. [ABSTRACT FROM AUTHOR]

Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding CSaR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lungand lamina propria-resident and alveolar macrophages, bone marrow-derived DCs, and lung-resident CD11b+ and monocytederived DCs, whereas intestinal and pulmonary CD103+ DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any CSaR1 expression in naive or activated CD4+ Th cells in vitro or in vivo. Mating the Hoxed GFP-C5aRl mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed CSaR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells. [ABSTRACT FROM AUTHOR]

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain–containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1–dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-Glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans–induced inflammasome responses in mouse dendritic cells. We show that the NLRP3–apoptosis-associated speck-like protein containing caspase recruitment domain protein–caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating β-glucan–induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting β-glucan–induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1β production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan– and C. albicans–induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components. [ABSTRACT FROM AUTHOR]

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/ CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ~8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans. [ABSTRACT FROM AUTHOR]

Activation of the complement system is primarily initiated by pathogen- and damage-associated molecular patterns on cellular surfaces. However, there is increasing evidence for direct activation of individual complement components by extrinsic proteinases as part of an intricate crosstalk between physiological effector systems. We hypothesized that kallikrein-related peptidases (KLKs), previously known to regulate inflammation via proteinase-activated receptors, can also play a substantial role in innate immune responses via complement. Indeed, KLKs exemplified by KLK14 were efficiently able to cleave C3, the point of convergence of the complement cascade, indicating a potential modulation of C3-mediated functions. By using in vitro fragmentation assays, mass spectrometric analysis, and cell signaling measurements, we pinpointed the generation of the C3a fragment of C3 as a product with potential biological activity released by the proteolytic action of KLK14. Using mice with various complement deficiencies, we demonstrated that the intraplantar administration of KLK14 results in C3-associated paw edema. The edema response was dependent on the presence of the receptor for C3a but was not associated with the receptor for the downstream complement effector C5a. Our findings point to C3 as one of the potential substrates of KLKs during inflammation. Given the wide distribution of the KLKs in tissues and biological fluids where complement components may also be expressed, we suggest that via C3 processing, tissue-localized KLKs can play an extrinsic complement-related role during activation of the innate immune response. [ABSTRACT FROM AUTHOR]

Allergen-IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut- or cow's milk-allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen-Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen-Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen-IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic response. [ABSTRACT FROM AUTHOR]

Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (CrlKO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the CrlKO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized CrlKO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses. [ABSTRACT FROM AUTHOR]

Th cell responses induced by Aspergillus fumigatus have been extensively investigated in mouse models. However, the requirements for differentiation and the characteristics of A. fumigatus-induced human Th cell subsets remain poorly defined. We demonstrate that A. fumigatus induces Th1 and Th17 subsets in human PBMCs. Moreover, we show that the cytokine IL-22 is not restricted to a specific Th subset, in contrast to IL-17A. The pattern recognition and cytokine pathways that skew these Aspergillus-induced Th cell responses are TLR4- and IL-1-, IL-23-, and TNF-α-dependent. These pathways are of specific importance for production of the cytokines IL-17A and IL-22. Additionally, our data reveal that the dectin-1/Syk pathway is redundant and that TLR2 has an inhibitory effect on Aspergillus-induced IL-17A and IL-22 production. Notably, blocking complement receptor (CR)3 significantly reduced Aspergillus-induced Th1 and Th17 responses, and this was independent on the activation of the complement system. CR3 is a known receptor for β-1,3-glucan; however, blocking CR3 had significant effects on Th cell responses induced by heat-killed Aspergillus conidia, which have minimal β-glucan expression on their cell surface. Collectively, these data characterize the human Th cell subsets induced by Aspergillus, demonstrate that the capability to produce IL-22 is not restricted to a specific T cell subset, and provide evidence that CR3 might play a significant role in the adaptive host defense against Aspergillus, although the ligand and its action remain to be elucidated. [ABSTRACT FROM AUTHOR]

Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A-D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22-30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22-30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22-30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22-30. The CR1 binding site for MBL/ficolins was mapped to CCP24-25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/Lficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25. [ABSTRACT FROM AUTHOR]

Complement is implicated in the pathogenesis of ischemia-reperfusion injury (IRI). The activation pathway(s) and effector(s) of complement in IRI may be organ specific and remain to be fully characterized. We previously developed a renal IRI model in decay-accelerating factor (DAF) and CD59 double-knockout (DAF-/-CD59-/-) mice. In this study, we used this model to dissect the pathway(s) by which complement is activated in renal IRI and to evaluate whether C3aR- or C5aR-mediated inflammation or the membrane attack complex was pathogenic. We crossed DAF-/-CD59-/- mice with mice deficient in various complement components or receptors including C3, C4, factor B (fB), factor properdin (fP), mannose-binding lectin, C3aR, C5aR, or Ig and assessed renal IRI in the resulting mutant strains. We found that deletion of C3, fB, fP, C3aR, or C5aR significantly ameliorated renal IRI in DAF-/-CD59-/- mice, whereas deficiency of C4, Ig, or mannose-binding lectin had no effect. Treatment of DAF-/- CD59-/- mice with an anti-C5 mAb reduced renal IRI to a greater degree than did C5aR deficiency. We also generated and tested a function-blocking anti-mouse fP mAb and showed it to ameliorate renal IRI when given to DAF-/-CD59-/- mice 24 h before, but not 4 or 8 h after, ischemia/reperfusion. These results suggest that complement is activated via the alternative pathway during the early phase of reperfusion, and both anaphylatoxin-mediated inflammation and the membrane attack complex contribute to tissue injury. Further, they demonstrate a critical role for properdin and support its therapeutic targeting in renal IRI. [ABSTRACT FROM AUTHOR]

