Complement Split Product C5a Research Articles

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 15‐Hydroxyeicosatetraenoic Acid by Purified Human Eosinophils and Neutrophils

In the presence of high concentrations of exogenous arachidonic acid (greater than or equal to 10 microM), eosinophils produced 15-hydroxyeicosatetraenoic acid (15-HETE) in the absence of stimuli. The calcium ionophore A23187, as well as the chemotaxins used in this study--complement split product C5a, platelet-activating factor (PAF), and N-formyl-methionyl-leucyl-phenylalanine (FMLP)--failed to increase 15-HETE production, indicating that eosinophil 15-lipoxygenase is already active. Production of 15-HETE from eosinophils increased with increasing concentrations of arachidonic acid, exogenously added. Maximal 15-HETE production was observed to be 1111 +/- 380 ng per 10(6) eosinophils at the concentration of 100 microM of arachidonic acid. With low concentrations of exogenous arachidonic acid (below 2 microM), eosinophils were considered to incorporate exogenous arachidonic acid into their cell membrane, and did not produce 15-HETE. In contrast, 15-HETE formation in highly purified neutrophils (eosinophils less than 1%) was negligible compared with that in eosinophils (300-fold less), suggesting that 15-HETE-forming activity in granulocytes is derived from the eosinophil 15-lipoxygenase pathway and that neutrophils may lack 15-lipoxygenase activity.

Inhibition of chemotactic migration of human neutrophilic granulocytes by recombinant human granulocyte-macrophage colony-stimulating factor

Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was analysed for effects on the migration of human neutrophilic granulocyte by the Boyden chamber assay. At concentrations ranging from 0.1 to 10 000 U/ml (or 10−12 to 10−7 mol/1) GM-CSF had neither chemokinetic nor chemotactic activity. When added to the cells in the upper compartment of the chamber GM-CSF dose-dependently inhibited the chemotactic migration towards the tripeptide f-Met-Leu-Phe and the complement split product C5a. Chemotaxis towards f-Met-Leu-Phe was inhibited more efficiently by GM-CSF than C5a-induced migration.

Chemotactic Responsiveness of Eosinophils Isolated from Patients with Inflammatory Skin Diseases

We determined the chemotactic responsiveness of peripheral eosinophilic granulocytes (eosinophils) isolated from patients with inflammatory dermatoses and healthy volunteers. Ten patients with atopic dermatitis, five patients with drug reactions, ten patients with psoriasis, and fourteen healthy volunteers were studied. Well characterized chemotaxins, the complement split product C5a, leukotriene B4 (LTB4), platelet activating factor (PAF), and N-formyl-methionyl-leucyl-phenylalanine (FMLP), were used as chemoattractants. Eosinophils from healthy volunteers showed strong migratory responses towards C5a and PAF but responded poorly to LTB4 and FMLP. When patients were grouped by disease severity, eosinophil chemotactic responses to PAF were significantly enhanced in severely affected patients (p less than 0.05); this was not true with C5a, LTB4 or FMLP. This enhanced eosinophil chemotaxis to PAF was not related to a specific disease. No correlation between eosinophil chemotactic activity and peripheral blood eosinophil count was observed. The increased responsiveness of circulating eosinophils towards PAF may be related to altered receptor expression during cutaneous inflammation.

15-Hydroxyeicosatetraenoic acid (15-HETE) specifically inhibits the LTB4-induced skin response

15-Hydroxyeicosatetraenoic acid (15-HETE), a 15-lipoxygenase product of arachidonic acid, inhibits leukotriene B4 (LTB4)-induced chemotaxis of polymorphonuclear leukocytes (PMNs) in vitro. In this study the effects of intradermal injections of LTB4 were determined in the absence or presence of 15-HETE. For comparison intradermal injections of purified human complement split product C5a were performed in the absence or presence of 15-HETE. The skin response was evaluated by measuring the diameter of the wheal, the area of the flare and by intensity of the erythema (erythema index). LTB4 and C5a were injected at the concentration of 200 ng/ml. At this concentration the maximal skin response of LTB4 and C5a were equivalent. In contrast to C5a reaction, which resolved within 1 h, LTB4-induced skin response lasted up to 18 h. In all subjects the skin response was significantly decreased when LTB4 was injected together with 300 ng of 15-HETE. The decrease of wheal, flare, and erythema index averaged 81.9%, 56.6%, 53.6%, respectively, when all parameters were obtained at the maximal skin response. In contrast, the C5a-induced skin response was not affected by addition of 15-HETE, even when the final dose of 15-HETE was increased 10 times to 3 micrograms. The LTB4-induced reaction could last up to 18 h after injection. After the addition of 300 ng of 15-HETE the skin response resolved after 1 h. The present results demonstrate that 15-HETE is a specific inhibitor of the LTB4-induced skin response and brings additional evidence in support of the ability of 15-HETE to regulate the proinflammatory effects of LTB4 in vivo.

Recombinant Human Tumour Necrosis Factor β (Lymphotoxin) Lacks Chemotactic Activity for Human Peripheral Blood Neutrophils, Monocytes, and T Cells

Human recombinant tumour necrosis factor beta (rhuTNF beta)/lymphotoxin was tested for human neutrophil granulocyte (PMN), monocytes (MO), and T-cell chemotactic activity by means of a modified Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-14)M)rhuTNF beta showed no chemotactic activity for PMN, MO, or T cells. In contrast, strong chemotactic migration was elicited in PMN and MO with the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and in T cells when complement split product C5a and leukotriene B4 (LTB4) were used as chemotaxins. The results of this study indicate that rhuTNF beta/lymphotoxin is not a chemotaxin for human PMN, MO, or T lymphocytes in vitro.

Phospholipase A in acute lung injury after trauma and sepsis: its relation to the inflammatory mediators PMN-elastase, C3a, and neopterin.

Inflammatory mediators involved in the pathogenesis of the adult respiratory distress syndrome (ARDS) are products of the humeral cascade systems like the complement cascade and substances released from neutrophil granulocytes and macrophages like proteases, O2-radicals and arachidonate products. Phospholipase A2 (PLA) was shown by Vadas et al. to be correlated with circulatory shock in the sepsis syndrome, the probably most important underlying disease of ARDS. In a clinical study in 48 patients at risk for ARDS after trauma and sepsis we found plasma PLA elevated (52 +/- 5 U/l) in sepsis, with a positive correlation to the complement split product C3a (r = 0.42, p less than 0.01) and neopterin (r = 0.49, p less than 0.05), which serves as a marker of macrophage stimulation. Elastase-alpha 1PI and C3a showed higher plasma levels in patients with ARDS compared with non-ARDS patients, whereas the neopterin and PLA concentrations were not different with regard to ARDS. The relation between PLA and neopterin shown in the study is consistent with the possibility of macrophages being a source of the plasma PLA, as reported in experimental studies.

Functional Properties of a Novel Neutrophil Chemotactic Factor Derived from Human Monocytes

Neutrophilic granulocytes (PMN) are attracted to sites of inflammation by chemotactic factors, the most potent of which are the complement split product C5a, the leukotriene B 4 and the bacterial chemotactic factor-related tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP). In addition to inducing directed migration, these agents increase the adherence of PMN to synthetic surfaces and endothelial cells; some stimulate an oxidative burst and the production of reactive oxygen derivatives, and they may be involved in the release of granule constituents. Here, we describe studies on the activities stimulated by a novel monocyte-derived chemotaxin (MOC). Human MOC attracted human PMN, but not monocytes or eosinophils. Like all chemotactic agents, it increased the adherence of PMN on nylon fibers. In contrast to other chemotactic factors it did not stimulate the release of superoxide anion regardless whether the cells were in suspension or adherent on nylon fibers. There was no release of the primary granule enzyme glucosaminidase or the secondary granule component vitamin B 12-binding protein in the absence or presence of cytochalasin B. The results suggest that MOC is a unique chemotactic agent with properties different from the most potent chemotactic factors C5a, LTB 4 and FMLP. The delayed release from macrophages suggests its involvement in protracted and chronic inflammatory reactions.

Modulation of Human Monocyte Functions during Acute Bacterial Infection

The in vitro functions of highly purified blood monocytes were studied in 11 patients suffering from acute bacterial infections (e.g. erysipelas, appendicitis, abscesses). Chemotaxis, superoxide-anion generation, and beta-glucuronidase release of the patients' monocytes in response to the receptor-dependent stimuli formyl-methionyl-leucyl-phenylalanine (FMLP), complement split product C5a, leukotriene B4 (LTB4), and opsonized zymosan particles were measured. All the patients were examined in a follow-up study during the course of illness. A group of 33 healthy volunteers served as control. The patients revealed a transient decrease in monocyte chemotactic migration in response to all stimuli between days 3 and 5 after onset of clinical symptoms. Superoxide-anion generation from patients' monocytes was found to be enhanced 3 days after impaired chemotaxis. Stimulated release of lysosomal beta-glucuronidase showed a decrease in the first days of the disease. However, spontaneous beta-glucuronidase release was enhanced between days 3 and 7 in the patients' monocytes. Serial measurements of monocyte responsiveness. These results indicate a distinct modulation of monocyte functions during the course of an acute bacterial infection. Changes in monocyte maturity and/or activation under inflammatory conditions may be responsible for these alterations in monocyte function.

15-Hydroxyeicosatetraenoic Acid (15-HETE) Specifically Inhibits LTB<sub>4</sub>-Induced Chemotaxis of Human Neutrophils

15-Hydroxyeicosatetraenoic acid (15-HETE), a 15-lipoxygenase (15-LO) product of arachidonic acid has the potential to inhibit leukotriene formation. In the present study the effect of 15-HETE on leukotriene B4 (LTB4)-induced polymorphonuclear leukocytes (PMN) and monocyte chemotaxis was investigated. LTB4-induced chemotaxis of PMNs was inhibited in a dose-dependent manner by 15-HETE. Maximum inhibition (68%) occurred at a 15-HETE concentration of 10(-4) M. The 15-LO product of eicosapentaenoic acid (15-HEPE) was approximately 10 times less potent in inhibiting LTB4-induced PMN chemotaxis. LTB4-induced chemotaxis of monocytes was unaffected by both 15-HETE and 15-HEPE. Using N-formyl-methionyl-leucyl-phenylalanine (FMLP) and complement split product C5a as chemoattractants, 15-HETE did not decrease PMN chemotaxis. Furthermore, 15-HETE itself did not affect random migration of leukocytes. The present results demonstrate that 15-HETE inhibits LTB4-induced chemotaxis of PMNs in vitro in a specific and selective way. Because 15-HETE not only inhibits formation, but also the effect of LTB4, it may be important in regulating LTB4-induced inflammation.

C5a-Specific Modulation of Phagocyte Functions in Patients with Localized Bacterial Infections

Chemotaxis, enzyme release and superoxide-anion (O − 2) generation of purified peripheral blood neutrophils (PMN) and monocytes were studied in a total of 87 patients suffering from localized bacterial infections. Shortly after disease onset, single or several PMN functions became non-responsive to the complement split product C5a. Functional activities elicited by the synthetic peptide f-met-leu-phe or leukotriene B 4 remained unaltered. C5a-specific impairment lasted one to several days and returned to normal with the subsidence of the disease. C5a elicited release of β-glucuronidase as well as generation of superoxide-anions was seen more often to be impaired as compared to chemotactic migration. In contrast to PMN, monocytes from the same patients failed to show paralleling C5aspecific functional alterations. There was no correlation between impairment of PMN functions and plasma levels of the complement split-products C3a and C4a as determined by RIA. It is concluded that in acute infectious disease a graded C5a-specific modulation of PMN functions may be present. This phenomenon is transient and not paralleled by functional alterations of monocytes or changes in plasma complement levels.

Methotrexate Inhibits the Human C5a-Induced Skin Response in Patients With Psoriasis

In this study we examined the effects of intradermal injections of human complement split product C5a in 10 patients with psoriasis in long-term treatment with methotrexate (MTX). The C5a was injected at the end of the weekly MTX cycle just before the intake of the first MTX dose and 3 h after the second of the 3 doses. The C5a-induced skin response was evaluated by measuring the diameter of the wheal and the area of the flare and by erythema index (EI), which was determined objectively by reflectance spectrophotometry. In all patients the skin response was significantly depressed when C5a was injected after MTX intake. The decrease of wheal, flare, and EI averaged 61.6%, 71.1%, and 57.5%, respectively, when all parameters were obtained at maximal skin response. The in vitro chemotaxis of peripheral blood neutrophils and monocytes from the patients toward C5a was markedly inhibited after intake of MTX (p less than 0.01). The skin biopsies obtained after C5a injection before intake of MTX revealed a perivascular inflammatory infiltrate and considerable dermal edema. After MTX intake the number of infiltrating leukocytes and the degree of dermal edema was markedly reduced. This study indicates that MTX is a potent inhibitor of C5a-induced skin inflammation, and that this inhibition may be caused by a direct effect on circulating neutrophils and monocytes. The results obtained in this work support the idea that anti-inflammatory effects of MTX may be partially responsible for its antipsoriatic effect.

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo.

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.

Chemiluminescence response of human B-cell lines.

Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cells share certain properties with monocytes: they are capable of presenting protein antigens to antigen-specific T-lymphocytes and of releasing an Interleukin 1-like factor. It was our interest to study whether transformed B-cells resemble monocytes by generating toxic oxygen radicals. Human B-cell lines were developed from human peripheral blood lymphocytes by EBV-transformation. The induction of the respiratory burst in the B-cells was assessed by chemiluminescence (CL) in the presence of lucigenin. B-cells were stimulated with phorbol-myristate-acetate (PMA), zymosan particles, the chemotactic peptide f-met-phe, the complement split product C5a and with a recently described granulocyte activating cytokine (GRAM). Stimulation with PMA elicited a distinct CL-response in the tested B-cell lines. The CL-signal was significantly reduced by superoxide dismutase, but not by D-mannitol and catalase. No significant response to any of the other stimuli was detected. Furthermore, none of the stimuli induced a luminol-enhanced CL signal, which, in contrast to lucigenin, is dependent on the presence of peroxidase. Our results indicate that EBV infected B-cells were able to generate significant amounts of reactive oxygen species, particularly superoxide. It appears that virus transformation uncovers genetic information which is usually not expressed in non-transformed B-cells.

Complement split product C5a mediates the lipopolysaccharide-induced mobilization of CFU-s and haemopoietic progenitor cells, but not the mobilization induced by proteolytic enzymes.

Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. The mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5. The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. The resistance of the mobilizing principle to heat treatment (56 degrees C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.

Subnormal sensitivity of neutrophils to complement split product C5a in rheumatoid arthritis: relation to complement catabolism and disease extent.

The capacity of circulating neutrophils for activation by complement was studied in outpatients with classical or definite rheumatoid arthritis during treatment with dextropropoxyphene only. Analysis of dose-response in the Boyden chamber assay of chemotaxis showed that sensitivity to the potent, complement derived anaphylatoxin, C5a, was markedly decreased, especially in those patients with few joints involved. In contrast, peak response to C5a was within the normal range. Increased complement 3c split products in plasma of the patients suggested involvement of complement cascade reactions. Subnormal sensitivity of neutrophils to phlogistic mediators released by complement may tend to limit their recruitment and potentially tissue destroying secretion locally in rheumatoid arthritis.

Subnormal activation of phagocytes by complement in chronic inflammatory bowel disease? Neutrophil chemotaxis to complement split product C5a.

The capacity of circulating phagocytes for activation by complement was investigated in consecutive, untreated cases of chronic inflammatory bowel disease. The major complement derived chemotactic factor, C5a, served as chemoattractant in dose response studies of neutrophil chemotaxis. A similar, significantly decreased sensitivity and peak response was revealed in patients with Crohn's disease and ulcerative colitis. This subnormal function of neutrophils could be shown even in cases of complete clinical remission. Chemotactic response to casein and spontaneous motility was within the normal range showing an unaffected basic cell function of neutrophils in the patients. The study shows a dysfunction of phagocytic cells, related to potentially important phlogistic mediators, in chronic inflammatory bowel disease.

A rapid technique for measuring leukocyte chemotaxis in vivo

A modification of connective tissue air bleb technique was used to develop a model system in inbred strains of mice for the study of chemoattractants in vivo. The method was developed using the well characterized n-formylated chemotactic peptide, f-Met-Leu-Phe, as the positive control. Injection of 0.1 ml of f-Met-Leu-Phe solutions from 10 −7 to 10 −10 M resulted in an influx of polymorphonuclear leukocytes within 2 h. Study of the kinetics of the response showed that the number of infiltrating cells reached a peak within 8 h and slowly declined over a 2-day period. The predominant infiltrating cell type during the first 24 h was the polymorphonuclear leukocyte. Between 24 and 48 h the polymorphonuclear leukocytes were replaced by monocytes. By utilizing an inbred mouse strain (DBA-1J and 2J) sufficient or deficient in C5 it was possible to distinguish compounds that were directly chemotactic from those that worked indirectly, or whose chemotactic potential could be enhanced by generation of the chemotactic complement split product C5a. The method was found to be technically simple, reproducible and semi-quantitative and represents a good model system to facilitate the comparison of chemotactic responses in vivo and in vitro.

Altered polymorphonuclear leukocyte responses in psoriasis: chemotaxis and degranulation.

Chemotactic activities of circulating polymorphonuclear leukocytes (PMN) were determined in twenty patients with psoriasis and twenty healthy control persons. After serial dilution of the complement split product C5a and the formylated tripeptide f-met-leu-phe (FMLP), chemotaxis profiles showed that PMN migration toward both chemotaxins was significantly increased in psoriasis. In addition, PMN from psoriatic patients responded to chemotaxins at much lower concentrations compared with controls. The liberation of (lysosomal) beta-glucuronidase was also determined in cytochalasin B-treated cells confronted with increased concentrations of the chemotaxins. Secretion of this marker enzyme started at lower concentrations in PMN derived from psoriatic patients. Our observations demonstrate migratory and secretory hyper-responsiveness of PMN from psoriatic patients. This may play a role in perpetuating the psoriatic tissue reaction.