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Model structure for the inflammatory protein C5a.

The complement cleavage product C5a is a potent stimulant of inflammatory processes; thus, inhibition of C5a activity is of therapeutic interest. The three-dimensional structure of the major portion of C5a was modeled from the homologous C3a crystal structure by comparative modeling techniques. The model shows that core residues of C5a are completely conserved, while external residues differ from C3a. Even though the amino-terminal 12 residues of C3a are disordered in the crystal, this sequence in C5a may form an amphipathic helix. The distribution of species sequence differences in the complete C5a structure suggests a possible receptor binding site.

Amino Acid Sequence↗

Rifampin affects polymorphonuclear leukocyte interactions with bacterial and synthetic chemotaxins but not interactions with serum-derived chemotaxins.

Three independent experimental approaches support the hypothesis that rifampin competes for receptors on polymorphonuclear leukocytes (PMLs) with small peptide chemoattractants, e.g., N-formylmethionylleucylphenylalanine (FMLP), but not with serum-derived chemoattractants (C5a). First, rifampin inhibited chemotaxis induced with FMLP but reversed the immobilization of PMLs that occurred at high FMLP concentrations. Second, rifampin competed with radiolabeled FMLP for binding sites on PMLs and displaced already-bound radiolabeled FMLP. Third, rifampin blocked and reversed the bipolar shape changes induced in PMLs by FMLP. These effects occurred at concentrations attained during rifampin therapy and were not due to rifampin toxicity. In contrast, no effect of rifampin was observed on serum-derived chemoattractants (C5a) in any of the three systems. The evidence suggests, therefore, that rifampin is a ligand for FMLP-type receptors on PMLs.

Binding, Competitive↗

Complement levels in pneumococcal pneumonia.

Levels of complement proteins and functional activity of the alternate complement pathway were assessed in 39 patients with pneumococcal pneumonia. Mean levels of C3 and properdin and the functional activity of the alternate pathway in acute sera were significantly (P less than 0.05) below normal, whereas levels of components of the early classical pathway were normal. Although levels of factor B were in the normal range, they correlated significantly with C3 levels; there was no significant relation between C3 levels and C4 or C1q levels. The 19 patients iwth pneumococcal pneumonia and bacteremia had significantly lower mean values of properdin and factor B than the 20 patients without bacteremia, suggesting a more severe depression of the alternate complement pathway with bacteremia. During convalescence, complement levels were normal or elevated in most of the patients, but mean levels of properdin remained significantly below normal in bacteremic patients. Functional activity of the alternate pathway also remained below normal. These results indicate that there is a selective depression of the alternate pathway in patients with pneumococcal pneumonia, and they are consistent with the concept that the alternate pathway has an important role in host defenses in pneumococcal infection.

Adult↗

Chemotaxigenesis by cell surface components of Staphylococcus aureus.

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.

Cell Membrane↗

Chemotaxigenesis by encapsulated Staphylococcus aureus M.

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.

Chemotactic Factors↗

Specificity and reversibility of chemotactic deactivation of human monocytes.

The chemotactic deactivation of human monocytes was studied to provide insight into the mechanism of chemotaxis. Deactivation was dependent on the dose of chemoattractant and time of incubation. A concentration in the cell suspension of 10(-8) M N-formylmethionylleucyl phenylalanine (FMLP) for 45 min at 37 degrees C led to 60% suppression of the subsequent specific chemotactic response. Higher concentrations of FMLP led to almost 100% specific suppression. Deactivation was specific under all conditions used. The response to a nonrelated chemoattractant, human serum-derived C5a, was unaffected by incubation in FMLP. Deactivation was also transient. If cells were deactivated at 37 degrees C with FMLP, they recovered within 6 h at 37 degrees C from this deactivation. Both phenomena, deactivation and recovery from deactivation, were temperature dependent. Monocytes could not be deactivated at 0 degrees C, and they did not recover from deactivation when kept at 0 degrees C. Thus, specific deactivation appears to require cellular metabolism, involving loss of receptors or blocking of a step between receptor occupancy and response.

