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Radioiodinated hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine (HNT) was employed as a photosensitive membrane-restricted probe to establish whether terminal complement proteins insert into membrane bilayers during cytolysis. The system studied consisted of natural membranes carrying intermediate (C5b-8) or fully assembled (C5b-9) cytolytic complexes of human complement prepared by two different methods. In one method, C5b-8 and C5b-9 were assembled on membranes de novo by incubating rabbit erythrocytes with C9-depleted or whole human serum, respectively. The probe was partitioned into lipid bilayers of the resulting MC5b-8 and MC5b-9 membranes by post-addition of HNT. Membranes were irradiated and photolabeled C5b-8 and C5b-9 were extracted, purified, and analyzed on polyacrylamide gels. Those constituents labeled by HNT within each complex were identified by autoradiography. The second method involved pre-addition of HNT to membranes carrying the precursive C5b-7 complex and subsequent conversion to MC5b-8 and MC5b-9 by addition of exogenous C8 and C9. After irradiation, C5b-8 and C5b-9 were again purified and analyzed for the presence of photolabel. Results from both methods were similar and indicated all constituents of each complex are labeled to a measurable extent. However, the C8 alpha subunit was predominantly labeled in C5b-8 and both C8 alpha and C9 were predominantly labeled in C5b-9. Because labeling by HNT is specific for intramembrane structural domains of proteins, these results provide direct evidence that constituents of terminal complement complexes insert into the lipid bilayer of cell membranes during lysis. Further, we conclude that in these complexes, C8 alpha and C9 are the primary contributors of inserted peptide domains.
The intratracheal instillation into rabbits of 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or native platelet-activating factor (PAF) was shown to induce a dose-dependent acute pulmonary inflammation characterized by accumulation of macrophages in the alveolar space, degenerative and necrotic changes of alveolar epithelium, and accumulation of polymorphonuclear leukocytes (PMNs) and platelets in the alveolar capillary lumens with degenerative changes of endothelial cells. Infiltration of alveolar septa by inflammatory cells and, in a later stage, pulmonary fibrosis were also observed. Intrabronchial instillation of lysoglyceryl ether phosphorylcholine (lyso-GEPC) produced no inflammatory changes or only mild ones. In comparison with acute inflammation induced by intratracheal instillation of C5a des Arg, which is mainly characterized by the presence of neutrophils, red blood cells, and fibrin in the alveolar space, AGEPC and native PAF seem to induce a more severe accumulation of macrophages in the alveolar space and septa and of platelet and PMNs in the lumens of alveolar capillaries. These results are compatible with the concept that during inflammatory reaction an intraalveolar release of PAF contributes to the development of pulmonary injury.
The C5a molecule is one of two spasmogenic fragments (i.e. C3a and C5a) released from serum components C3 and C5 during complement activation. These fragments are called anaphylatoxins because their ability to stimulate mast cell histamine release, smooth muscle contraction, and increased vascular permeability may lead to a fatal reaction resembling anaphylactic shock in experimental animals. In addition, the C5a molecule, which is a glycoprotein, is perhaps the most potent of all humoral chemoattractants for polymorphonuclear leukocytes. Most of the structural analyses in this study were performed on the desArg 74 form of human C5a (C5adesArg). C5adesArg represents a natural form of C5a that is recovered from activated serum when no inhibitors are added to block the action of serum carboxypeptidase. The complete primary structure of the human C5a polypeptide portion is reported here. A partial characterization of intact human C5a has been previously reported (Fernandez, H. N., and Hugli, T. E. (1976) J. Immunol. 117, 1688--1694). The polypeptide portion of C5a contains 74 amino acids, accounting for a molecular weight of 8,200 while the carbohydrate portion accounts for approximately 3,000. The carbohydrate portion of C5a exists as a single complex oligosaccharide unit attached to an asparagine at position 64. An unusual feature of the C5a molecule is its large content of half-cystine, which accounts for more than 9% of its total residues. Two repeating Cys sequences occur in the linear structure and 6 of the 7 half-cystines in C5a are located at nearly identical positions to those in the human C3a molecule. In fact, sequence similarities between C3a and C5a indicate their common genetic ancestry. The role of C5a and C5adesArg as chemotactic factors prompted comparisons of their structural features with those of the chemotactically active formyl-Met peptides (Schiffman E., Corcoran, B. A., and Wahl, S. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1059--1062). Removal of the COOH-terminal arginyl residue from C5a reduces chemotactic activity; therefore, the terminal portion of this molecule appears to play an active role in stimulating leukocyte migration. Hence the COOH-terminal sequence of C5a was examined for structural similarities to that of the formyl-Met peptides. Since methionine assumes a special functional importance in the formyl-Met peptides, attention is focused on the single methionyl residue in C5a. This methionyl residue, located near the COOH terminus of the molecule, may play an active role in the functional expression of C5a as a chemotactic factor. Although human and pig C3a show a close structural and functional relationship to C5a they lack the ability to excite leukotaxis, and this difference may correlate with the absence of a methionyl residue near the COOH terminus of C3a.
