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The syntheses of 4-(2-phenyl-1H-indol-3-yl)cyclohexane-1-carboxylic acids are described. These compounds express potent in vitro inhibition of the human classical complement pathway, and qualitative SAR have been determined. Several of the in vitro active compounds also suppressed the complement dependent reverse passive Arthus reaction (RPAR) in guinea pigs.
We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37 degrees C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37 degrees C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 +/- 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 +/- 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 microg/ml of C1q significantly increased GMC-apoptosis up to 39.4 +/- 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab')2-ER4G (F(ab')2-ER4G-GMC), while ER14-GMC or F(ab')2-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse K-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.
MN12H2 is a bactericidal antibody directed against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. Binding of MN12H2 to PorA at the meningococcal surface activates the classical complement pathway resulting in bacterial lysis. We have determined the crystal structure of the unliganded MN12H2 Fab fragment in two different crystal forms and compared it with the structure of the Fab in complex with a P1.16-derived peptide. The unliganded Fabs have elbow bend angles of 155 degrees and 159 degrees, whereas the liganded Fab has a more closed elbow bend of 143 degrees. Substantial differences in quaternary and tertiary structure of the antigen binding site are observed between the unliganded and liganded MN12H2 Fab structures that can be attributed to peptide binding. The variable light and heavy chain interface of the liganded Fab is twisted by a 5 degrees rotation along an axis approximately perpendicular to the plane of the interface. Hypervariable loops H1, H2, and framework loop FR-H3 follow this rotation. The hypervariable loop H3 undergoes conformational changes but remains closely linked to hypervariable loop L1. In contrast with the binding site expansion seen in other Fab-peptide structures, the MN12H2 binding site is narrowed upon peptide binding due to the formation of a "false floor" mediated by arginine residue 101 of the light chain. These results indicate that PorA epitope P1.16 of N. meningitidis is recognized by the complement-activating antibody MN12H2 through induced fit, allowing the formation of a highly complementary immune complex.
Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events including complement activation play important roles. Cardioprotection by complement inhibition inter alia C1-esterase-inhibitor (C1-INH) was examined in several experimental models and under clinical conditions with ischemia and reperfusion. C1-INH reduced local anaphylatoxin release revealing the importance of the classical complement pathway. Inhibition of local complement activation was accompanied by improvement of myocardial function and perfusion of the previously ischemic myocardium. Leukocyte endothelial cell-cell interaction was strikingly reduced in the reperfused tissues as afflection of anti-inflammatory effect.
When an eight-year-old boy with a syndrome compatible with disseminated neisseria infection was found to lack C7, studies on the role of antibody and complement in the interaction of polymorphonuclear leukocytes (PMNLs) and Neisseria were initiated with use of a luminol-enhanced chemiluminescence assay. The chemiluminescent response to opsonized Neisseria meningitidis was markedly lower than the response to opsonized zymosan or Streptococcus pneumoniae but was similar to that obtained with Haemophilus influenzae type b. IgG antibody to N. meningitidis was shown to enhance the chemiluminescent response. The chemiluminescent response of PMNLs to N. meningitidis was normal when the bacteria were incubated with sera deficient in C5, C6, or C7 but was absent in serum lacking C2. Thus, both antibody and the early-acting proteins of the classical complement pathway appear to be essential for maximal stimulation of PMNL oxidative metabolism by N. meningitidis, although the late-acting components of complement are not.
After three serial passages of Haemophilus influenzae type b strain Fuju in rats, we recovered a variant, which differed in colonial morphology, serum sensitivity, and lipopolysaccharide configuration from the parent strain. The parent organism (Fuju) appeared iridescent and transparent on Levinthal agar, and the rat-passaged variant (rat3 Fuju) appeared iridescent and opaque. The transparent, parent strain Fuju was sensitive to the complement-mediated bactericidal activity of normal rat serum, and the opaque, rat-passaged strain rat3 Fuju was serum resistant. Serum killing of the serum-sensitive strain appeared to be mediated by the classic complement pathway. Both serum-resistant and serum-sensitive strains were killed equally well by immune rat and human serum. These two strains did not differ in the amount of capsular polysaccharide that they elaborated nor in their major outer membrane protein patterns on SDS-PAGE. Lipopolysaccharide isolated from these two strains demonstrated different electrophoretic mobility patterns. Furthermore, the organisms showed different reactivities with two monoclonal antibodies directed against determinants of Hib lipopolysaccharide. Thus, the difference in susceptibility to complement-mediated bactericidal activity of normal rat serum displayed by these two strains is associated with their phenotypes, appears to be unrelated to differences in major outer membrane proteins or in the amounts of capsular polysaccharide elaborated, and is associated with differences in surface lipopolysaccharides.
We investigated serum bactericidal reactions against Neisseria meningitidis (serogroups A, B, C, D, Y, W-135, 29E, X, and Z) in the sera of two healthy adults with properdin deficiency. Bactericidal reactions mediated via the classic complement pathway (unchelated system) were not impaired in properdin-deficient serum. The properdin-deficient sera supported alternative pathway-mediated killing (Mg++EGTA-chelated system) of some, but not all, of the strains investigated. Vaccination of the properdin-deficient individuals with serogroup A and C polysaccharide clearly increased the concentrations of antibody to meningococci. At least some of the antibodies induced by vaccination supported the bactericidal activity of properdin-deficient serum. Some antibodies to meningococci, probably of the IgM class, promoted alternative pathway-mediated bactericidal reactions in the absence of properdin. By contrast, presensitizing meningococci with IgG enhanced the alternative pathway-mediated reactions, but this was strictly a properdin-dependent effect.
The clinical application of xenotransplantation between distantly related species is currently prevented by the occurrence of hyperacute rejection (HAR). Controversy exists over the importance of natural xenoreactive antibody (NAb)-mediated activation of the classical complement pathway vs. direct activation of the alternative C pathway in this process. In order to evaluate HAR of xenografts (Xgs) in the absence of NAb, this study utilized K strain leghorn chickens that were bursectomized (Bx) on day 17 in ovo (n = 18) to prevent B cell development and production of NAb. Aged-matched untreated siblings served as controls (n = 13). Based on pretransplant antibody levels, the Bx chickens were divided into two groups: totally Bx (Total Bx, n = 9) and partially Bx (Part Bx, n = 9). Chickens then underwent heterotopic cardiac xenotransplantation using PVG rats as donors, where the Xg was connected to the circulation of the chicken recipient utilizing cannulae. For the control group, Xg survival was 28 +/- 3 min (mean +/- SEM), while Part Bx prolonged survival to 80 +/- 15 min. Total Bx extended rat Xg survival to 102 +/- 11 min, with 5 of 9 Xgs functioning well at the time of termination of the study (90-120 min). Three chickens in the Total Bx group with rat cardiac Xgs that were functioning at 120 min were given a 1 ml i.v. injection of heat inactivated control chicken serum. This led to loss of Xg function within 10 min, confirming the important role for NAb in HAR in this species combination. Histologic examination of the Xgs following perfusion revealed significant arterial endothelial injury in the control and Part Bx groups but not in the Total Bx group. Conversely, Xgs from the Total Bx group showed marked venous congestion, which was not seen in the other two groups. This study demonstrates that: (1) Bx effectively eliminates NAb; (2) Xg survival is significantly prolonged in the absence of NAb in this rat-to-chicken xenogeneic combination; (3) the presence of NAb is associated with arterial endothelial injury; and (4) in the absence of NAb, marked venous congestion and injury occurs, which is possibly mediated by alternative C pathway activation or other humoral mechanisms.
The nature of the beta inhibitor in guinea-pig serum and its mechanism of neutralization of influenza virus have been investigated. This inhibitor was shown to be a mannose-binding lectin serologically related to human serum mannose-binding protein. Ca(2+)-dependent binding of the guinea-pig lectin to influenza virus or to mannan could be detected with polyclonal or monoclonal antibodies against human mannose-binding protein in an ELISA. Furthermore, the monoclonal antibody inhibited both the haemagglutination-inhibiting and virus-neutralizing activities of the guinea-pig lectin. The lectin was active against influenza viruses of both type A and type B. In haemagglutination inhibition it acts independently of complement, apparently by sterically hindering access to the receptor-binding site on the viral haemagglutinin through binding of the lectin to carbohydrate side-chains in the vicinity of this site. Neutralization by the lectin, however, was shown to require activation of the classical complement pathway. To our knowledge, the neutralization of influenza virus by a serum lectin plus complement represents a previously unrecognized mechanism of complement-dependent viral inactivation that may be important in first-line host defence against a variety of enveloped viruses.