Anaphylatoxins↗

Chemotaxis of purified human monocytes in vitro: lack of accessory cell requirement.

This study was undertaken to determine whether cell cooperation, either among monocytes or between monocytes and lymphocytes, is a prerequisite for monocyte chemotactic responsiveness. We compared Ficoll-Hypaque-separated mononuclear cells and a preparation of 99% pure monocytes obtained by chemotaxis in a newly designed separation chamber. Monocytes of both preparations migrated to chemoattractants without a lag phase, and no further increase in migrated cells was observed after 70 min. The cell dose-response was linear for both preparations over a wide range of cell concentrations in the cell input well of the chemotaxis chamber, suggesting that no monocyte-monocyte interaction was required. Since only 20 to 60% of the monocytes purified by chemotaxis migrated a second time, the possibility of a requirement for an accessory cell was tested. The addition to purified monocytes of several different mononuclear cell preparations comprising lymphocytes or nonmigrating monocytes had no effect on monocyte migration. These experiments show that normal human blood monocytes in vitro do not require stimuli from other cells to respond to chemoattractants. Their behavior is profoundly different from that of mouse peritoneal macrophages, which exhibit a time lag in vitro before migration toward an attractant and become more responsive with either increasing cell concentration or addition of purified lymphocytes.

Cell Communication↗

Dissemination of gonococcal infection is associated with delayed stimulation of complement-dependent neutrophil chemotaxis in vitro.

Gonococci isolated from patients with uncomplicated gonorrhea or disseminated infection were examined for their ability to stimulate neutrophil chemotaxis in vitro. A neutrophil chemotactic response was not observed when as many as 10(9) colony-forming units of gonococci were incubated in buffer alone. However, a striking response was observed when 4 x 10(7) colony-forming units were incubated in 10% pooled normal human serum. Activation of complement was required for chemotaxis as demonstrated by complement consumption and failure of chemotactic activity generation in serum treated with heat or EDTA. Chromatography of activated serum demonstrated a single peak of chemotactic activity with an apparent molecular weight of 15,000 and was shown to be due to C5a. Examination of the kinetics of chemotactic factor generation demonstrated that local isolates stimulated a rapid response (about 60% maximal in 5 min), whereas the response to disseminated isolates was delayed (50% maximal in 20 to 30 min). Chemotactic activity generated by both types of isolates was suppressed at early time periods in agammaglobulinemic serum, indicating that immunoglobulins contribute to the generation of activity. Both pathways of complement activation were utilized by the two types of gonococci, but there was preferential dependence on the alternative pathway for disseminated strains and on the classical pathway for local isolates. We suggest that delayed stimulation of complement-dependent neutrophil migration may account in part for the infrequency of genital symptoms and may contribute to the mechanism of dissemination in patients with systemic gonococcal infection.

Chemotaxis, Leukocyte↗

Chemotactic and phagocytic responses of human alveolar macrophages to activated complement components.

Human alveolar macrophages (AMs) migrated toward and aggregated to C5a but not toward C5a-deficient serum. Human AMs from cigarette smokers migrated significantly farther than did AMs from nonsmokers. Human AMs phagocytized Escherichia coli opsonized with activated C3. Immunoglobulin G was not required for phagocytosis. Human AMs demonstrate chemotaxis and aggregation to C5a. Further, human AMs can phagocytize bacteria coated with activated C3.

Chemotaxis↗

Complement activation and stimulation of chemotaxis by Chlamydia trachomatis.