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Homogeneous anaphylatoxins C3a (human or porcine), C5a (porcine), and the porcine classic anaphylatoxin, a mixture of C5a and C5a des Arg, isolated from complement-activated serum, were shown to induce acute pulmonary injury in the guinea pig following intrabonchial instillation. The gross physiologic response to these factors is characterized by respiratory distress with rapid, shallow breathing. Administration of 8--17 micrograms/kg of porcine classic anaphylatoxin proved lethal in 50% of the animals treated. The acute response (less than 20 minutes after instillation) of pulmonary tissue to insult by the anaphylatoxins is characterized by constriction of the smooth muscle walls in both bronchioles and pulmonary arteries and by focal atelectasis. Aggregates of platelets and leukocytes in pulmonary vessels and in other organs such as the chambers of the heart were commonly observed after intrabronchial administration of the anaphylatoxins. Although C3a was never lethal in guinea pigs even when doses as high as 500 micrograms/kg were administered by the intrabronchial route, this anaphylatoxin did induce the same pattern of acute pulmonary injury as C5a. In vitro experiments employing guinea pig platelets indicated that these cells aggregate in the presence of 10(-10) M porcine C5a but are not affected by C3a (human or porcine) even at levels up to 10(-6) M. Hence, platelet aggregation as observed in vivo may be directly affected by C5a, but in the case of C3a, secondary mediators must be involved. Anaphylatoxin preparations were also shown to induce contraction of guinea pig lung strips in vitro: this effect was not inhibited by antihistamines at concentrations that blocked contraction to exogenous histamine. The in vivo response to anaphylatoxin could be blocked with high doses of the antihistamine chlorpheniramine but not by corresponding doses of diphenhydramine.
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a potent inducer of systemic anaphylactoid reactions in animals. It was found to be similarly potent in contracting smooth muscle of guinea pig ileum and lung and in enhancing vascular permeability when injected subcutaneously into these animals. This factor, therefore, possesses in vitro and in vivo bioactions that resemble those of C3a and C5a anaphylatoxins. However, platelet-activating factor induces a slowly developing, sustained contractile wave in ileum that is not inhibited by an antihistaminic compound, pyrilamine, whereas C3a and C5a stimulate rapid transient contraction that is abrogated by the antihistamine. Furthermore, platelet-activating factor desensitized the ileum to restimulation by itself but not by C3a or C5a; conversely, C3a and C5a desensitized the ileum to themselves but not to platelet-activating factor. Thus, platelet-activating factor possesses a distinctive set of anaphylactic actions. It stimulates a slow wave of muscle contraction and can act independently of histamine release and receptors for the C3a and C5a anaphylatoxins.
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Studies were undertaken to determine the incidence and nature of serum chemotaxis inhibitors in patients with Crohn's disease and ulcerative colitis. Sera from patients with active Crohn's disease were shown to contain chemotactic factor-directed inhibitory activity. The molecular size of the inhibitor estimated by gel filtration is similar to the 7S gamma-globulins and it is precipitable by 40% ammonium sulfate. Less marked inhibitory activity is present in the same fractions of sera from patients with ulcerative colitis and from healthy controls, suggesting that it represents increased activity of one or more normal serum globulins. Leukocyte-directed serum inhibitors have also been demonstrated in patients with Crohn's disease and ulcerative colitis, particularly in patients with active disease. In Crohn's disease the presence of serum chemotactic factor inhibitors may explain the diminished chemotaxis of neutrophils into skin window chambers reported previously. The inhibitors are nonspecific and are probably secondary to the disease state but may have some bearing on the nature of the mucosal inflammation.
The C3a and C5a polypeptides release, under conditions of complement activation, causes a change in PMN shape and adhesiveness with formation of leukoemboli in microvascular districts. Those modified granulocytes produce oxygen radicals with toxic activity. This mechanism seems to play a role in different pathological situations like pulmonary distress in hemodialysis, leukapheresis, cardiopulmonary by-pass and extent of necrosis in acute myocardial infarction. The same mechanism has been largely investigated in Adult Respiratory Distress Syndrome (A.R.D.S.). In this condition the leukoembolization toxicity seems to be the most important pathogenetic factor of the alveolo-capillary membrane damage which is the base of the disease.
We have previously shown that complement (C) activated plasma causes granulocyte (PMN) aggregation in vitro and that C5a is responsible. The C-induced aggregation of PMNs treated with cytochalasin-B (CB) is markedly enhanced and irreversible, and the magnitude of the response is proportional to the log (concentration of activated plasma), allowing use of this technique to detect C5a and hence C-activation. To compare the sensitivity of granulocyte aggregometry to that of more standard methods of detecting C-activation, we produced graded C-activation in vitro by treating fresh serum with varying amounts of zymosan. Aggregometry was the most sensitive index of C-activation, detecting C-activation, produced by 0.02 mg zymosan/ml of serum--1/10 that required to produce C-activation detectable by C3 immunoelectrophoresis (the next most sensitive technique). Granulocyte aggregometry may also be used to detect in vivo C-activation. We have found aggregating activity in plasmas from patients with systemic lupus erythematosus, immune vasculitis, transfusion reactions, and other conditions associated with in vivo C-activation, but not in the plasmas of normal subjects.
The present study was conducted to gain insight into the process of assembly of the C5b-7 complex on the phospholipid bilayer. The C5b-6 complex, C7, and 2 different forms of the C5b-7 complex (the C5b-7 complex either bound to artificial phospholipid bilayers or in fluid phase) were digested with trypsin, and the resulting products were analyzed by SDS-polyacrylamide gel electrophoresis. The alpha'-chains of C5b and C6 in the C5b-6 complex and C7 were susceptible to trypsin. The formation of the C5b-7 complex from the C5b-6 complex and C7 was accompanied by alteration in susceptibility to trypsin to C7: the C7 subunit of the fluid phase complex as well as of the complex bound to phospholipid vesicles is not susceptible to trypsin. On the other hand, the alpha'-chains of C5b and C6 in the C5b-7 complex bound to phospholipid vesicles as well as in fluid phase remained accessible and susceptible to trypsin, and cleaved as in the C5b-6 complex. Upon ultracentrifugation in a sucrose density gradient, the proteolyzed C5b-7 complex in fluid phase dissociated into subunits. However, none of the subunits in the proteolyzed C5b-7 complex bound to phospholipid vesicles dissociated from vesicles upon sucrose density gradient ultracentrifugation. Thus, the molecule of the C5b-7 complex became stable and resistant to proteolytic dissociation upon binding to the phospholipid bilayer. It is proposed that each subunit of the C5b-7 complex participates in the stable interaction of the complex with the phospholipid bilayer. In addition, the possible significance of the alteration in susceptibility to trypsin of C7 associated with the formation of the C5b-7 complex is discussed.
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The C5b-9 monomer having the sedimentation coefficient of 23S was extracted from the rabbit erythrocyte membranes that had been treated with a limiting amount of C9-deficient human serum and of 125I-C9. Upon proteolysis by trypsin and chymotrypsin, the C9 subunits of this complex were cleaved by these enzymes at multiple sites, yielding fragments with m.w. ranging fro 40,000 to 19,000. The uncomplexed C9 was also cleaved by both enzymes at multiple sites. By contrast, the C9 subunits of the C5b-9 dimer were found to be totally insusceptible to chymotrypsin under the conditions studied (37 degrees C; 24 hr) and only partially susceptible to trypsin (33% of the C9 subunits were cleaved by trypsin into 2 fragments during incubation at 37 degrees C for up to 24 hr). Therefore, these results indicate that, although the binding of C9 molecules to the C5b-8 complex (C5b-9 monomer formation) does not significantly affect the susceptibility to proteases of the C9 molecules, C5b-9 dimer formation markedly limits the accessibility of proteases to the C9 subunit molecules. A implication of this finding to a role for C9 in C5b-9 dimerization is discussed.