Altered expression of CD88 and associated impairment of complement 5a-induced neutrophil responses in human immunodeficiency virus type 1-infected patients with and without pulmonary tuberculosis.

The effect of infection with human immunodeficiency virus type 1 (HIV patient group), infection with Mycobacterium tuberculosis (TB patient group), and coinfection with both of these organisms (HIV/TB patient group) on the expression of CD88 on polymorphonuclear leukocytes (PMNL) was determined by using a receptor-specific monoclonal antibody and flow cytometry. A significant reduction in the fluorescence intensity of CD88 on PMNL was observed in the HIV and HIV/TB groups, compared with both the healthy donor (HD) and TB groups. Furthermore, when degranulation of PMNL was induced by ligation of CD88 by complement 5a (C5a), a large proportion of patients in the HIV and the HIV/TB groups was found to have reciprocal degranulation responses. Patients in the 2 HIV groups also were found to have significantly reduced C5a-induced chemotactic responses and significantly elevated peripheral levels of C5a des Arg, compared with the HD and TB groups. These differences may contribute to the increased susceptibility of HIV-1-infected individuals to secondary microbial infections.

Biocompatibility evaluation of polyamide hemofiltration.

INTRODUCTION: Postdilution hemofiltration with a polyamide membrane is a renal replacement technique widely used, but very little information is available regarding the biocompatibility of this treatment. In this paper we report the results of an acute study of the biocompatibility of polyamide hemofiltration. PATIENTS AND METHODS: Complement activation such as C3a and C5a Des Arg (RIA), granulocyte degranulation like alpha 1 elastase intradialytic increase (ELISA) and the expression of high affinity membrane receptors for IL-2 (anti-TAC) were determined. Beta 2-microglobulin (RIA) intradialytic decrease, as well as its convective removal, was evaluated. The nature of protein layer adsorbed onto the polyamide membrane, at the end of the dialytic session was investigated with a new immunohistochemical technique. Cell-associated cytokine concentration (like IL-1 beta and IL-1Ra - ELISA) was determined on mononuclear cell lysates. RESULTS: A low degree of complement activation was detected with the polyamide membrane when data were adjusted for hemoconcentration and for 1 m2 of membrane surface area. An important convective removal not only of Beta 2-microglobulin (258+/-20 mg/session), but also of the activated anaphylatoxins (225+/-76 ng/ml for C3a and 22.5+/-4 ng/ml for C5a) was revealed. A marked deposition of all coagulation factors with no detectable amount of immunoglobulins and complement factors was revealed on the polyamide membrane at the end of the dialytic session. No intradialytic (for IL-1beta) (from 14. 1+/-3.0 to 13.5+/-2.9 pg/2.5 x 10(6) cell) and interdialytic (for IL-1Ra) (from 4572+/-1076 to 5408+/-615 pg/2.5 x 10(6) cell) cell-associated cytokine expression was induced by hemofiltration. DISCUSSION AND CONCLUSION: Polyamide hemofiltration is a highly biocompatible technique due to the use of a synthetic membrane with a sterile reinfusion fluid and the convective removal of the activated anaphylatoxins and Beta 2-microglobulin.

Quantification of C5a/C5a(desArg) in bovine plasma, serum and milk.

Complement activation generates two potent inflammatory mediators from C5, C5a and its derivative C5a(desArg), which results from the removal of the C-terminal arginine by ubiquitous carboxypeptidases. In this paper we describe the purification of milligram amounts of bovine C5a(desArg) by a simplified procedure, and the preparation of mouse monoclonal antibodies (MAbs) to C5a/C5a(desArg) which do not recognize native C5. A MAb was used to develop a sandwich ELISA which made it possible to quantify levels of C5a/C5adesArg in bovine biological fluids. Small amounts (means +/- SEM) of C5a/C5a(desArg) were found in EDTA-plasma (0.58 +/- 0.06 ng.mL-1). The anticoagulant EDTA was more efficient than citrate or heparin in inhibiting in vitro activation of the complement system. Complement activation occurred during coagulation since the baseline concentration of C5a/C5a(desArg) (15.4 +/- 4.1 ng.mL-1) was higher than in plasma. Zymosan, a potent activator of the complement cascade, was used to generate C5a/C5a(desArg). The time-course of the reaction and the dose-effect of zymosan were investigated. Optimal conditions were incubation at 39 degrees C for 1 or 2 h with 2 mg of zymosan per mL of serum. The maximal concentration of C5a/C5a desArg attained in zymosan-activated serum was 4.28 +/- 0.14 micrograms.mL-1. Normal milk (from healthy, uninflamed mammary glands) contained on average 0.12 ng of C5a/C5a(desArg).mL-1 (range 0.02-0.19 ng.mL-1). The maximal amount of C5a/C5a(desArg) which was generated in milk with zymosan was 1.1 ng.mL-1 (range 0.68-2.17 ng.mL-1). In milk from quarters with subclinical infections by coagulase-negative staphylococci, values were 0.18 ng.mL-1 and 2.37 ng.mL-1 for spontaneous and zymosan-generated C5a/C5a(desArg) concentrations, respectively. In milk from Escherichia coli endotoxin-induced mastitis, C5a/C5a(desArg) concentrations (means of four cows) before and after zymosan activation reached 6.5 ng.mL-1 and 55 ng.mL-1, respectively. These results indicate that a C5-convertase can operate in normal milk, that only minute amounts of C5a/C5a(desArg) can be generated (less than 1/1,000 of plasma potential), but that much higher concentrations are reached in milk during endotoxin-induced inflammation. The ELISA made it possible to determine normal ranges of C5a/C5a(desArg) in bovine blood plasma and in milk, and is a valuable tool to define the variations of its concentrations in exudates during inflammatory reactions.

