Interaction of human anaphylatoxin C3a with rat mast cells demonstrated by immunofluorescence

Specific interaction of human C3a with rat peritoneal mast cells was demonstrated by means of the indirect immunofluorescence technique using anti-C3a. Anti-C3b, an antiserum exclusively directed against the native structure of C3, anti-C3c and anti-C3d as well as antisera against other complement components and against IgE did not show fluorescence.     

The active site of human C4a anaphylatoxin

The human C4 activation peptide C4a has recently been shown to be biologically active and to share common tissue receptors with human C3a anaphylatoxin. Human C3a and C4a each induce contraction and cause cross-desensitization of isolated guinea-pig ileal strips. The essential active site of C3a is comprised in the model peptide containing the five COOH-terminal residues, Leu-Gly-Leu-Ala-Arg. The anaphylatoxic activities of the corresponding C4a pentapeptide, Ala-Gly-Leu-Gln-Arg, and several other synthetic peptides related to the COOH-terminal sequence of human C4a were examined. The C4a pentapeptide induced contraction of guinea-pig ileum at 1 X 10(-3) M and produced a wheal and flare reaction in human or guinea-pig skin when 2-5 mumols were injected intradermally. The corresponding C3a pentapeptide is 500-fold more active, since it induces contraction of guinea-pig ileum at 3-4 X 10(-6) M and only 4-10 nmole induce a visible skin reaction. Although the C4a pentapeptide is relatively inactive compared to the C3a pentapeptide, two analogs of these peptides, Leu-Gly-Leu-Gln-Arg and Ala-Gly-Leu-Ala-Arg, each exhibited significantly greater activity than Ala-Gly-Leu-Gln-Arg and each analog desensitized ileal smooth muscle towards contraction by either C3a or C4a. Thus it is a combination of two amino acid substitutions, the Ala for Leu-73 and Gln for Ala-76, in the COOH-terminal pentapeptide of C3a that accounts for the markedly reduced activity of C4a. The contribution of the COOH-terminal portion of C4a on its activity was further documented by examining the C4a octapeptide, Lys-Gly-Gln-Ala-Gly-Leu-Gln-Arg and a trialanyl analog, Ala-Ala-Ala-Ala-Gly-Leu-Gln-Arg. The C4a octapeptide, C4a (70-77), exhibited 5-fold greater biologic activity than the C4a pentapeptide, while the trialanyl analog was 40-fold more active. Anaphylatoxic activities of the C4a-(73-77) pentapeptide, C4a-(70-77) octapeptide, and the trialanyl octapeptide analog and their ability to specifically block the action of C3a and C4a on smooth muscle tissue support the conclusion that, as in C3a, the essential active site of C4a resides at its COOH terminus. Since C4a functions as an anaphylatoxin and significant quantities of this mediator may be generated in individuals with hereditary angioneurotic edema (HANE), the hypotheses that the kinin-like activity promoting edema in HANE patients is derived solely from component C2 and/or kininogens should be reappraised. The activities previously assigned to C4a and now confirmed by synthetic C4a analog peptides suggest that the kinin-like activity generated in HANE plasma may be derived in part from C4a.     

Antagonistic peptides against human anaphylatoxin C5a.

Multivalent synthetic peptides derived from C5a were prepared in order to examine their effects on the C5a receptor (C5aR). Multiple antigen peptide (MAP) of the C5a C-terminal region (MAP61-74) bound to cells expressing C5aR with high affinity. On the other hand, N-terminal peptides (MAP3-16 and MAP12-26) and one with a sequence from the mid-portion of C5a (MAP37-53) did not bind to the cells. In addition, MAP61-74 inhibited Ca2+ mobilization and release of beta-hexosaminidase by C5a from dibutyryl cAMP-activated U937 cells. This Ca2+ mobilization was also inhibited by MAP12-26 and Mono61-74, the monomeric C-terminal peptide. Taken together, these data indicate that C5a binds to the C5aR via its C-terminal region. Furthermore, MAP61-74, a 14mer peptide that has additional amino acids at the N-terminal compared with the C-terminal octapaptide, can bind to C5aR and can be considered an antagonist of C5a which may prove useful as an agent for controlling the allergic response caused by complement activation.     

Complement gene expression is regulated by pro-inflammatory cytokines and the anaphylatoxin C3a in human tenocytes

Interplay between complement factors, regulatory proteins, anaphylatoxins and cytokines could be involved in tendon healing and scar formation. The expression and regulation of complement factors by cytokines or anaphylatoxins are completely unclear in tendon.Hence, the gene expression of the anaphylatoxin receptors C3aR, C5aR and cytoprotective complement regulatory proteins (CRPs) was analysed in human tendon, cultured primary tenocytes and to directly compare the general expression level, additionally in human leukocytes. Time-dependent regulation of complement by cytokines and the anaphylatoxin C3a was assessed in cultured tenocytes.Gene expression of the anaphylatoxin receptors C3aR, C5aR and the CRPs CD46, CD55 and CD59 was detected in tendon, cultured tenocytes and leukocytes, whereas CD35 could only be found in tendon and leukocytes. Compared with cultured tenocytes, complement expression was higher in tendon and compared with leukocytes C3aR, C5aR, CD35 and CD55, but not CD46 and CD59 gene expression levels were lower in tendon. C3aR mRNA was up-regulated by both TNFα and C3a in cultured tenocytes in a time-dependent manner whereby C5aR gene expression was only induced by C3a. IL-6 or C3a impaired the CRP gene expression. C3a stimulation lead to an up-regulation of TNFα and IL-1β mRNA in tenocytes. Degenerated tendons revealed an increased C5aR and a reduced CD55 expression.The expression profile of the investigated complement components in tendon and cultured tenocytes clearly differed from that of leukocytes. Tenocytes respond to the complement split fragment C3a with CRP suppression and enhanced pro-inflammatory cytokine gene expression suggesting their sensitivity to complement activation.     

Plasma clearance of the human C5a anaphylatoxin by binding to leucocyte C5a receptors.

The C5a anaphylatoxin is a potent complement-derived mediator of inflammation with chemotactic activity. In this study the possible role of specific high-affinity binding sites for C5a on peripheral blood leucocytes for the removal of C5a from human blood plasma was investigated. The addition of purified granulocytes or mononuclear cells to complement-activated plasma resulted in the rapid and dose-dependent removal of up to 80% of plasma C5a, as determined by ELISA. The specific role of leucocyte C5a receptors (C5aR) in the plasma clearance of C5a was demonstrated by the inhibition of C5a uptake by the preincubation of cells with the C5aR-specific monoclonal antibody S5/1. Furthermore, U937 cells which had been induced by db-cAMP to express C5aR, but not undifferentiated U937 cells, were capable of removing C5a from plasma. The inhibition of C5aR internalization by monensin did not affect C5a uptake by leucocytes. The co-incubation with leucocytes had no effect on the plasma clearance of complement activation products C3a or terminal complement complex (TCC), as determined by this in vitro assay. The binding of the C5a anaphylatoxin to cellular receptors represents an effective control mechanism that protects the organism from systemic effects of this potent phlogistic mediator.     

Quantitation of the anaphylatoxin C3a in the presence of C3 by a novel sandwich ELISA using monoclonal antibody to a C3a neoepitope

C3a levels in plasma are usually measured by a competitive inhibition radioimmunoassay (RIA) using 125I-labelled C3a-desArg and antibodies to C3a capable of detecting C3a determinants which are also present on the native C3. Therefore, prior to the assay native, non-cleaved C3 has to be removed completely from the C3a-containing sample by precipitation. We developed a new rapid two-site sandwich ELISA system for the quantitation of C3a-desArg in plasma. This immunoassay uses a monoclonal antibody (mAb H466) reacting with C3a-desArg but not with C3. The reactivity of mAb H466 with a neoantigenic determinant of C3a-desArg permitted the direct quantitation of C3a-desArg without removal of C3 from the sample. The mAb H466 was used as a capture antibody and bound C3a-desArg was detected with a second peroxidase-labelled anti-C3a mAb. The lower limit of detection of C3a-desArg in this ELISA was 1 ng/ml. The C3a-desArg levels measured in the plasma samples of various patients were found to differ over a wide range. A good correlation was observed between the results obtained in the RIA and those obtained in the ELISA (r = 0.95). High levels of C3a-desArg were detected in plasma from patients with multiple trauma and patients undergoing haemodialysis. The C3a-desArg assay described should facilitate the routine quantitation of C3a in samples of plasma.     

Complement proteins and C3 anaphylatoxin in the tears of patients with conjunctivitis

Previous studies in our laboratory have demonstrated pollen-specific IgG antibodies in the tears of patients with vernal conjunctivitis (VC) and elevated tear IgG levels in patients with contact lens-induced giant papillary conjunctivitis (GPC). Tear secretions were examined for complement (C) proteins to determine the role of this effector system in the pathogenesis of these ocular disorders. The tears of VC (15) and GPC (10) patients with active disease had elevated tear levels of both C3 and factor B. By use of transferrin as a marker for the leakage of plasma proteins into the tears, most C3 was locally produced by the conjunctival tissues. Although immune complexes could not be detected in the tear secretions, increased levels of C3 des Arg were present in the tears that suggested complement activation with the generation of anaphylatoxins. These studies suggest that complement may be important in the inflammatory ocular process of VC and GPC and that the generation of anaphylatoxins (C3a), even by nonimmune mechanisms, may contribute to basophil and mast cell activation with the release of inflammatory mediators into the tear secretions.     

Expression of the anaphylatoxin receptors C3aR and C5aR is increased in fatal asthma

BACKGROUND: The mechanisms leading to death from asthma are not completely understood. Recent studies suggest the involvement of the anaphylatoxins C3a and C5a, generated during complement activation, and their receptors C3aR and C5aR in the pathogenesis of asthma. OBJECTIVE: The aim of our study was to investigate the expression of C3aR and C5aR in fatal asthma. METHODS: We analyzed lung tissue from 14 subjects who died of asthma (fatal asthma; FA) and 14 subjects who died of nonpulmonary causes (controls) and bronchial biopsy specimens from 16 subjects with mild intermittent asthma (MIA). C3aR and C5aR expression was evaluated by immunohistochemistry, and a semiquantitative analysis of the intensity of staining was performed according to a visual analogue scale (score, 0-3). RESULTS: C3aR was expressed on airway epithelium, smooth muscle, submucosal, and parenchymal vessels. C5aR was expressed on myeloid cells infiltrating the submucosa and on airway epithelium. Statistical analysis demonstrated higher expression of C3aR on submucosal vessels in FA compared with controls and MIA (median [minimum-maximum], controls, 0.24 [0-1.48]; MIA, 0.0 [0-1.00]; FA, 1.56 [0.13-3]; P = .002). C3aR was also increased on parenchymal vessels in FA (controls, 0.56 [0-2.00]; FA, 1.81 [0.5-3]; P = .0004). C5aR expression on airway epithelium was increased in FA compared with controls and MIA (controls, 1.25 [0.25-3]; MIA, 1.00 [0-2.00]; FA, 3.00 [1.13-3.00]; P = .001). CONCLUSION: The results of our study suggest a role of complement in FA.     

Cutaneous depression of Ib reflex pathways to motoneurones in man

Summary Variations in the H-reflex of soleus (Sol), quadriceps (Q) and short head of biceps femoris (Bi) muscles in normal man were used to investigate the effect of volleys in low threshold cutaneous afferents from the ipsilateral limb on transmission of Ib effects from ankle and Q muscles to these different motoneurone (MN) pools. Stimulation of cutaneous afferents from the foot sole and the toes (but not from the thigh, knee or calf), which did not modify the size of the test reflexes when applied alone, strongly depressed Ib reflex pathways to MNs supplying muscles operating at the knee. The very brief central latency of this depression suggests that tactile cutaneous afferents from the foot have oligosynaptic spinal connexions with the interneurones intercalated in the Ib pathways to MNs. The same cutaneous stimuli did not at all modify Ib inhibition of Sol MNs from triceps surae. These findings are discussed with regard to the role of the different muscles in human locomotion. It is suggested that during the stance phase of heel bipedal locomotion, the cutaneous depression of Ib reflex pathways to MNs supplying muscles operating at the knee might operate in association with the strong Ia connexions from ankle to knee muscles described in the previous paper.This work was supported by grants from Pierre et Marie Curie University (Paris VI) and from the Institut National de la Santé et de la Recherche Médicale (INSERM, 78.1.269.6)Attachée de recherches à l'INSERM     

Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology.     

Acute neurohormonal responses to hypoxaemia in man

We have studied the integrated neuroendocrine and haemodynamic effects of acute hypoxaemia in ten healthy volunteers studied on two separate occasions. After reaching a resting haemodynamic state, subjects breathed either room air or a nitrogen/oxygen mixture which rendered arterial oxygen saturation between 75% and 80%. Measurements of pulmonary and systemic haemodynamics were made and blood samples taken at baseline and after 30 min breathing air or the hypoxic gas. Blood was assayed for plasma sodium and potassium, renin-angiotensin-aldosterone system activity, natriuretic peptides, cortisol and catecholamines. Hypoxaemia significantly increased heart rate, cardiac output and mean pulmonary artery pressure (P _pa), but not mean arterial pressure compared with normoxaemia. Although plasma renin activity, angiotensin II and cortisol were unaffected by hypoxaemia, plasma aldosterone fell significantly in comparison with normoxaemia. This was associated with an increase in plasma atrial natriuretic peptide (ANP) but not b-type natriuretic peptide (BNP) during hypoxaemia whilst no changes were observed during normoxaemia. The increase in plasma ANP correlated positively with the increase inP _pa. During hypoxaemia there is therefore dissociation of the renin-angiotensin-aldosterone system where plasma aldosterone decreased, despite there being no effects on plasma renin activity and angiotensin II or on plasma cortisol. This dissociation may be due to increased levels of ANP but not BNP having specific inhibitory effects on aldosterone biosynthesis. ANP increased in proportion to the degree of pulmonary vasoconstriction induced by hypoxaemia which may indicate a counter-regulatory role.     

Catecholamine responses to histamine infusion in man

To evaluate the effects of histamine-induced hypotension on plasma catecholamine levels, eight normal men, aged 20 to 40 years, were infused with incremental doses of histamine starting at 0.2 microgram/kg/min at a 30 degree tilt position with monitoring of blood pressure (BP) and heart rate. