anti-C5a Antibody Research Articles

Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and cardioplegia-induced coronary endothelial dysfunction

Objective: Because C5a induces tissue injury by activating polymorphonuclear leukocytes, the hypothesis was that inhibition of C5a activity would reduce cardioplegia-related injury. Methods: Pigs were placed on cardiopulmonary bypass. The hearts were arrested for 1 hour with hyperkalemic cardioplegia. Pigs were then separated from bypass, and the hearts were reperfused for 2 hours. Anti-porcine C5a monoclonal antibody (1.6 mg/kg, intravenously; n = 6) was administered 20 minutes before the onset of cardiopulmonary bypass. Six pigs received saline solution vehicle. Reactivity of coronary arterioles was studied in vitro with videomicroscopy. Microvessels from uninstrumented pigs served as controls for vascular studies. Results: Endothelium-dependent relaxation to adenosine diphosphate (percent relaxation of precontraction) was reduced after cardioplegic reperfusion (63% ± 14% vs 77% ± 10% in control at 10 μmol/L; P = .01). This impairment in endothelium-dependent relaxation was improved with anti-porcine C5a monoclonal antibody (80% ± 22%; P = .01 vs saline solution), as was the impaired endothelium-dependent relaxation to clonidine (64% ± 12% control; 26% ± 17% saline solution; 55% ± 24% anti-porcine C5a monoclonal antibody at 10 μmol/L; P = .01 saline solution vs control or anti-porcine C5a monoclonal antibody). Myeloperoxidase activity was significantly decreased (0.2 ± 0.2 units/g protein; P = .04) in the anti-porcine C5a monoclonal antibody group compared with 5.2 ± 2.7 in the saline solution group. CH 50 2 hours after bypass was not statistically different (0.57 ± 0.41 unit and 0.65 ± 0.41 unit, respectively) between the anti-porcine C5a monoclonal antibody and saline solution groups. Despite less myocardial polymorphonuclear leukocyte infiltration after C5a inhibition, maximum rate of rise of left ventricular pressure, percent segmental shortening, and blood flow through the left anterior descending coronary artery were similar in the anti-porcine C5a monoclonal antibody and saline solution groups. Conclusions: Inhibition of C5a limits neutrophil-mediated impairment of endothelium-dependent relaxation after cardiopulmonary bypass and cardioplegic reperfusion, but it has no effect on short-term myocardial functional preservation. (J Thorac Cardiovasc Surg 1998;116:1060-8)

Complement activation by the hydroxyl radical during intestinal reperfusion.

This study examines the hypothesis that hydroxyl radical (OH.) generation during intestinal reperfusion activates the complement system forming the potent chemotaxin C5a. Anesthetized Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IIR). Complement (C) activation was assessed by measuring total plasma C activity and C5a-related chemotaxis and leukoaggregation. Dimethylthiourea and the iron chelator deferoxamine were utilized to assess the role of the OH. in the activation of C in this model. Sham-operated animals served as controls. Total plasma C activity of animals sustaining IIR was 64% of controls (p < .05). Plasma of animals sustaining IIR induced greater chemotaxis and leukoaggregation than plasma from sham-operated groups (p < .05). Treatment of IIR plasma with anti-C5a antibody ameliorated the enhanced leukoaggregation characteristic of IIR plasma. Pretreatment with dimethylthiorea and deferoxamine prevented reperfusion-induced activation of complement and inhibited the chemotactic activity of plasma from IIR animals. These data are consistent with the hypothesis that IIR activates complement and that the OH. generated during reperfusion may be one mechanism by which C is activated in this injury model.

C3a activates reactive oxygen radical species production and intracellular calcium transients in human eosinophils.

