Complement-deficient Mice Research Articles

Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3(-/-) mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3(-/-) mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3(-/-) mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3(-/-) blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR(-/-) mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

TheBordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement-mediated killing

Bordetella pertussis is a human-restricted Gram-negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non-polar in-frame deletion of the bpsA-D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end-product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps-expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement-deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.

RETRACTED ARTICLE: TGF-β signaling regulates neuronal C1q expression and developmental synaptic refinement

Immune molecules, including complement proteins C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement localizes to synapses and refines the developing visual system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. Previous work implicated an astrocyte-derived factor in regulating neuronal C1q expression. Here we identify retinal TGF-β as a key regulator of neuronal C1q expression and synaptic pruning in the developing visual system. Mice lacking TGF-β receptor II (TGFβRII) in retinal neurons have reduced C1q expression in RGCs, reduced synaptic localization of complement, and phenocopy refinement defects observed in complement-deficient mice, including reduced eye specific segregation and microglial engulfment of RGC inputs. These data implicate TGF-β in regulating neuronal C1q expression to initiate complement- and microglia-mediated synaptic pruning.

Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation

ABSTRACTLevels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells.

Mesenchymal stem cells are injured by complement after their contact with serum

AbstractDespite the potent immunosuppressive activity that mesenchymal stem cells (MSCs) display in vitro, recent clinical trial results are disappointing, suggesting that MSC viability and/or function are greatly reduced after infusion. In this report, we demonstrated that human MSCs activated complement of the innate immunity after their contact with serum. Although all 3 known intrinsic cell-surface complement regulators were present on MSCs, activated complement overwhelmed the protection of these regulators and resulted in MSCs cytotoxicity and dysfunction. In addition, autologous MSCs suffered less cellular injury than allogeneic MSCs after contacting serum. All 3 complement activation pathways were involved in generating the membrane attack complex to directly injure MSCs. Supplementing an exogenous complement inhibitor, or up-regulating MSC expression levels of CD55, one of the cell-surface complement regulators, helped to reduce the serum-induced MSC cytotoxicity. Finally, adoptively transferred MSCs in complement deficient mice or complement-depleted mice showed reduced cellular injury in vivo compared with those in wild type mice. These results indicate that complement is integrally involved in recognizing and injuring MSCs after their infusion, suggesting that autologous MSCs may have ad-vantages over allogeneic MSCs, and that inhibiting complement activation could be a novel strategy to improve existing MSC-based therapies.

Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases.

Complement-Dependent Injury and Protection in a Murine Model of Acute Dextran Sulfate Sodium-Induced Colitis

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. C1q/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 C1q/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 C1q/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.

Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?

AbstractTo explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035–41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.

The Vaccinia Virus Complement Control Protein Modulates Adaptive Immune Responses during Infection

Complement activation is an important component of the innate immune response against viral infection and also shapes adaptive immune responses. Despite compelling evidence that complement activation enhances T cell and antibody (Ab) responses during viral infection, it is unknown whether inhibition of complement by pathogens alters these responses. Vaccinia virus (VACV) modulates complement activation by encoding a complement regulatory protein called the vaccinia virus complement control protein (VCP). Although VCP has been described as a virulence factor, the mechanisms by which VCP enhances VACV pathogenesis have not been fully defined. Since complement is necessary for optimal adaptive immune responses to several viruses, we hypothesized that VCP contributes to pathogenesis by modulating anti-VACV T cell and Ab responses. In this study, we used an intradermal model of VACV infection to compare pathogenesis of wild-type virus (vv-VCPwt) and a virus lacking VCP (vv-VCPko). vv-VCPko formed smaller lesions in wild-type mice but not in complement-deficient mice. Attenuation of vv-VCPko correlated with increased accumulation of T cells at the site of infection, enhanced neutralizing antibody responses, and reduced viral titers. Importantly, depleting CD8(+) T cells together with CD4(+) T cells, which also eliminated T helper cell-dependent Ab responses, restored vv-VCPko to wild-type levels of virulence. These results suggest that VCP contributes to virulence by dampening both antibody and T cell responses. This work provides insight into how modulation of complement by poxviruses contributes to virulence and demonstrates that a pathogen-encoded complement regulatory protein can modulate adaptive immunity.

