Anaphylatoxins and infectious and non-infectious inflammatory diseases.

In recent years a plethora of data has accumulated directing toward an important role of polypeptides C3a and C5a and its degradation product C5adesArg, summarized as anaphylatoxins (ATs), in microbial host defense and immune regulation. The ATs exert their various biologic functions by interacting with specific C3a- and C5a-receptors present on cells of myeloid origin, epithelial cells, smooth muscle cells as well as on activated B- and T-cells. Activation of AT receptors mediates signal transduction pathways triggering a variety of proinflammatory events. However, by interacting with the cytokine- and chemokine network C3a and C5a exhibit also anti-inflammatory properties. In this review the focus is on the pathogenetic role of the ATs in sepsis, immune complex disease, delayed type hypersensitivity and asthma. Discussed are data from animal models in which the ATs are blocked by specific C3a or C5a inhibitors or from mice with genetic deletions of the specific receptors of either C3a or C5a/C5adesArg.

Phagocytosis and killing of Staphylococcus aureus by bovine neutrophils after priming by tumor necrosis factor-alpha and the des-arginine derivative of C5a.

OBJECTIVES: To evaluate effects of proinflammatory mediators on phagocytosis and killing of Staphylococcus aureus, the oxidative burst (OB), and expression of receptors for opsonins by bovine neutrophils. SAMPLE POPULATION: Neutrophils from 10 cattle. PROCEDURE: Neutrophils were primed with recombinant bovine tumor necrosis factor-alpha (TNF-alpha) or the des-arginine derivative of bovine C5a (C5a(desArg)) and mixed with S aureus. Phagocytosis and OB were measured by use of flow cytometry. Rate of phagocytosis and intracellular killing were evaluated. Expression of receptors for immunoglobulins and the C3bi fragment of complement were estimated by use of flow cytometry. RESULTS: Priming of neutrophils by TNF-alpha improved phagocytosis of S aureus with a concentration-dependent effect. Phagocytosis of preopsonized washed bacteria was increased by activation of neutrophils with C5a(desArg). Phagocytosis was optimal when neutrophils primed with TNF-alpha were activated with C5a(desArg). The OB of phagocytizing neutrophils was highest when TNF-alpha and C5a(desArg) were used in combination. Bactericidal activity of neutrophils was stimulated by priming with TNF-alpha or C5a(desArg). Binding of bovine IgM or IgG2 to bovine neutrophils was not stimulated byTNF-alpha, C5a(desArg), or both, and aggregated IgG1 did not bind to neutrophils regardless of their activation state. Both TNF-alpha and C5a(desArg) increased expression of beta2 integrins (CD18), with the highest expression when they were used in combination. CONCLUSIONS AND CLINICAL RELEVANCE: The mediators TNF-alpha and C5a(desArg) stimulated phagocytic killing by neutrophils and potentiated each other when used at suboptimal concentrations. Bovine neutrophils have enhanced bactericidal activities at inflammatory sites when TNF-alpha, C5a(desArg), or both are produced locally.

Surprisingly high levels of anaphylatoxin C5a des Arg are extractable from psoriatic scales.

Water-soluble extracts from psoriatic scales and normal human skin were prepared using either phosphate-buffered saline, pH 7.2, or 0.1 M carbonate buffer, pH 10.8. Anaphylatoxin C5a des Arg was quantified using a novel sandwich enzyme-linked immunosorbent assay (ELISA) using neoepitope-specific monoclonal antibodies. Alkali was about five to eight times more efficient than PBS in extracting C5a des Arg from scales, probably via dissociation of bound C5a des Arg. C5a des Arg concentration in scales from three patients suffering from psoriasis vulgaris varied between 2.5 and 4.6 ng/mg scale. No C5a des Arg was detectable in normal skin extracts. The biological activity of alkali-extractable C5a des Arg, i.e. chemotaxis, was preserved. The concentration of C5a des Arg relative to the concentration of albumin was taken as a parameter of the degree of complement activation in the psoriatic lesions, and was found to be more than six times higher than values attained in serum after maximum complement activation by zymosan. We conclude that complement activation may play a quantitatively important role in the inflammatory process in psoriasis.

Production of monoclonal antibodies against the human anaphylatoxin C5a des Arg and their application in the neoepitope-specific sandwich-ELISA for the quantification of C5a des Arg in plasma.

A panel of ten murine monoclonal antibodies (MAbs) was raised against the human anaphylatoxin C5a des Arg. The MAbs were shown to abrogate or significantly inhibit the chemotactic activity in zymosan activated serum. MAb 4A2E10E2 and MAb 3G3C4 were used as capture and detecting antibody, respectively, in a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of C5a des Arg. This ELISA was shown to be very sensitive (detection limit 20 pg/ml) and could be applied directly to plasma/serum samples. The lack of interference by plasma components, in particular C5, suggested specificity for an epitope on C5a (des Arg) which is concealed in native C5 and exposed on the activation fragment only, i.e., a 'neoepitope'. The mean C5a des Arg level in EDTA-plasma from 25 healthy individuals assessed at a 1/20 dilution was 11.2 ng/ml (SD 3.4; range 6.4-16.8 ng/ml). The applicability of the assay was investigated in patients treated with haemodialysis using different membranes. Markedly elevated plasma levels of C5a des Arg were found in blood returning from the dialyzer following contact with cuprophane membranes.

Effect of psoralen + ultraviolet-A on the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg.

It was shown that psoralen + UV-A inhibits the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg. This reaction required oxygen and there is a high possibility that the active oxygen species was in a singlet state. Oxygen did not act directly on C5a des Arg, but rather produced oxidized psoralen which inhibited C5a des Arg activity. The effect was dependent on the concentration and types of psoralen and on the UV-A dose. Among the psoralens, the inhibitory effect of 4,5',8-trimethylpsoralen was the strongest, followed by 8-methoxypsoralen and 3-carbethoxypsoralen and finally 4,4',6-trimethylangelicin. Psoralen + UV-A failed to inhibit the chemotactic activity towards chemotactants other than anaphylatoxin C5a such as casein, the cultured filtrate of E. coli, platelet-activating factor, leukotriene B4, 5-hydroxy-6,8,11,14-eicosatetraenoic acid and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid.

Gcglobulin functions as a cochemotaxin in the lower respiratory tract. A potential mechanism for lung neutrophil recruitment in cigarette smokers.

Activation of the complement pathway with generation of the potent chemotaxin C5a may play a significant role in the neutrophil accumulation seen in the lungs of patients with smoking-associated diseases. Although C5a is rapidly degraded to the less potent chemotaxin C5a des Arg, binding of this peptide with its cochemotaxin Gcglobulin (GcG) can restore its chemotactic potency. Therefore, modulation of Gcglobulin levels in smoking-induced lung disease could affect the accumulation of neutrophils seen in this disorder. To test this hypothesis GcG was measured by an enzyme-linked immunosorbent assay in bronchoalveolar lavage fluids (BALF) obtained from non-smokers, asymptomatic smokers, and patients with chronic obstructive pulmonary disease (COPD). Antigenic amounts of GcG in BALF were increased in COPD patients and asymptomatic smokers compared with nonsmokers (6.35 +/- 1.02 and 5.15 +/- 1.07 versus 2.82 +/- 0.37 micrograms/mg albumin, p less than 0.05). In addition, we found that BALF enhanced C5a des Arg-mediated chemotaxis (48.3 +/- 5.6 versus 11.2 +/- 1.6 cells/high power field, p less than 0.05), an effect that was not seen in the presence of GcG antibody. Furthermore, BALF GcG was similar to serum GcG using Western blot analysis, and the interaction of GcG with C5a des Arg was not inhibited by cigarette smoke. These data demonstrate that elevation of BALF GcG levels occurs in smoking-associated lung disease and that this protein is biologically active and capable of increasing C5a des Arg-mediated chemotaxis. This suggests that modulation of GcG levels may be important in smoking-associated lung diseases.

The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg.

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 micrograms/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4 degrees C followed by washing and warming to 37 degrees C caused a 344% increase (n = 26) in the rate of superoxide anion production in response to zymosan-activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il-1 beta) or granulocyte-macrophage-colony stimulating factor (GM-CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37 degrees C. Only recombinant human tumour necrosis factor-alpha (rh-TNF-alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh-TNF-alpha. The priming effect could be markedly reduced (74.8%, n = 6) by employing the use of polyclonal antibody to TNF-alpha in the preincubation step; assaying for TNF-alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF-alpha present. Rh-TNF-alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity. The results show that TNF-alpha produced by LPS stimulated MNC after 5 h binds to a PMN surface receptor in the cold and warming of the cells to 37 degrees C allows for an immediate and dramatic response to ZAS stimulation. This suggests that TNF-alpha is the important cytokine upregulating PMN responses to other physiological mediators, including C5a des arg during the early phases of an inflammatory reaction.

A sensitive enzyme immunoassay for the quantitation of human C5a/C5a(desArg) anaphylatoxin using a monoclonal antibody with specificity for a neoepitope.

The direct quantitation of C5a/C5a(desArg) in human plasma was achieved by a specific and highly sensitive ELISA which is based on the neoepitope-specific monoclonal antibody C17/5. The error-prone removal of C5 from plasma prior to the assay is therefore not required. With a detection limit of 20 pg C5a/ml plasma, the sensitivity of this assay allowed to define normal ranges (1.94 +/- 1.49 ng C5a/nl, mean +/- SD) of C5a/C5a(desArg) in human blood plasma. This assay will also be applicable to the quantitation of C5a in specimens with low protein content where precipitation-based assays fail to accurately determine C5a. In addition, the mAb C17/5 turned out to efficiently block the binding of C5a/C5a (desArg) to its cellular receptor and therefore provides a valuable tool in the delineation of C5a effects in complex biologic systems in the presence of native C5, such as under in vivo conditions.