Concentrations of C3a and C4a anaphylatoxins in suction blister fluids were determined by radioimmunoassay in patients with psoriasis and normal controls. Comparison of anaphylatoxin levels between serum samples and blister contents in the same subjects revealed that the levels of both C3a and C4a anaphylatoxins were significantly higher in the former than the latter even in those raised on normal skin, suggesting that the classic complement pathway is activated during suction procedure. Therefore we cannot regard suction blister fluid to be simply representative of undisturbed interstitial tissue fluid as far as the complement system is concerned. There was no difference in anaphylatoxin levels between those from uninvolved skin of psoriatic patients and those from normal controls. However, significantly high anaphylatoxin levels were noted in fluids of suction blisters raised on lesional skin as compared with those produced on uninvolved skin in psoriatic patients.
Many chronic ITP patients have increased amounts of platelet-associated IgG, C3, C4 and C9, suggesting in vivo complement activation. In this study, we assessed the ability of various antiplatelet antibodies (APA) to activate and deposit complement proteins and to cause platelet lysis in vitro. Platelet sensitization with rabbit APA, anti-P1A1 antibody (one patient), anti-HLA antibody (two patients) and ITP autoantibodies (four patients) resulted in the deposition of C4 and C3 onto platelets in an amount proportional to the quantity of antibody-containing sera used to sensitize the platelets. Although C9 deposition onto platelets could not be quantitatively demonstrated on platelets sensitized with ITP serum, platelet lysis (51Cr release) was noted after incubation with each of three ITP sera and complement. When compared, ITP autoantibodies, anti-HLA and anti-P1A1 antibodies activated complement to a similar degree. We conclude that some autoantibodies in chronic ITP activate the classical complement pathway. The demonstration of in vitro platelet lysis by autoantibodies and complement suggests that in vivo platelet lysis may occur in some chronic ITP patients.
Sera from 59 patients with hypogammaglobulinaemia (hypogamma) were assessed for lymphocytotoxic activity. The activity was compared with that found in sera from patients with systemic lupus erythematosus (SLE) in titre, class of antibody, target cell, and complement requirements. Sera from patients with hypogamma showed cytotoxic activity when compared with sera from normal donors; however, the activity was far less than that in sera from SLE patients, The cytotoxic titre of both hypogamma and SLE sera was greater against T cells than non-T cells. Sera showing activity could be used to sensitize lymphocytes and render them susceptible to subsequent lysis, but IgM was not detectable on the surface of the sensitized cells. Following sucrose density gradient fractionation of the sera the lymphocytotoxic activity was recovered in those fractions that contained IgM. Lymphocytotoxic activity was dependent on classical complement pathway activity. In all the experiments the hypogamma sera showed the same pattern of activity as the SLE sera, but the activity was always far weaker than that found in SLE sera. The evidence does not suggest that lymphocytotoxicity is likely to be more important in the pathogenesis of hypogamma.
Mannan-binding protein is synthesized by the liver and functions in first-line host defence by opsonizing mannose-rich microorganisms due to activation of the classical complement pathway independent of Clq, and by an intrinsic ability to opsonize and mediate phagocytosis. We have investigated whether the increased susceptibility to bacterial infections in patients with cirrhosis could be explained by low plasma concentrations of mannan-binding protein and impaired complement-dependent opsonization. We examined 51 patients with compensated alcoholic cirrhosis, 34 who were decompensated and 16 healthy controls. Irrespective of group, we found a significant correlation (p < 0.05) between plasma mannan-binding protein concentration and deposition of the complement opsonin C4 on mannan from baker's yeast. In contrast to what was expected, this kind of opsonization and plasma levels of mannan-binding protein were significantly increased in the patients with decompensated cirrhosis (p = 0.01 and p = 0.007, respectively). A significant correlation (0 < 0.05) was found between mannan-binding protein and erythrocyte sedimentation rate, fibrinogen and haptoglobin in these patients. Though the correlations were weak (rho = 0.49, rho = 0.48 and rho = 0.40, respectively), the elevated levels of mannan-binding protein in the patients with decompensated cirrhosis may reflect an acute phase reaction. It is concluded that plasma levels of mannan-binding protein are increased in patients with decompensated cirrhosis and that complement-dependent opsonization of mannan does not seem to be compromized in patients with alcoholic cirrhosis.