Complement activation during storage of whole blood, red cells, plasma, and buffy coat.

BACKGROUND: The process of separating whole blood into components and the storage of blood components may cause the release of toxic metabolites from the complement cascade. The aim of this study was to determine whether the storage of blood components leads to the activation of the complement cascade and the release of anaphylatoxins. STUDY DESIGN AND METHODS: Blood from 12 healthy volunteers was collected and stored either as whole blood or as components: red cells in saline-adenine-glucose-mannitol solution, plasma, and buffy coat. The concentrations of anaphylatoxins and other complement proteins in the various blood components were intermittently analyzed during a 5-week storage period. RESULTS: Increasing levels of anaphylatoxins were demonstrated during the storage of whole blood and plasma. Elevated concentrations of the anaphylatoxins C3a and C5a were observed during the storage of whole blood. Increased C5a levels were observed after 7 days of storage. High concentrations of C3a were found in plasma after 14 days of storage. Low or non-detectable levels of C3a; C5a, and other complement components were found in red cells stores in saline-adenine-glucose-mannitol solution. CONCLUSION: The study demonstrated activation of complement during the storage of whole blood and plasma but not in red cells in storage solution. The transfusion of larger volumes of stored whole blood or plasma may contribute to the risk of development of organ dysfunction. Therefore, it is advisable to use red cells in storage solution.

Complement activation on the nasal mucosal surface--a feature of the immediate allergic reaction in the nose.

Complement is a system of functionally linked serum proteins that interact to exert biologic effects in inflammatory and immunologic processes. As part of a larger study with a potential topical antiallergic drug, we measured C3a des Arg and C5a des Arg in 13 patients with seasonal allergic rhinitis and in five nonatopic controls after placebo treatment. After 1 week of placebo treatment, a nasal allergen challenge with increasing doses of pollens was performed in both allergic subjects and controls. A symptom score method was used, and in returned nasal lavage fluid, the activity of C3a des Arg and C5a des Arg was measured. We found that allergen challenge in the allergic subjects induced nasal symptoms concomitantly with increased levels of C3a des Arg and C5a des Arg (P < 0.05). No increases either in symptoms or in the very low base-line levels of C3a des Arg and C5a des Arg were observed in the nonallergic controls. We conclude that the activation of the complement cascade is one part of the vasculature exudative response during the immediate allergic reaction in the upper airways. Because of their biologic potency, these proteins may be an essential part of the exudative response which perpetuates the ongoing inflammatory reaction.

Quantification of chemotactic peptides (C5a anaphylatoxin and IL-8) in psoriatic lesional skin.

BACKGROUND AND DESIGN: Psoriatic scale extracts contain a unique chemotactic peptide fraction that is likely to be involved in the induction of rhythmic transepidermal leukocyte chemotaxis. Recent studies have identified the presence of two unrelated chemotactic peptides in this fraction, ie, C5a/C5a des Arg and interleukin 8 (IL-8), and its related cytokines. To investigate their relative contribution to the transepidermal leukocyte migration as well as their interrelationship in psoriatic lesions, we have quantified concentrations of immunoreactive C5a/C5a des Arg and IL-8 in psoriatic lesional scale extracts and those from related sterile pustular dermatoses such as subcorneal pustular dermatosis and pustulosis palmaris et plantaris. RESULTS: The concentrations of C5a/C5a des Arg and IL-8 were more significantly increased in the horny-tissue extracts from lesional skin than in those from noninflammatory orthokeratotic skin (P < .01). The increase of C5a/C5a des Arg concentration was specific to the lesional scale extracts, but showed a rather wide range of variation. By contrast, IL-8 concentration, although consistently increased in the lesional scale extracts, was also moderately increased even in noninflammatory scale extracts prepared from ichthyosis vulgaris. The elevation of IL-8 levels in psoriatic lesions was also confirmed by measuring their levels in cutaneous tissue fluid samples collected from suction blisters. However, unexpectedly, some control samples obtained from normal skin also showed a moderate increase in the IL-8 level. Neutrophil chemotactic activity correlated significantly only with the levels of C5a/C5a des Arg in the scales (P < .05). No such significant correlation was found between chemotactic activity and IL-8 or between C5a/C5a des Arg and IL-8. CONCLUSION: Based on these results, we speculate that, although IL-8 may exert a synergistic effect with C5a/C5a des Arg in the induction of transepidermal leukocyte chemotaxis, it constitutes a proinflammatory cytokine that is involved in the production of the persistent inflammatory changes characterized by a T-lymphocyte infiltration. In contrast, C5a/C5a des Arg seems to be generated only in the inflammatory lesional skin under specific circumstances that preferentially favor complement activation and also seems to play a major role in the induction of cyclic transepidermal leukocyte chemotaxis from "squirting papillae."

Complement activation and polymorphonuclear neutrophil leukocyte elastase in sepsis. Correlation with severity of disease.

Complement activation is necessary for an adequate immune and inflammatory response to infections. Activation releases anaphylatoxins that cause vasodilation, increase vascular permeability, and trigger release of polymorphonuclear neutrophil leukocyte (PMN) lysosomal enzyme and oxygen radicals. Under normal circumstances, an orderly progression of such events has a beneficial antimicrobial effect. The same mechanism, however, when uncontrolled, may damage host tissues. To provide information about the clinical importance of such events in sepsis, different complement parameters (C3, C4, and the desarginated forms of C3a [C3a(des)-Arg] and C5a [C5a(des)-Arg]), PMN elastase, and malondialdehyde (a by-product of membrane peroxidation by oxygen radicals) were measured daily in 26 septic patients and correlated with two objectively assessed and previously validated severity scores (acute physiology and chronic health evaluation [APACHE II] and Sepsis Severity Score [SSS]). Nonsurvivors (n = 12) had significantly greater and longer lasting complement activation than that in survivors, as reflected by higher levels of catabolic peptides (C3a(des)-Arg) and lower levels of native proteins (C3 and C4). C3a(des)-Arg, C3, C4, and the C3a(des)-Arg-C3 ratio were correlated with Sepsis Severity Scores. Polymorphonuclear neutrophil leukocyte elastase levels were higher in nonsurvivors and were correlated with C3a(des)-Arg and the C3a(des)-Arg-C3 ratio. Malondialdehyde levels were significantly higher in all patients than in controls, without, however, any relationship to severity of disease or clinical outcome. Since the higher and more persistent the complement activation and polymorphonuclear neutrophil leukocyte stimulation, the worse the patient's prognosis, we conclude that these mechanisms may be important in the clinical development of sepsis.

Anaphylatoxin generation and distribution during in vitro LDL apheresis.

The extent of anti-apo-B IgG-Sepharose-induced complement activation in serum and plasma (heparin 2 U/ml and ACD-B 1:20) was investigated using an in vitro model of LDL apheresis. The total volume of serum or plasma loaded to the chromatography column was collected in defined aliquots. The washing, desorption and regeneration fluids were processed in the same way. From the obtained values of generated complement split products C3a (desarg), C4a (desarg), C5a (desarg) and complement proteins C3, C4, C5, the conversion rates of the precursor were calculated. In the experiments with serum, 19% of C3, 8% of C4 and 2.3% of C5 were converted by the immunoadsorbent, whereas with plasma 7, 6, and 0.6%, respectively, were found. Furthermore, only 60-74% of total anaphylatoxins were found in the effluent during the loading process. The residual 26-40% was removed from the column with the subsequent washing fluids. Therefore, in the clinical routine, only a reduced part of generated anaphylatoxins will be retransfused to the patient. The fact that C5 is converted to the most limited extent to its biologically active fragment additionally contributes to the understanding of the good clinical tolerability of the LDL apheresis.

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.

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.

Hypersensitivity reactions during haemodialysis: role of complement fragments and ethylene oxide antibodies.

IgE-antibodies against ethylene oxide (EtO) and activation of the complement system have been suspected as causative factors for acute hypersensitivity reactions at the onset of haemodialysis. The present study was conducted to determine which of the two mechanisms is mainly responsible for the occurrence of reactions. According to clinical criteria, 13 of the 129 patients studied were identified as having suffered from at least one episode of acute hypersensitivity. Seven of them experienced severe symptoms, including five with elevated circulating serum EtO antibodies. Of the remaining six patients who experienced moderate symptoms, only one had elevated serum EtO antibodies, and of the 114 patients not exhibiting symptoms, two had enhanced serum concentrations of EtO antibodies. In-vivo complement activation was determined during haemodialysis by measurement of both C3adesarg and C5adesarg fragments. No differences in the onset levels and the degree of complement activation were found between patients suffering from severe, moderate, or no hypersensitivity reactions. In-vitro activation of the complement cascade by zymosan, measured in plasma samples obtained from patients prior to dialysis, revealed no differences in the extent of C3adesarg generation. Finally, we determined the activity of the C3a- and C5a-inactivating enzyme, carboxypeptidase N1 (CN1), in plasma samples of patients both with and without hypersensitivity reactions. No difference in the CN1 activity between the two groups of patients was found. These data lead us to conclude that sensitisation to ethylene oxide is responsible for the majority of severe hypersensitivity reactions in haemodialysis patients. However, a small subgroup of patients having anaphylactoid reactions displayed neither elevated serum EtO antibodies with excessive complement activation nor a tendency towards reduced inactivation of anaphylatoxins.