In vitro effects of organic solvents on immunity indicators in serum

Serum treatment in vitro with organic solvents (chloroform, ether, toluene) failed to produce an effect on immunoglobulin levels and activity. After chloroform and ether treatment, no complement activity could be determined, with chloroform-treated serum beginning to express anticomplement activity against autologous, allogenic and xenogenic sera. The classical pathway of complement activation (C1, C4, C2, C3) was primarily inhibited, whereas the alternative pathway remained unaffected. Chloroform-treated sera exhibited significantly declined levels of C1-INH, C3 and C4 as well as of circulating immune complexes. Toluene did not influence any of the parameters tested, while ether blocked complement activity without affecting either the concentration or activity of the other components under investigation. The obtained findings are discussed from the aspect of organic solvent applications in preparing immune products and determining immunity indicators in the serum or other biological fluids.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

Characteristics of the beta-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.     

Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

Importance of carbohydrate in the interaction of Tamm-Horsfall protein with complement 1q and inhibition of classical complement activation

Tamm-Horsfall protein (THP) binds strongly to complement 1q (C1q), a key component of the classical complement pathway. The goals of this study were to determine whether THP altered the activation of the classical complement pathway and whether the carbohydrate portion of THP was involved in this glycoprotein's binding to C1q and alteration of complement activation. The ability of THP to prevent complement activation in diluted serum or plasma incubated at 37 degrees C was assessed using both a haemolytic assay with antibody-sensitized sheep RBC and a C4d ELISA. Both these methods showed that THP inhibited activation of the classical complement pathway in a dose-dependent manner. Glycosidases were used to remove most of the carbohydrate from THP. This partially deglycosylated THP bound human IgG with a higher affinity (KD1 = 1.4 nmol/L; KD2 = 0.31 micromol/L) than did intact THP (KD1 = 33.4 nmol/L; KD2 = 31.0 micromol/L). An ELISA showed that removal of carbohydrate from THP reduced, but did not eliminate, the ability of this protein to inhibit binding of C1q to intact THP. Haemolysis assays using antibody-sensitized sheep RBC showed that removal of THP carbohydrate eliminated the ability of THP to protect against complement activation. In conclusion, THP inhibited the activation of the classical complement pathway that occurred in diluted serum or plasma. The carbohydrate moieties of THP appeared to be important in this inhibitory activity.     

Identification of RNA as a complement inhibitory component in an extract of Ehrlich ascites tumor cells.

A factor capable of inhibiting complement was obtained from intact Ehrlich ascites tumor cells by mild extraction with phosphate-buffered saline (PBS). The inhibitor caused a decrease in extent of lysis of EAC14 with a concomitant extension of Tmax. EA, EAC1, EAC4 and EAC142 were all less susceptible to complement-mediated lysis after treatment with the tumor cell extract. Partial purification of a complement inhibitor was accomplished. The inhibitor was rich in RNA and its activity was totally destroyed by RNAase but not DNAase. RNA from mouse tissues, yeast, and Escherichia coli also inhibited complement hemolytic activity. The partially purified material only inhibited lysis of EAC1 and EAC14. Slow inhibition of fluid phase C1 was also demonstrated. In addition, RNA-rich partially purified tumor cell extract was capable of precipitating with purified human C1q.     

Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement.

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.     

Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

Substituted isocoumarins as inhibitors of complement serine proteases.

Inhibition of complement proteins D, B, C2, C1s, C1r, I, and the catalytic fragments Bb and C2a by substituted isocoumarins was investigated. 3,4-Dichloroisocoumarin, a general serine protease inhibitor, inhibited factor D, C1r, and C1s moderately with second-order inhibition constants (kobs/[I]) of 40 to 190 M-1 s-1, but it did not inhibit C2, factor B, C2a, or Bb. The best inhibitor for factors D and B was 4-chloro-7-guanidino-3-methoxyisocoumarin with kobs/[I] values of 250 and 290 M-1 s-1, respectively. Most isocoumarins did not inhibit C2 or C2a; only 4-chloro-3-isothiureidoalkoxyisocoumarins were slightly inhibitory. 3-Alkoxy-4-chloro-7-guanidinoisocoumarins inhibited C1r and C1s moderately. The best inhibitor for C1r and C1s was 4-chloro-3-(3-isothiureidopropoxy)isocoumarin with kobs/[I] values of 6,600 and 130,000 M-1 s-1, respectively. Fifty amino acid or peptide thioesters containing Arg or other amino acids at the P1 site were tested as substrates of factor I, however none was hydrolyzed. Isocoumarins substituted with chloro and basic groups such as guanidino and isothiureidoalkoxy inhibited factor I activity with its natural substrate C3b, but kobs/[I] values were low. 4-Chloro-3-ethoxy-7-guanidinoisocoumarin inhibited activation of the alternative pathway and, to a lesser extent, of the classical pathway in serum. Several other substituted isocoumarins also inhibited cobra venom factor-initiated activation of the alternative pathway in serum.     

PRELP protein inhibits the formation of the complement membrane attack complex

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.     

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.     

Complement-mediated hemolytic activity of succinylated concanavalin A: preparation and activity of cell intermediates

Succinylated concanavalin A (SCon A) lyses sheep erythrocytes (E) in the presence of complement, whereas the native tetravalent lectin, Con A, is inactive. We have studied the ability of E-SCon A (ES) to interact with early acting guinea pig (gp) or human (hu) complement components (C1, C2, C4) and found that cell intermediates ESC1, ESC4, ESC14, and ESC142 can be generated that are analogous to intermediates conventionally prepared with E and rabbit IgM (pentameric) anti-Forssman antibody. Titration of gp or hu C1, C4, and C2, and quantification of the number of activated C1 molecules bound to ESC4 by the C1 fixation and transfer test showed in each case that an average of one effective site per cell was sufficient to cause cell lysis. Determination of tmax for optimal formation of ESC142 sites depended on the species combination of components used to make the intermediates, and the decay of ESC142 and EAC142 sites or sites generated with ESC4, EAC4, and trypsin-activated C2 were similar. The sugar alpha-D-methylglucopyranoside (alpha-MGP) inhibited binding of SCon A to E and eluted the lectin from ES, whereas galactose was nearly inactive, consistent with lectin sugar-binding selectivity. In contrast, both sugars were ineffective in eluting SCon A or C4hu from ESC4hu, indicating that C4hu blocked the interaction between lectin and alpha-MGP, perhaps by steric hindrance. SCon A is a divalent functional analogue of IgM anti-Forssman antibody that may be a uniquely suited reagent specific for cell membrane glycoconjugates for studying the mechanism of binding and activation of complement components.     

The T-cell receptor and cytotoxicity An anti=idiotype antiserum that inhibits a graft-versus-host reaction does not inhibit cell-mediated cytotoxicity.

An anti-idiotype serum was prepared by injecting BALB anti-AKR serum into (BALB × AKR) F₁ (F1) mice. This serum gave a precipitation band against a hyperimmune BALB anti-AKR serum and inhibited a BALB anti-AKR serum plus complement (C)-mediated cytotoxic reaction. When BALB spleen cells were treated with this serum plus C, their local graft-versus-host reaction against F1 mice was specifically inhibited, proving that T-cell receptors have V regions similar to those of immunoglobulins. On the contrary, the same anti-idiotype serum could not inhibit a cell-mediated Cytotoxicity (CMC) reaction of the same specificity. This would indicate that CMC may be mediated by a population of T-cells which do not necessarily recognize the H-2 antigens through the V region of their receptors.     

Functional analysis of Ficolin-3 mediated complement activation

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.     

The Role of Salivary and Intestinal Complement System Inhibitors in the Midgut Protection of Triatomines and Mosquitoes

Saliva of haematophagous arthropods contain biomolecules involved directly or indirectly with the haematophagy process, and among them are encountered some complement system inhibitors. The most obvious function for these inhibitors would be the protection of the midgut against injury by the complement. To investigate this hypothesis, Triatoma brasiliensis nymphs were forced to ingest human serum in conditions in which the protection of midgut by the inhibitors is bypassed. In these conditions, the anterior midgut epithelium was injured by the complement, causing cell death. Once some insects such as Aedes aegypti have no salivary inhibitors, we hypothesized the existence of intestinal inhibitors. The inhibitory activity was investigated in the intestine of A. aegypti as well as in the saliva and intestine of other three triatomine species (T. brasiliensis, T. infestans and Rhodnius prolixus) using an immunological method able to determine the level of deposition of some complement factors (C1q, C3b, or C4b) on the surface of complement activator molecules linked to microplates. This methodology permitted to identify which points along the activation phase of the complement cascade were inhibited. As expected, soluble contents of A. aegypti''s intestine was capable to inhibit C3b deposition by the classical and alternative pathways. Saliva or soluble intestinal contents, obtained from triatomines were unable to inhibit C1q deposition by the classical pathway. C4b deposition by the classical pathway was inhibited by the intestinal contents from the three triatomines. On the other hand, only T. brasiliensis saliva inhibited C4b deposition. Both, saliva and intestinal contents from all triatomines were able to inhibit C3b deposition in the classical and alternative pathways. None of the material extracted from the intestinal cell membranes from the triatomines inhibited C3b deposition in the classical pathway. The existence of complement inhibitors may have important biological consequences which are discussed in detail.     

Human astrovirus coat protein inhibits serum complement activation via C1, the first component of the classical pathway

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.     

Complement-mediated release of histamine from human leukocytes.

Activation of either the alternative or classical pathway of complement generated a factor which induced release of histamine from both non-allergic and allergic human basophils. This factor probably is derived from the complement system since 1) its formation was associated with loss of C3 activity in human serum, 2) chemotactic factor, probably also a complement product, was generated simultaneously, 3) heat inactivation blocked its formation, 4) anti-C3 and anti-C5 blocked formation of the factor, and finally 5) anti-C5 inhibited the activity of the factor once it had been formed. It appears that both complement-mediated and allergen-mediated release of histamine from basophils are secretory, non-cytolytic pathways since both were maximal at 37 degrees C, required the presence of divalent cations, and were inhibited by theophylline. One consistent difference between these two mechanisms was noted: complement-initiated release of histamine occurred more quickly.     

Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.