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid-deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development. [ABSTRACT FROM AUTHOR]

Complement receptor 1 (CR1) expressed on the surface of phagocytic cells binds complement-bound immune complexes (IC), playing an important role in the clearance of circulating IC. This receptor is critical to prevent accumulation of IC, which can contribute to inflammatory pathology. Accumulation of circulating IC is frequently observed during malaria, although the factors contributing to this accumulation are not clearly understood. We have observed that the surface expression of CR1 on monocytes/macrophages and B cells is strongly reduced in mice infected with Plasmodium yoelii, a rodent malaria model. Monocytes/macrophages from these infected mice present a specific inhibition of complement-mediated internalization of IC caused by the decreased CR1 expression. Accordingly, mice show accumulation of circulating IC and deposition of IC in the kidneys that inversely correlate with the decrease in CR1 surface expression. Our results indicate that malaria induces a significant decrease on surface CR1 expression in the monocyte/macrophage population that results in deficient internalization of IC by monocytes/macrophages. To determine whether this phenomenon is found in human malaria patients, we have analyzed 92 patients infected with either P. falciparum (22 patients) or P. vivax (70 patients), the most prevalent human malaria parasites. The levels of surface CR1 on peripheral monocytes/macrophages and B cells of these patients show a significant decrease compared with uninfected control individuals in the same area. We propose that this decrease in CR1 plays an essential role in impaired IC clearance during malaria. [ABSTRACT FROM AUTHOR]

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry-/-DAF-/- mice were viable on a C3-/- background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF-/- mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre+-Crryflox/flox mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre+-Crryflox/flox bone marrows showed platelets from C3-/- but not C3+/+ recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre+-Crryflox/flox mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre+-Crryflox/flox mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre+-Crryflox/flox mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies. [ABSTRACT FROM AUTHOR]

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that Clq and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS 0-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CDllb/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CDllc/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and 0-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via Clq to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. [ABSTRACT FROM AUTHOR]

Complement plays a key role in the pathophysiology of many inflammatory diseases, and in this study, we investigated the role of complement in the pathogenesis of inflammatory bowel disease. Compared to wild-type mice, mice deficient in C3 or factor B were protected from acute dextran sulfate sodium (DSS)-induced colitis. Clq/mannose-binding lectin (MBL) double-deficient mice, however, exhibited more severe colitis than wild-type mice. When mice were allowed to recover after DSS treatment, all Clq/MBL-/- mice died by day 2 of recovery period, and, surprisingly, all C3-/- and factor B-/- mice died by day 5. Serum endotoxin levels were significantly increased in complement-deficient mice prior to death, particularly in Clq/MBL-/- mice, and antibiotic treatment prevented the lethal effect of DSS in all complement-deficient mice. In contrast to complement deficiency, targeted complement inhibition with either complement receptor 2 (CR2)-Crry (blocks all pathways at C3 activation) or CR2-factor H (blocks alternative pathway) was highly protective at treating established acute colitis. Endotoxin levels remained low in complement-inhibited mice, and complement inhibition also reduced inflammatory cytokines, leukocyte infiltration, and tissue injury while improving wound repair and mucosal healing. CR2-factor H provided more effective protection than CR2-Crry. Thus, complement has both pathogenic and protective roles in acute DSS-induced colitis, and whereas the alternative pathway appears to play a key role in tissue inflammation and injury, the classical/lectin pathway provides important protection in terms of host defense and wound repair. Targeted inhibition of the alternative pathway may represent a therapeutic modality for treating acute phases of inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Activation of the alternative pathway of complement plays a critical role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in mice. Endogenous factor H, a potent inhibitor of the alternative pathway, is increased in the airways of sensitized and challenged mice, but its role in regulating inflammation or AHR has been unknown. We found that blocking the tissue-binding function of factor H with a competitive antagonist increased complement activation and tissue inflammation after allergen challenge of sensitized mice. Conversely, administration of a fusion protein that contains the iC3b/C3d binding region of complement receptor 2 linked to the inhibitory region of factor H, a molecule directly targeting complement-activating surfaces, protected mice in both primary and secondary challenge models of AHR and lung inflammation. Thus, although endogenous factor H does play a role in limiting the development of AHR, strategies to deliver the complement-regulatory region of factor H specifically to the site of inflammation provide greater protection than that afforded by endogenous regulators. Such an agent may be an effective therapy for the treatment of asthma. [ABSTRACT FROM AUTHOR]