The stimulus for the migration of polymorphonuclear leukocytes (PMNs) in acute chlamydial infection was studied in vitro by examining the chemotaxigenic effect of L2 and DE Chlamydia trachomatis elementary bodies (EB) upon the plasma of three healthy donors. In each individual experiment, chemotactic response was assessed with PMNs and plasma from the same respective donor, and no specific antibodies against C. trachomatis were detected in the plasma of any donor. Chemotaxis was observed in an agarose plate assay and was quantitated as the chemotactic differential, or CD (directed migration of PMNs minus random movement of PMNs). For each donor, the mean CD was significantly greater (P less than 0.005) when plasma preincubated for 2 h with L2 EB was used as the chemoattractant than when (i) plasma alone, (ii) plasma preheated to 56 degrees C for 30 min before incubation with L2 EB, or (iii) L2 EB in phosphate-buffered saline (PBS) was used as the potential chemoattractant. Similarly, in the one donor in whom DE EB were studied, the mean CD was also significantly greater (P less than 0.005) for plasma preincubated with DE EB as compared with (i) plasma alone or (ii) DE EB in PBS. Complement activation by C. trachomatis EB was assessed by radioimmunoassay for C5a des-arginine in all chemoattractant preparations used in the chemotaxis assay. Mean C5a des-arginine levels were high in plasma samples preincubated with L2 EB (171.00 +/- 10.64, 107.00 +/- 4.76, and 89.70 +/- 1.74 ng per ml) or DE EB (37.40 +/- 15.76 ng per ml) but were undetectable (less than 4.0 ng per ml) in (i) plasma alone, (ii) preheated plasma incubated with L2 EB, and (iii) PBS containing L2 EB. Thus, L2 EB and DE EB of C. trachomatis exert a chemotaxigenic effect upon normal antibody-negative plasma, and this effect is at least in part a result of complement activation and generation of the potent chemotaxin C5a.

Chemotaxis, Leukocyte↗

Localization of the streptococcal C5a peptidase to the surface of group A streptococci.

Immunofluorescent staining was used to determine that the streptococcal C5a peptidase (SCP) exists as a cell surface antigen on group A streptococci. The ability of hyperimmune serum to neutralize cell-associated SCP activity provided further evidence for the location of SCP. Quantification of SCP during growth in vitro by indirect enzyme-linked immunosorbent assay showed that approximately 90% of the measurable antigen is cell bound.

Antigens, Surface↗

Characterization of murine monoclonal antibodies that recognize neutralizing epitopes on human C5a.

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complement fragment C5a. These MAbs were characterized for their ability to immunoprecipitate 125I-labeled C5a, bind C5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes. Four of these MAbs had affinity constants for C5a in the 1 X 10(9) to 3 X 10(9) M-1 range. These MAbs blocked C5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively administered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeutic use in states of complement activation.

Antibodies, Monoclonal↗

Mouse genetic locus Lps influences susceptibility to Neisseria meningitidis infection.

We surveyed a number of inbred mouse strains for susceptibility to meningococcemia. Mice of all strains became bacteremic after intraperitoneal injection of a serogroup C, serotype 2a human disease isolate, but the strains differed in levels of bacteremia, indicating influences of the host genome on susceptibility. There was no significant correlation between level of bacteremia and differences at major histocompatibility or immunoglobulin loci; the Salmonella susceptibility locus, Ity; the complement C5 locus, Hc; the antibody response locus, xid; or the transferrin locus, Trf. However, the Lps locus, which influences a range of host cellular responses to endotoxin and affects susceptibility to Salmonella typhimurium, did influence susceptibility to meningococcemia. There were significant differences in levels of bacteremia between C3H/HeJ (Lpsd) mice and each of the other strains (all Lpsn). We confirmed the association of the Lpsd genotype with susceptibility by using coisogenic strains from two widely separated mouse lineages: C3H and B10. Lpsd mice experienced a 1,000-fold proliferation of bacteria and were bacteremic for days before clearing the infection. In contrast, Lpsn mice cleared the bacteremia in less than 1 day. There was no difference in meningococcal growth in vitro in serum from C3H/HeJ and coisogenic C3H/HeN (Lpsn) mice, suggesting that the Lps-related difference in susceptibility may involve a cellular response.

Animals↗

Aeromonas salmonicida resistance to complement-mediated killing.