Infectivity of Borrelia burgdorferi sensu lato is unaltered in C3-deficient mice

B. burgdorferi, B. afzelii, and B. bavariensis show resistance to mouse and human complement. B. garinii and B. valaisiana are sensitive to mouse and human complement. We evaluated whether the absence of C3 in mice influenced infectivity and pathogenicity of different Borrelia species. C3 knockout mice (C3−/−) and syngeneic C57Bl/6 wild-type (WT) mice were challenged with 5 different Borrelia species. After 2 weeks, quantitative PCR (qPCR), culture, histopathology, and immunofluorescence were performed on heart, joint, brain, bladder, and skin. Spirochaetes were detected by qPCR after infection with B. burgdorferi, B. afzelii, or B. bavariensis strains. In joints of C3−/−, but not WT mice challenged with B. burgdorferi, spirochaetes were detected by qPCR. No other significant differences between C3−/− and WT mice were seen. Histopathology demonstrated concordance between borrelia load and inflammation score. Only after B. burgdorferi and B. afzelii infection, spirochaetes were detected by immunofluorescence microscopy. B. burgdorferi was cultured from heart, joint, bladder, and skin from all mice within 2 weeks. B. afzelii and B. bavariensis grew only from heart tissue from both C3−/− and WT mice after 2–6 weeks. The infectivity and pathogenicity of complement-resistant Borrelia strains is unchanged in complement-deficient mice. Complement-susceptible strains do not become infectious in the absence of C3.

Properdin homeostasis requires turnover of the alternative complement pathway

Properdin is a plasma protein and is also released from neutrophil granules following stimulation. At inflammatory sites it can bind bacteria and apoptotic bodies to trigger alternative pathway (AP) activation. Principles governing properdin homeostasis are unknown. We monitored properdin during AP activation and in complement-deficient mice. There was a >90% reduction of properdin in the Crry single-knockout mice (Crry SKO). These membrane complement regulatory protein-deficient mice feature accelerated AP turnover, leading to reduced C3 and fB. Injecting cobra venom factor into wild-type mice activated the AP and led to the consumption of C3, fB, and properdin. However, and unexpectedly, properdin was also deficient in C3(-/-), fB(-/-), and fD(-/-) mice. It was present in C1q(-/-), C4(-/-), and C5(-/-) mice. These findings implicate AP turnover in the maintenance of basal levels of properdin in the blood. To explore the mechanism, classical pathway-activating immune complexes were infused. Within 10 min, properdin was partially restored in fB(-/-) but not in C3(-/-) mice. Markedly reduced properdin in mice deficient in an AP component and its partial restoration by activating C3 suggest a requirement for continuous C3 activation via AP tickover to maintain properdin homeostasis. The mechanism underlying this C3-dependent process was not identified. Engagement of C3a and C5a receptors was ruled out. These findings represent an instructive example of how a positive regulator of an innate immune recognition and effector pathway is controlled. A rationale for such a means to supply properdin for immune reactions is proposed.

Cholesterol Lipids of Borrelia burgdorferi Form Lipid Rafts and Are Required for the Bactericidal Activity of a Complement-Independent Antibody

Borrelia burgdorferi, the agent of Lyme disease, is unusual as it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). We find that the bactericidal actionof CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterialcholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi and in model membranes from B. burgdorferi lipids. The order and size of the microdomains are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.

Murine complement deficiency ameliorates acute cigarette smoke-induced nasal damage

Objective Exposure to cigarette smoke is a risk factor for chronic rhinosinusitis. Current literature confirms complement fragments are activated in human nasal mucosa. The mechanism(s) responsible for this activation is unclear. We investigated the effects of cigarette smoke on nasal mucosa in vitro and via a model of cigarette smoke exposure by using animals deficient in complement components. Study Design Prospective, controlled animal and in vitro human cell line study. Setting University laboratory. Subjects and Methods Human respiratory epithelial cells were exposed to five, 10, and 20 percent cigarette smoke extract (CSE) in vitro in the presence or absence of human serum. Complement activation was assessed by enzyme-linked immunosorbent assay and immunofluorescent techniques. Complement-deficient (C3 –/–, n = 6; factor B –/–, n = 50) and sufficient mice (wild type, n = 10) were exposed to the smoke of four cigarettes per exposure for two exposures per day for three days. Mice were sacrificed 12 hours after the last exposure, and the nasal cavity was surgically removed. Histological characteristics were analyzed by the use of a subjective scale and quantitative image analysis scoring systems. Results In vitro analysis of respiratory cell cultures demonstrated that exposure of serum to CSE resulted in complement activation. Furthermore, immunofluorescent staining for C3d could only be demonstrated in CSE-exposed cultures. In vivo analysis demonstrated that complement deficiency, either C3 or factor B deficiency, resulted in a significant reduction in histological evidence of damage as compared with wild-type control mice (wild type vs C3 –/–, P = 0.02; wild type vs factor B –/–, P = 0.07; no significant difference between C3 –/– vs factor B –/–). Conclusion These data demonstrate that cigarette smoke activates the complement system. Furthermore, complement deficiency protected against smoke-induced mucosal damage in this small series.

