Inflammatory properties of human C5a and C5a des Arg/ in mast cell-depleted human skin.

C5a and its degradation product, C5a des Arg, elicit immediate cutaneous inflammatory reactions after intradermal injection. Histologically, these reactions are characterized by neutrophil-rich leukocytic infiltrates, leukocytoclasis, edema, and dermal mast cell degranulation. It has not been possible to assess in vivo the relative contributions of resident mast cells and circulating leukocytes to this reaction because the accumulation of leukocytes and degranulation of mast cells occur simultaneously after injection of these anaphylatoxins. To assess the role of mast cells in these inflammatory reactions, we have examined the reactivity of human skin selectively depleted of dermal mast cells by local corticosteroid treatment. Corticosteroid-treated skin became virtually devoid of dermal mast cells within 4-6 wk as assessed by light microscopy, immunofluorescence with fluorescein-conjugated avidin, or electron microscopy. Mast cell-depleted skin demonstrated normal vasopermeability and vasodilatory responsiveness to intradermal injection of histamine, but the reactivity of these sites to the mast cell secretagogue, morphine, was absent. Moreover, no clinical reactions were detectable in mast cell-depleted human skin after intradermal challenge with 50 ng of either C5a or C5a des Arg, despite the fact that biopsies of these sites revealed substantial, neutrophil-rich infiltrates. These infiltrates were qualitatively and quantitatively identical to C5a or C5a des Arg-induced infiltrates in mast cell replete skin. This experimental approach in vivo has allowed the independent analysis of the anaphylactogenic and chemoattractant activities of human C5a and C5a des Arg in human skin, demonstrated the importance of dermal mast cells in these clinical responses, and shown that leukocytes can accumulate at these injection sites directly in response to these mediators.

Endotoxin-induced shock in the rat. A role for C5a.

Administration of endotoxin from gram-negative bacteria to rats results in systemic hypotension, an increased hematocrit, and decreased numbers of circulating leukocytes (polymorphonuclear), monocytes, and platelets. These potentially lethal physiologic changes may be partially attributed to complement activation and generation of anaphylatoxins by the endotoxin (LPS). We demonstrated an elevation in the plasma levels of both C3a and C5a in LPS-treated rats. Injection of 5 micrograms C5ades Arg (rat) into rats produced effects similar to those induced by LPS, including decreased mean arterial pressure (systemic hypotension) and decreased numbers of circulating polymorphonuclear leukocytes, monocytes, and platelets. Unlike the response to LPS, C5a did not increase the hematocrit, indicating little effect on vascular permeability at the doses used. When LPS-treated animals were pretreated with F(ab')2 fragments of rabbit anti-rat C5a, no changes were measured in the circulating cell counts compared with LPS alone; however a significant improvement in the mean arterial pressure and a decrease in hematocrit was observed. We conclude that LPS-induced (septic) shock in the rat may result, in part, from the effects of complement activation and particularly from the generation of C5a. The influence of C5a on the LPS effect in the rat appears to enhance both the hypotensive (mean arterial pressure) and vascular permeability (hematocrit) responses. These results appear to support and confirm earlier observations that anti-human C5a increased survival in a septic-shock monkey model by eliminating circulating C5a and presumably thereby reducing the effects of endotoxin on blood pressure. Our results demonstrate that C5a plays a significant role in the hemodynamic changes associated with endotoxin-induced shock. Neutralization of C5a with specific antibodies may reduce the hypotensive response to endotoxin sufficiently to prevent lethal septic shock both in animals and in man.

Chemotactic factor generation and cell accumulation in acute lung injury induced by endotracheal acid instillation.

We studied the time course of chemotactic factor generation and inflammatory cell accumulation in the rabbit aspiration pneumonia model. Two major potent chemotactic factors, leukotriene B4 (LTB4) and C5a, in bronchoalveolar lavage fluid (BALF) were measured by radioimmunoassay, and cell analysis was also done. The level of LTB4 increased only in the early phase (2-6 h) after endotracheal acid instillation. The level of C5a increased gradually almost in parallel with the total protein level in BALF, and reached a maximum at 24 h. Neutrophil accumulation occurred early and reached a maximum at 24 h. In contrast, the number of alveolar macrophages increased from days 1 to 7. These findings suggest that the increases in LTB4 and C5a are responsible for accumulation of neutrophils and that C5a may be an important chemotactic factor for alveolar macrophage.