The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using different strains and their isogenic mutants that had been previously characterized for their surface components. We found that the classical complement pathway is involved in serum killing of susceptible A. salmonicida strains, while the alternative complement pathway seems not to be involved. All of the A. salmonicida strains are able to activate complement, but the smooth strains (with or without the A-layer) are resistant to complement-mediated killing. The reasons for this resistance are that C3b may be bound far from the cell membrane and that it is rapidly degraded; therefore, the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants are serum sensitive because they bind more C3b than the smooth strains, and if C3b is not completely degraded, then the lytic complex (C5b-9) is formed.

Aeromonas↗

Subnormal sensitivity of neutrophils to complement split product C5a in rheumatoid arthritis: relation to complement catabolism and disease extent.

The capacity of circulating neutrophils for activation by complement was studied in outpatients with classical or definite rheumatoid arthritis during treatment with dextropropoxyphene only. Analysis of dose-response in the Boyden chamber assay of chemotaxis showed that sensitivity to the potent, complement derived anaphylatoxin, C5a, was markedly decreased, especially in those patients with few joints involved. In contrast, peak response to C5a was within the normal range. Increased complement 3c split products in plasma of the patients suggested involvement of complement cascade reactions. Subnormal sensitivity of neutrophils to phlogistic mediators released by complement may tend to limit their recruitment and potentially tissue destroying secretion locally in rheumatoid arthritis.

Adult↗

Complement activation and HLA-B27.

The efficiency of complement activation was studied in sera from HLA-B27 positive and negative subjects (27 with previous yersinia arthritis and 35 controls). Activation of complement with zymosan induced higher mean levels of the anaphylatoxin C3a in HLA-B27 positive sera (mean (SD) 7.40 (1.66) mg/l) than in HLA-B27 negative sera (6.41 (1.79) mg/l). Similarly, higher levels of C3d,g, another C3 breakdown fragment, were obtained in HLA-B27 positive sera after Escherichia coli 0111:B4 lipopolysaccharide treatment (17.6 (3.7)% v 15.0 (3.8)%). The differences occurred irrespective of previous arthritis, complement C4 or Bf phenotype, or variation in background complement levels. The findings suggest that an increased responsiveness to complement activators may contribute to the pathogenesis of HLA-B27 associated inflammatory diseases.

Adult↗

Defective release of C5a related chemo-attractant activity from complement in Crohn's disease.

Complement was studied in 20 untreated cases of Crohn's disease and in 20 healthy volunteers by an in vitro activation of the cascade reaction. Total haemolytic complement was normal in all patients. In contrast, activation of the alternative pathway lead to a decreased release of C5a related chemo-attractant activity together with a subnormal utilisation of the main complement component C3. This abnormality of complement function was not related to the activity of the disease, site of involvement or to disease duration. The results suggest that an inadequate stimulation of important neutrophil functions may result when bacterial lipopolysaccharides and other macromolecules activating the alternative pathway penetrate the gut mucosa. A delayed clearance from the tissue of such foreign material could be a further pathogenic factor in Crohn's disease leading to granulomatous inflammation by a foreign body reaction.

Chemotaxis, Leukocyte↗

Subnormal activation of phagocytes by complement in chronic inflammatory bowel disease? Neutrophil chemotaxis to complement split product C5a.

The capacity of circulating phagocytes for activation by complement was investigated in consecutive, untreated cases of chronic inflammatory bowel disease. The major complement derived chemotactic factor, C5a, served as chemoattractant in dose response studies of neutrophil chemotaxis. A similar, significantly decreased sensitivity and peak response was revealed in patients with Crohn's disease and ulcerative colitis. This subnormal function of neutrophils could be shown even in cases of complete clinical remission. Chemotactic response to casein and spontaneous motility was within the normal range showing an unaffected basic cell function of neutrophils in the patients. The study shows a dysfunction of phagocytic cells, related to potentially important phlogistic mediators, in chronic inflammatory bowel disease.

Adult↗