Whereas C5a is a well-established potent activator of eosinophils, the functional role of C3a in the activation of eosinophils is, so far, poorly understood. Here, the activation of human eosinophils stimulated with C3a was analyzed and compared to C5a activation. Flow-cytometrical measurements revealed that stimulation of eosinophils by C3a resulted in a transient elevation of the intracellular calcium concentration ([Ca2+]i) in a dose-dependent manner. In addition, the production of reactive oxygen radical species (ROS) of eosinophils after C3a and C5a stimulation was measured by lucigenin-dependent chemiluminescence and quantified by superoxide dismutase-inhibitable reduction of ferricytochrome C. Half maximal and maximal ROS production in response to C3a was observed at 50 ng/ml and 1000 ng/ml, respectively, whereas C3a-desArg was inactive. To ensure that C3a stimulation was not caused by contamination with C5a, monoclonal antibodies were used to demonstrate the specificity of C3a. The effect of C3a was completely abolished in the presence of monovalent antigen-binding fragments of a functionally blocking anti-C3a monoclonal antibody. In addition, blockade of the C5a receptor by the monoclonal anti-C5a receptor antibody S5/1 totally inhibited the C5a-evoked ROS production, whereas the C3a response in the presence of S5/1 was unaffected. Finally, desensitization experiments revealed a homologous desensitization of C3a after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. Furthermore, the C3a- and C5a-induced production of ROS of eosinophils was totally inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi-proteins). In summary, these results demonstrate that C3a is a potent activator for eosinophils initiating transient [Ca2+]i changes and production of reactive oxygen species. C3a therefore may play a part in the pathophysiology of diseases with eosinophil and complement activation.

Inflammatory mechanisms in the passive cutaneous anaphylactic reaction in the rabbit: evidence that novel mediators are involved.

1. We have examined the mechanisms of local oedema formation in the passive cutaneous anaphylactic (PCA) reaction in the rabbit. 2. IgE-containing antiserum was injected i.d. and allowed to sensitize skin sites for periods up to 240 h. Antigen (bovine gamma globulin) was injected i.d. or i.v. and local oedema formation assessed by the accumulation of i.v. injected 125I-labelled rabbit serum albumin. Potential inhibitors were mixed with antigen prior to i.d. injection or were administered i.v. 3. Maximum oedema formation was observed when a sensitization period of 48-72 h was used. Oedema formation in the PCA reaction was of short duration with a t 1/2 of approximately 15 min. No evidence of late oedema formation (up to 6 h) was found. 4. Local oedema formation in the PCA was reduced by indomethacin suggesting that vasodilator, oedema-potentiating prostaglandins were released. However, it was likely that other vasodilators were also generated. 5. Antihistamines were poor inhibitors of oedema formation as were PAF antagonists, a 5-lipoxygenase inhibitor, a kallikrein inhibitor, a bradykinin antagonist and anti-C5a antibody. 6. Local oedema formation in the PCA was partially reduced by neutrophil depletion and colchicine suggesting that neutrophil-dependent mediators were involved. 7. Exudate fluid from anaphylactic reactions in the rabbit peritoneal cavity contained permeability-increasing activity when injected into rabbit skin. This activity is now being characterized. 8. A vasodilator prostaglandin appears to be released in the rabbit PCA reaction but none of the established permeability-increasing mediators appears to be involved. Thus, there may be novel inflammatory mediators generated in this reaction which may have relevance for human allergic skin diseases.

Binding of Fluorescein-Labeled Anaphylatoxin C5a to Human Peripheral Blood, Spleen, and Bone Marrow Leukocytes

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a-F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12-myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN-gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low-density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a- F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12- myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN- gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low- density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

The sequential generation of neutrophil chemoattractant proteins in acute inflammation in the rabbit in vivo. Relationship between C5a and proteins with the characteristics of IL-8/neutrophil-activating protein 1.

An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.

Partial characterization of a C5a-inhibitor in peritoneal fluid.

We have recently described a 40-kDa protein in peritoneal fluid that neutralized the chemotactic activity of the C fraction C5a. It was deficient in peritoneal fluids of patients suffering from familial Mediterranean fever. Further characterization of the inhibitor with the use of 125I-rC5a binding to dibutyryl cAMP-induced U937 cells revealed dependence on the peritoneal fluid concentration, on the time of incubation and on temperature and pH. Fractionation of 125I-C5a on Sephadex G-50 column, before and after incubation with peritoneal fluid, revealed similar fractionation patterns despite loss of biologic activity of the treated C5a (but not its binding to polyclonal anti-C5a antibody). Analysis of rC5a by SDS-PAGE before and after treatment with partially purified C5a inhibitor, revealed slight modification of the inhibitor-treated C5a. Using various protease inhibitors (i.e., PMSF) suggested that the C5a inhibitor is a serine protease. It neutralized C5a by means of limited proteolysis which did not change C5a immunologic properties and changed only slightly its m.w. but abolished its receptor binding and chemotactic functions. It is suggested that the C5a inhibitor plays a role in the regulation of inflammation in serosal tissues and that its deficiency in familial Mediterranean fever may explain the attacks of sterile inflammation characteristic of this disease.