Protein synthesis dependent and independent mechanisms of neutrophil emigration. Different mechanisms of inflammation in rabbits induced by interleukin-1, tumor necrosis factor alpha or endotoxin versus leukocyte chemoattractants.

Inflammation constitutes the body's principal mode of defense against infection and other harmful agents. Neutrophil leukocytes are the primary effector cells in this process. The role of protein synthesis in neutrophil emigration into acute inflammatory lesions was examined. Local intradermal injections of actinomycin D, cycloheximide or puromycin could inhibit in a dose- and time-dependent manner neutrophil emigration induced by interleukin-1, tumor necrosis factor alpha or endotoxin, but not by the leukocyte chemoattractants C5a des arg (zymosan-activated plasma), n-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. Maximal inhibition, measured at the time of peak emigration, was greater than 90%. The onset of neutrophil emigration induced by the cytokines or by endotoxin was delayed by 30 to 60 minutes in comparison to the leukocyte chemoattractants. These results demonstrate at least two mechanisms of neutrophil emigration: one with a slower onset and dependence on local RNA transcription and translation and the other rapid in onset and independent of protein synthesis.

The cardiac and renal effects of the complement fragment C5a des Arg are partly mediated by the release of histamine and arachidonic acid metabolites.

This report suggests that the release of various inflammatory mediators such as histamine, LTC4, D4 and E4, and TXA2 measured as the stable metabolite TXB2 are partly responsible for the various cardiac and renal effects of the complement fragment C5a des Arg anaphylatoxin, in addition to its direct vasoconstrictor activity.

Complement activation and anaphylatoxin (C3a and C5a) formation in preeclampsia and by amniotic fluid.

The aim of this study was to determine whether the biologically active complement peptides C3a and C5a are formed in pregnancy and whether amniotic fluid can activate complement. C3a and C5a are formed when complement is activated. They increase smooth-muscle contraction, vascular permeability, and histamine release from mast cells and basophils. Thirty pregnant women were studied, 16 with uncomplicated and 14 with preeclamptic pregnancies. The plasma C3a and C5a concentrations before delivery were significantly higher in the preeclamptic than in the normal group. The concentrations returned to normal within 1 week. Plasma, serum, and amniotic fluid from 12 pregnant women (eight uncomplicated and four preeclamptic pregnancies) were drawn in connection with delivery. Amniotic fluid was incubated in fresh autologous serum at 37C for 15 minutes. A dose-dependent formation of C3a and C5a was registered with increasing amounts of amniotic fluid.

Complement levels in septic primates treated with anti-C5a antibodies.

During gram-negative sepsis it is known that endotoxin activates complement by the alternate pathway. The complement anaphylatoxin C5a, a result of this activation, is thought to play a key role in attracting and activating neutrophils in the lungs, leading to the adult respiratory distress syndrome. Complement levels were measured in primates made septic by Escherichia coli infusions. Anti-human C5a antibodies were administered to study their effect on neutrophil-mediated lung injury. Control (I), septic (II) and septic + anti-C5a antibody (III) groups (n = 4) were studied. The antibody-treated group (III) demonstrated a significant attenuation of septic shock and pulmonary edema as has been previously reported. All complement profiles were corrected for varying hemoglobin concentrations. C3, C4, and C5 levels were measured by radial immunodiffusion and were depleted in both septic groups. Once the levels were depleted from the plasma, they did not recover. The depletion of C4 indicates that classical pathway activation also occurred. C3a, C4a, and C5a levels were measured by radioimmunoassay. Significantly increased peak levels were reached in the septic groups 15 min after initiation of the E. coli infusion. There were no significant differences in early peak C3a and C4a levels between groups II and III. However, the mean peak C5a level in group III (anti-C5a antibodies) was 42% lower than that in group II, and after this early peak, C5a levels were not elevated above control levels in group III. The antibody to human C5a was thus shown to be cross-reactive with primate C5a and was specific since C3a and C4a levels were not decreased in group III.(ABSTRACT TRUNCATED AT 250 WORDS)

1H nuclear magnetic resonance assignments and secondary structure of porcine C5ades Arg.