MBL‐associated serine protease 1 (MASP‐1) is necessary for thrombin substrate cleavage in vitro

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Previous research established a connection between the complement and coagulation systems; however no studies have specifically investigated the molecular mechanisms underlying this interaction. Thus, the purpose of this study was to elucidate the role of complement, specifically the lectin pathway, in thrombin‐like activity in vitro. MBL‐complexes were captured on mannan‐coated plates using serum collected from wild‐type (WT) and a variety of complement deficient mice. The captured MBL‐complexes were incubated with a chromogenic thrombin substrate, S2238, for 1.5hrs at 37°C, and cleavage was measured using a fluorometer. MBL‐complexes captured from WT, C2/fB−/−, MBL‐A−/− or MBL‐C−/− sera showed no significant difference in S2238 cleavage between groups. In contrast, complexes captured from MBL null and MASP‐1/3−/− mouse sera demonstrated significantly decreased S2238 cleavage compared to WT (P<0.01). Recombinant human (rh)MBL alone also failed to cleave S2238, yet cleavage could be restored when rMASP‐1 was added back to either MASP1/3−/− sera or rhMBL. Taken together, these findings indicate that the MBL‐complex with MASP‐1 has thrombin‐like activity, and may suggest a key role for lectin complexes in thrombus formation and coagulopathy in vivo. Supported by HL56086, HL92469 and HL99130.

Enhanced Susceptibility to Acute Pneumococcal Otitis Media in Mice Deficient in Complement C1qa, Factor B, and Factor B/C2

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

Mobilization studies in complement-deficient mice reveal that optimal AMD3100 mobilization of hematopoietic stem cells depends on complement cascade activation by AMD3100-stimulated granulocytes

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) induced by granulocyte-colony stimulating factor (G-CSF) and C5 cleavage plays an important role in optimal egress of HSPCs. In the current work, we explored whether CC is involved in mobilization of HSPCs induced by the CXCR4 antagonist, AMD3100. To address this question, we performed mobilization studies in mice that display a defect in the activation of the proximal steps of CC (Rag−/−, SCID, C2.Cfb−/−) as well as in mice that do not activate the distal steps of CC (C5−/−). We noticed that proximal CC activation-deficient mice (above C5 level), in contrast to distal step CC activation-deficient C5−/− ones mobilize normally in response to AMD3100 administration. We hypothesized that this discrepancy in mobilization could be explained by AMD3100 activating C5 in Rag−/−, SCID, C2.Cfb−/− animals in a non-canonical mechanism involving activated granulocytes. To support this granulocytes i) as first egress from BM and ii) secrete several proteases that cleave/activate C5 in response to AMD3100. We conclude that AMD3100-directed mobilization of HSPCs, similarly to G-CSF-induced mobilization, depends on activation of CC; however, in contrast to G-CSF, AMD3100 activates the distal steps of CC directly at the C5 level. Overall, these data support that C5 cleavage fragments and distal steps of CC activation are required for optimal mobilization of HSPCs.

Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Abstract 367▪▪This icon denotes an abstract that is clinically relevant.We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures:No relevant conflicts of interest to declare.

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype

Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.

Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generationin vitro

The alternative pathway (AP) of complement alone is capable of mediating immune complex-induced arthritis in the collagen antibody-induced arthritis (CAIA) model in mice. Whether the classical pathway (CP) or lectin pathway (LP) alone can mediate CAIA is not known. Using mice genetically deficient in different complement components, our results reported herein establish that the CP and LP alone are each incapable of mediating CAIA. A lower level or absence of C3 and/or C5 activation by the CP may be possible explanations for the importance of the AP in CAIA and in many murine models of disease. In addition, other investigators have reported that CP C5 convertase activity is absent in mouse sera. To address these questions, we employed an in vitro system of adherent immunoglobulin (Ig)G-induced complement activation using plates coated with murine anti-collagen monoclonal antibody (mAb). These experiments used complement-deficient mouse sera and wild-type mouse or normal human sera under conditions inactivating either the CP (Ca(++) deficiency) or the AP (mAb inhibitory to factor B). Robust generation of both C3a and C5a by either the AP or CP alone were observed with both mouse and human sera, although there were some small differences between the species of sera. We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP.

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs

Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA.