Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans.

Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5+/+) and congenic C57BL/6 (B6 C5-/-, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5-/- mice when compared to B6 C5+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5+/+ mice than in B6 C5-/- mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5+/+ than B6 C5-/- mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5+/+ and B6 C5-/- were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host.

Basal physiological parameters of two congenic mice strains: C5 deficient C57BL/6 and C5 sufficient A/J.

To investigate the in vivo role of complement component C5 it is common to compare the inflammatory response between C5-normal and C5-deficient inbred mice strains. Nevertheless, it should be expected that differences in the genetic backgrounds between those strains may affect several physiological parameters, complicating the correct interpretation of results. The use of congenic mice, developed by backcrossing, is therefore preferred. Still, several physiological parameters may be distinctive in the normal and deficient strains and therefore require careful analysis before animals are selected for investigation. We generated two congenic mouse strains: C57BL/6 (B6) C5(-), derived from the parental B6 C5(+) strain and A/J C5(+) mice derived from the parental A/J C5(-) strain. After confirmation by nucleotide sequencing, immunodiffusion and hemolytic activity analysis, several basal physiological parameters were analyzed in the congenic and parental strains before antigen exposition. Serum levels of liver alanine aminotransferase, alkaline phosphatase, albumin, triglycerides, cholesterol and uric acid were found to be different in C5-sufficient and C5-deficient mice from one or both genetic backgrounds (B6 and/or A/J). On the other hand, serum levels of liver aspartate aminotransferase, glucose and urea were not affected by the presence of C5 in either strain. Furthermore, in some cases, C5-dependent variations in these parameters were more evident in mice of the same gender. We conclude here that C5-deficient mice strains may present distinct systemic behaviors which should be taken in consideration before differences in the immune responses are attributed solely to the lack of circulating C5.

Early activation of the host complement system is required to restrict central nervous system invasion and limit neuropathology during Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus, family Togaviridae, that is responsible for sporadic outbreaks in human and equid populations in Central and South America. In order to ascertain the role that complement plays in resolving VEEV-induced disease, complement-deficient C3(-/-) mice were infected with a VEEV mutant (V3533) that caused mild, transient disease in immunocompetent mice. In the absence of a functional complement system, peripheral inoculation with V3533 induced much more severe encephalitis. This enhanced pathology was associated with a delay in clearance of infectious virus from the serum and more rapid invasion of the central nervous system in C3(-/-) mice. If V3533 was inoculated directly into the brain, however, disease outcome in C3(-/-) and wild-type mice was identical. These findings indicate that complement-dependent enhancement of peripheral virus clearance is critical for protecting against the development of severe VEEV-induced encephalitis.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5.

OBJECTIVE: Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. MATERIAL: AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. METHODS AND RESULTS: While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. CONCLUSIONS: The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

Behçet's disease patients present high levels of deglycosylated anti-lipoteichoic acid IgG and high IL-8 production after lipoteichoic acid stimulation.

OBJECTIVES: Lipoteichoic acid (LTA), induces some of the clinical symptoms of Behçet's disease (BD) in a rat animal model. These results led to the hypothesis that LTA may also trigger BD in humans. We investigated the humoral and cellular immune response against LTA and lipopolysaccharide (LPS) in patients with BD, and compared these responses with those of patients with active chronic oral ulcers (OU) and normal controls. METHODS: Samples were obtained from 12 active BD, 12 inactive BD, 12 active OU and 12 normal controls. Anti-LTA, anti-LPS antibodies levels and the capacity of immune complexes anti-LTA IgG-LTA to activate complement were studied. Exposed mannose residues in anti-LTA IgG were analyzed in the four groups. The interleukin-8 (IL-8) production by peripheral blood mononuclear cells cultures after LTA and LPS stimulation was also studied in all groups. RESULTS: The capacity to bind mannan binding protein (MBP) of anti-LTA IgGs was significantly higher in BD and active OU patients relative to normal controls (p < 0.001). However, only active BD patients generated significantly higher levels of C5a than controls (p < 0.0001). The IgGs purified from the sera of BD patients showed a high specificity for LTA from Streptococcus sanguis or Streptococcus faecalis. LTA also stimulates the secretion of IL-8 in peripheral blood mononuclear cells isolated from active BD patients. Anti-LPS IgA and IgG titers were significantly higher only in active OU patients relative to normal controls (p < 0.0018). CONCLUSION: These results suggest a mechanism involving LTA from streptococci in the pathogenesis of BD.