Sequence-specific assignments of the 1H nuclear magnetic resonance spectrum of porcine C5ades Arg are described. Assignments were facilitated by comparison of spectra obtained in H2O with partially exchanged spectra obtained in 2H2O. The sequence-specific assignments thus obtained were used to characterized the regular secondary structure in the protein, which is helical in the regions 2 to 11, 16 to 27, 35 to 41 and 45 to 64. The structure is very similar to that of human and bovine C5a.

Analysis of the interaction of human C5a and C5a des Arg with human monocytes and neutrophils: flow cytometric and chemotaxis studies.

C5a and C5a des Arg are potent complement-derived mediators that bind receptors on peripheral blood leukocytes and tissue-specific cellular elements to elicit and amplify inflammatory and immunomodulatory reactions. To study the interactions of C5a and C5a des Arg with these cells, fluorescein conjugates of these ligands were prepared by a new technique and their binding to monocytes, neutrophils, platelets, and endothelial cells was studied with flow cytometry. Fluoresceinated C5a produced neutrophil myeloperoxidase release and chemotaxis and also bound rabbit anti-C5a antibody much like native anaphylatoxin; likewise, fluoresceinated C5a des Arg demonstrated retention of biologic and antigenic activities. Both fluorescein-conjugated C5a and C5a des Arg bound to monocytes and neutrophils in a concentration-dependent, saturable, and homogeneous manner, but 10- to 15-fold higher concentrations of C5a des Arg were required to attain saturable binding of these leukocytes. Ligand binding was specifically inhibited by native purified human C5a in a concentration-dependent manner, while it was unaffected by C3a or N-formyl-methionyl-leucyl-phenylalanine-lysine. There was no evidence of a C5a receptor-negative subpopulation of monocytes or neutrophils. Moreover, comparative binding experiments with leukocytes from multiple normal volunteers showed that a greater percentage of monocytes than neutrophils bound C5a at less than saturable concentrations of ligand (P less than 0.05, 0.5 to 5.0 nM). A representative half-maximal binding of fluorescein-conjugated C5a (C5a des Arg) binding to monocytes and neutrophils was 1.2 nM (30 nM) and 2.6 nM (68 nM), respectively. In contrast, fluorescein-conjugated C5a did not specifically bind to human platelets or umbilical vein endothelial cells.

C5 neoepitopes appearing during activation.

After activation of serum complement, a neoantigen was reported to appear on a 54,000-dalton fragment of C5. On the other hand, a monoclonal antibody (MoAb), No. 568, revealed that an epitope on the beta-chain of C5 became masked after the formation of SC5b-9 complex. Murine MoAbs were obtained to human C5a-des-Arg. Some of them are specific only to C5a-des-Arg but not to C5a or C5. Using these MoAbs, a sandwich immunoassay was devised for detection and measurement of C5a-des-Arg. The neoepitopes for these MoAbs were analyzed by their reactivities to synthetic peptide derivatives of human and porcine C5a, and the structural changes of C5a were suggested to occur after the removal of the carboxyl terminal arginine residue.

Identification of two endogenous neutrophil-activating peptides in psoriatic skin and inflammatory cells: C5ades arg and NAP.

Migration of polymorphonuclear leukocytes (PMN) into the upper layers of involved epidermis represents a characteristic feature of psoriasis. By analysis of psoriatic scale material we were able to identify two potent proinflammatory peptides, which are present in the upper epidermis from psoriatic lesions. Both factors (C5ades arg and NAP) show strong chemotactic activity for human neutrophils in vitro as well as in vivo. Whereas C5ades arg is a known mediator activated by either alternative or classical activation of the complement cascade, NAP represents a newly detected peptide with a molecular weight of 8,000 daltons which is produced by a variety of cells participating in the psoriatic tissue reaction.

The effect of L-glutamic acid on airway function and reactivity in the rabbit.

The ingestion of monosodium glutamate in sensitive individuals has been reported to cause severe asthma. We therefore studied the effects of L-glu on airway function and histamine (H) responsiveness in the rabbit. Histamine dose response curves (HDR's) were performed by measuring total lung resistance (RL) after inhalation of saline and increasing concentrations of H (1-30 mg/ml). The concentration of H producing a 20% increase in RL (PC20H) was obtained by interpolation. To assess the effects of L-glu, 8 rabbits were infused with L-glu (0.2 g/kg/hr) or saline in random order (14 days apart) for 4 hours followed by an HDRC. To look at possible late effects, a repeat HDRC was also performed in 6 rabbits 12 hours after completion of the L-glu infusion. In order to see whether rabbits rendered hyperresponsive responded to L-glu, the above protocol was performed in 7 rabbits following the inhalation of 3 micrograms of the activated complement fragment C5a des Arg. The L-glu infusions increased the plasma levels approx. ten-fold (mean +/- SEM 0.119 +/- 0.012 base-line, 1.272 +/- 0.061 mmol/l post infusion). L-glu did not increase the PC20H or baseline RL in either the normal rabbits at 4 or 12 hours or in the C5a des Arg treated rabbits at 4 hours. It is concluded that L-glu does not cause bronchoconstriction or an increase in airway responsiveness to H in the rabbit.

Group B streptococci inhibit the chemotactic activity of the fifth component of complement.

Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts.

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.

Accentuated formation of the terminal C5b-9 complement complex in patient plasma precedes development of the adult respiratory distress syndrome.

Extensive studies have been conducted to determine the pathogenesis of the adult respiratory distress syndrome (ARDS) by investigating the role of complement, a mediator of inflammation. Complement activation products have been detected in blood samples from patients during ARDS. However, the individual complement components that have been assessed only indicated generalized inflammation, and none could unequivocally discriminate the onset of this acute inflammatory lung injury. In this two-year prospective study of 87 septic patients, 22 of whom developed ARDS (25%), we determined complement activation by quantifying the terminal complement complex (TCC), C5b-9. The TCC is a stable complement by-product formed following activation of either the classical or alternative pathways. Our results show that plasma TCC concentrations increased an average of 110% (p = 0.002) two days prior to the onset of ARDS and also transiently increased an average of 45% (p = 0.01) immediately preceding its resolution. Furthermore, plasma TCC concentrations were a more sensitive measure of this acute inflammatory lung injury than levels of C3a desarginine, C4a desarginine, C5a desarginine, and total hemolytic complement activity. We conclude that a temporal association exists between accentuated formation of plasma TCC and the development and also resolution of septic ARDS. Therefore, we suggest that researchers include plasma TCC concentrations in clinical studies when they could use a potential early indicator for ARDS.

Complement activation and the toxicity of stroma-free hemoglobin solutions in primates.

The toxicity of hemoglobin solutions was studied in the context of their ability to activate serum complement (C). Three bovine polymerized hemoglobin solutions (BPHSs) with different degrees of purity were used for experiments in vitro and in vivo. BPHS-1 contained bacterial endotoxins (E) (5 EU/ml) and stromal phospholipids (PLs) (1.2 mg/dl), BPHS-2 contained only PLs (2.0 mg/dl), while BPHS-3 was completely free of both contaminants. C-activation was studied by the direct measurement of C3a, C4a, and C5a des Arg fragments, using commercially available RIA kits. During 1 hour of incubation with fresh monkey plasma, BPHS-1 and -2 activated both pathways of C, while BPHS-3 caused no activation of any factor. In vivo, Hb solutions were used to replace one-third of blood volume in three groups of six Coebus monkeys each, while fresh homologous plasma was used in a control group of four animals. Impure solutions activated the alternative pathway of C and caused significant reactions of the circulating blood (thrombocytopenia, leukopenia, and disseminated intravascular coagulation) associated with multiorgan dysfunction (cardiac arrhythmias, hypoxemia, reduction of renal clearance of endogenous creatinine, and elevation of liver enzyme SGPT). The pure solution neither activated C nor caused any reaction in the circulating blood. However, it caused a moderate degree of direct tissue injury, evidenced by transient reduction of creatinine clearance and elevation of SGPT. These observations suggest that impure and pure Hb solutions carry separate mechanisms of toxicity. Complement, activated by toxic impurities, plays an active role in the toxicity of impure solutions. C-activation in vitro could be used as a screening test of biocompatibility.