Lesions in Alzheimer disease are characterized by the assembly of a variety of cells and proteins associated with the immune system. Activated microglia express high levels of MHC glycoproteins and receptors for complement. Small numbers of T-lymphocytes infiltrate tissue. Proteins of the classical complement pathway are closely connected with beta-amyloid deposits. Several materials associated with senile plaques, including beta-amyloid protein itself, bind C1q in vitro and activate the pathway. The membrane attack complex of complement, as well as proteins which defend against that complex, colocalize with dystrophic neurites. These data imply that an autodestructive process is occurring in Alzheimer's disease, and that anti-inflammatory drugs might be an effective form of therapy. Some epidemiological evidence and results of a pilot clinical trial support this hypothesis.
Deficiencies of the early components of the classical complement pathway impair the actions of innate and humoral immunity and may lead to increased susceptibility to infections. We have studied the genetic basis of total C4B deficiency in a Finnish patient with recurrent meningitis, chronic fistulas and abscesses. The maternal chromosome carried a four-gene deletion including the C4B gene, and a conversion from C4B to C4A gene was found on the paternal chromosome resulting in complete deficiency of C4B. In the converted C4A gene, mutation screening did not reveal any amino acid changes or prominent mutations, yet a large number of nucleotide variations were found. Further, the patient was heterozygous for structural deficiency of mannan binding lectin (MBL) associating with medium levels of serum MBL. Our data provides new information on the genetic instability of the C4 gene region, and on the association of homozygous C4B deficiency and variant MBL genotype with increased susceptibility to recurrent and chronic infections. Importantly, plasma therapy induced a prompt clinical cure with long-term effects.
In an attempt to delineate the staphylococcal cell surface components of importance in chemotaxigenesis, we incubated intact Staphylococcus aureus H, crude cell walls, purified cell walls, peptidoglycan, teichoic acid, and cell membranes with human sera. The results reported indicate that both crude cell walls and purified cell walls, as well as peptidoglycan, were potent chemotaxigens. These particles led to the generation in normal human serum of a factor that was chemotactic for human polymorphonuclear leukocytes. Cell wall peptidoglycan and teichoic acid both appeared to play a role in chemotaxigenesis. Kinetic studies employing C2-deficient serum and immunoglobulin-deficient serum revealed that optimal chemotaxigenesis required the presence of an intact classical complement pathway, as well as antibody. Granulocyte aggregometry studies showed that significant levels of C5a were generated in normal serum and that this activated complement component appears to be a major chemotactic factor produced in serum upon interaction with staphylococcal cell wall components.
Although encapsulated Staphylococcus aureus M is not opsonized by normal human serum, as a chemotaxigen this organism behaved similarly to an unencapsulated variant strain. For optimal chemotaxigenesis, an intact classical complement pathway was required, and C5a appeared to be the major chemotaxin.
Killing of Salmonella minnesota and Salmonella typhimurium S and R strains in serum of nonimmune humans and guinea pigs was drastically reduced in the selective absence of C1q, C1r, Ca2+, C4, or C2, the components of the classical complement pathway. Binding of C1 and C1q to the S form and six different core-deficient R mutant strains became stronger the shorter the lipopolysaccharide molecule. C1 and C1q had, under physiological conditions, no affinity to the serum-resistant S forms, whereas these components were bound by the serum-sensitive R forms with high affinity. However, a mixture of the individual complement components C1-C9, which rapidly lysed sensitized erythrocytes, did not kill the serum-sensitive bacteria. Isolated C1 bound to these bacteria cleaved fluid-phase C4 but did not convert C2. C2 turnover could be detected only when serum was used as a source of C1 or C4, indicating that an additional serum component is necessary for the antibody-independent bactericidal effect. Functional tests indicated that this factor is a euglobulin which mediates binding of C4 to the bacteria even in the absence of C1 or after treatment with EDTA. Binding of C4 followed by the generation of C4b sites as acceptors for C2 was a prerequisite for the killing of the bacteria. The factor could not be replaced by immunoglobulin G or immunoglobulin M, nor was it blocked by preincubation with anti-immunoglobulin G or anti-immunoglobulin M.