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. Additionally, the antibody did not prevent activation of the parent C5 molecule since C5 was depleted in group III.

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.

Chemotactic activity for polymorphonuclear and mononuclear leukocytes in rheumatoid synovial fluids.

In order to why polymorphonuclear leukocytes (PMNs) are predominant and mononuclear leukocytes (MNLs) are few in rheumatoid synovial fluids, chemotactic factor(s) for PMNs and MNLs were studied in the synovial fluids of rheumatoid arthritis (RA-SF) and osteoarthritis (OA-SF) using both Boyden's and agarose methods. The RA-SF showed strong chemotactic activity for human peripheral blood PMNs compared with non-rheumatoid OA-SF. The chemotactic activity for PMNs was well correlated with the number of PMNs in RA-SF, suggesting that it was a natural mediator for PMN emigration into rheumatoid joint cavity. The major chemotactic factor for PMN in RA-SF was of apparent molecular weight of 14,000 and its activity was suppressed to less than 10 percent by anti-C5a antibody, but it failed to show any anaphylatoxin activity which was an attribute of C5a. It was, therefore, suggested to be C5a-like molecule but not C5a itself. The possibility that the factor may be a C5a des-Arg was discussed. On the contrary, the chemotactic activity for MNLs was not found neither in RA-SF nor OA-SF. These findings may explain the fact that PMNs are predominant in rheumatoid synovial fluids.

Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates.

In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS). First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro. We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis). Three groups of primates, control, septic, and anti-C5a antibody treated septic, were studied (n = 4 in each group). A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe sepsis and ARDS. Primates were killed 4 h after completion of the E. coli infusion. Septic animals not treated with anti-C5a antibody had 75% mortality (3/4), decreased oxygenation, severe pulmonary edema, and profound hypotension. Septic primates treated with anti-C5a antibodies did not die and did not develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05). They also had a marked recovery in their mean arterial blood pressure (P less than 0.05). This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in nonhuman primates.

Analysis of the binding of fluorescent C5a and C3a to human peripheral blood leukocytes.

Fluorescein-labeled human C5a and C3a were prepared and utilized to analyze the binding of C5a and C3a to human neutrophils and mononuclear cells. The fluorescein derivatives of C5a (Fl-C5a) and C3a (Fl-C3a) contained approximately one fluorescein molecule per molecule of protein. Fl-C5a retained biologic activity as determined by neutrophil O2- production, enzyme release, receptor binding, and reaction with rabbit anti-C5a antibody. Fl-C3a was biologically active as measured by contraction of guinea pig ileal strips, and maintained 87% of its antigenic character when reacted with rabbit anti-human C3a. The binding of Fl-C5a and Fl-C3a to human neutrophils and mononuclear cells was assessed with the use of flow cytometry. Fl-C5a bound to greater than 90% of neutrophils, with an average ED50 ranging from 2.8 to 6.8 nM, depending on the method of analysis. Fl-C5a binding to neutrophils was specific and was not inhibited by the presence of formyl-methionyl-leucyl-phenylalanine (f-MLP), C3a, or casein. Fl-C5a binding was totally blocked by an excess of C5a. C5a des arg partially inhibited the binding of Fl-C5a to neutrophils, but was 1000-fold less effective than C5a. Similar experiments with mononuclear cells showed that Fl-C5a was bound by monocytes but not by lymphocytes. Fl-C5a binding to monocytes was blocked totally by C5a but not by C3a or f-MLP. Comparative binding studies with neutrophils, monocytes, and lymphocytes showed that Fl-C5a was bound by an average of 93% +/- 4 of neutrophils, 68% +/- 9 of monocytes, and 6% +/- 3 of lymphocytes. Fl-C3a did not show significant binding to neutrophils, monocytes, or lymphocytes. These studies demonstrate that fluorescein derivatives of C5a and C3a can be prepared with retention of biologic activity, and provide a means to evaluate the binding of C5a to individual cells.