The complement system is involved in acute inflammation but not in the hemorrhage produced by a Bothrops atrox snake venom low molecular mass proteinase.

Low molecular weight hemorrhagins were purified from crude Bothrops atrox snake venom by gel filtration followed by ionic strength chromatography. The protein fractions obtained, designated HI-1 to HI-8, contained proteins with molecular masses lower than 30 kDa. HI-5, the most representative among of these fractions, exhibited, in vitro, proteolytic and C inactivating properties, as analyzed by proteolysis of a protein substrate, and C system consumptive activities as assayed by reduction of the hemolytic C activity in normal human serum and by cleavage of partially purified component C3. HI-5 hemorrhagin injected i.m. into C-sufficient BALB/c mice induced a local inflammation characterized by edema, accumulation of polymorphonuclear leucocytes (PMN) and hemorrhage. In contrast, when injected into BALB/c mice previously C-depleted, the number of PMN per tissue section, but not hemorrhage, was significantly reduced (129.668 +/- 31.341 cells per microscopic field) as compared with the control C-sufficient mice (812.168 +/- 111.194 cells per microscopic field). The observations were confirmed by using C5-deficient mice instead of C-depleted mice. The average number of PMN per tissue section in C5-defficient A/J mice was 72.666 +/- 19.416 cells per microscopic field. These data indicate that the C system is involved in PMN accumulation, but not in the hemorrhage, at the local induced lesions by low molecular mass B. atrox hemorrhagins. HI-5 apparently is not contaminated with other direct or indirect inflammation mediators, PMN accumulation and hemorrhage, however, an independent phenomenon, could be mediated by the same hemorrhagin proteinase domain.

Contribution of C5-mediated mechanisms to host defence against Echinococcus granulosus hydatid infection.

The aim of this work was to investigate the contribution of complement C5-mediated mechanisms, with an emphasis on inflammation, to host defences against Echinococcus granulosus hydatid disease. Thus, we compared the systemic and local inflammatory responses induced by the parasite, and the outcome of infection, between congenic C5-sufficient (B10.D2 n/SnJ) and C5-deficient (B10.D2 o/SnJ) mice challenged with protoscoleces. Indirect evidence of in-vivo complement activation during the establishment phase was obtained; infection induced serum amyloid P and eosinophil responses which were dependent on C5. Early recruitment of polymorphonuclear cells was not dependent on the presence of C5. The higher capacity of C5-sufficient mice to recruit eosinophils was also observed during the cystic phase of infection, and mice recruiting more eosinophils developed lower parasite masses. Analysis of the outcome of infection after 8 months showed that C5-sufficient mice were more resistant to infection than C5-deficient mice in terms of individuals with no cysts; this trend was not statistically significant. In addition, C5-deficient mice developed higher numbers of large (> 5 mm in diameter) cysts and higher cyst weights than C5-sufficient mice indicating that C5-mediated mechanisms are detrimental for parasite growth. Taken together, our results suggest that complement, through C5-mediated effectors, contributes to host defences by both restricting the establishment of infection and controlling the growth of established cysts. This contribution may, at least partially, be associated with the ability of C5a to promote eosinophil infiltration.

Potential pathogenicity of deglycosylated IgG cross reactive with streptokinase and fibronectin in the serum of patients with rheumatoid arthritis.

OBJECTIVE: Fibronectin (FN) and the streptococcal plasminogen activator streptokinase (SK) share the epitope LTSRPA. This epitope is not reactive in native FN and it reacts with anti-SK antibodies only after plasmin digestion of the protein. To investigate a potential correlation between the high levels of anti-LTSRPA antibodies in sera of patients with rheumatoid arthritis (RA) and the perpetuation of the immune response characteristic of this disease, we analyzed their capacity to activate complement and the process of binding to the serum lectin mannan binding protein (MBP). METHODS: We used a radioimmunoassay to evaluate immune complexes between anti-LTSRPA IgG and FN, plasmin degraded FN, or the LTSRPA peptide for their capacity to activate complement C5 to C5a. Purified human serum lectin MBP was used to quantify the degree of exposed mannose or N-acetylglucosamine residues in the Fc region of anti-LTSRPA IgG of patients with RA and healthy controls. RESULTS: Anti-LTSRPA IgG from patients with RA have a greater capacity to activate complement C5 to C5a when bound to either the LTSRPA peptide or plasmin degraded FN in vitro. We found a very strong correlation between the complement activating capacity of the RA immune complexes and their binding to MBP. CONCLUSION: The enhanced capacity of RA anti-LTSRPA IgG immune complexes to activate complement C5 to C5a is directly correlated with their binding capacity to MBP. As MBP binding depends on exposed mannose or N-acetylglucosamine residues in the Fc region of the IgG molecule, these studies suggest that defective glycosylation of circulating anti-SK IgG may play a role in the etiology of RA.

Binding of complement to trypomastigotes of a Brazil strain of Trypanosoma cruzi: evidence for heterogeneity within the strain.

Binding of the complement components C3 and C5 to epimastigote and trypomastigote stages of the Brazil strain of Trypanosoma cruzi was examined using radioligand binding and flow cytometric assays. Fibroblast-derived trypomastigotes bound approximately 40% fewer molecules of [125I]C3 per parasite than did epimastigotes. The predominant molecular species of C3 deposited on fibroblast-derived trypomastigotes was the inactive form iC3b. Addition of parasite-specific antisera failed to enhance the number of molecules of [125I]C3 per parasite or the proportion of active to inactive C3b. Flow cytometric studies revealed that only 50% of trypomastigotes (fibroblast-derived or blood-form) bound C3. In contrast to results of the [125I]C3 binding studies, flow cytometric analysis showed that the percentage of trypomastigotes binding C3 actually increased upon incubation with parasite-specific antisera. C5 was found also to bind to only a percentage of trypomastigotes.

In-vitro lysis of sensitized Trypanosoma cruzi by platelets: role of C3b receptors.

Incubation of Trypanosoma cruzi bloodstream trypomastigotes (Btrys) with C5-deficient blood in the presence of anti-T. cruzi immune mouse serum (IMS) or its IgG fraction resulted in an immediate formation of small clumps in which one could easily see platelets adhered to the parasites. After 4 h of incubation most of the clumps had disappeared and the number of the parasites was considerably reduced. Twenty-four hours later there were only a few remaining parasites. This same sequence of events was also observed when the parasites were incubated with isolated platelets in presence of IMS or its IgG fraction. When the parasites were incubated with plasma in the presence of IMS or its IgG fraction but in the absence of platelets there was strong agglutination of the parasites but no reduction in their number. Incubation of the parasites with platelets and IMS or its IgG fraction in C3-depleted plasma prevented adherence of the platelets to the parasites and their subsequent lysis. It is concluded that platelets are able to induce in-vitro lysis of the Btrys and that this phenomenon is dependent on the platelet' receptor for C3b.

Studies on anti-component 5 antibodies in animals infected with Trypanosoma cruzi

Foi usado um teste imunoenzimático competitivo para investigar a presença de anticorpos anticomponente 5 nos soros de sariguês, cäes, coelhos e ratos infectados com o Trypanosoma cruzi. Neste teste, foi utilizado um anticorpo monoclonal contra o antígeno 5 que é específico do T. cruzi. Também foram testados os soros de 51 pacientes venezuelanos com doença de Chagas. Apesar desses anticorpos näo serem detectados nos soros de cäes, ratos e sariguês infectados com o T. cruzi, alguns soros de coelhos infectados apresentaram resultados positivos porém em níveis próximos aos do limite de positividade do teste. Esses achados surgerem que a resposta imune em animais naturalmente ou experimentalmente infectados com o T. cruzi é diferente daquela que é observada na doença de Chagas humana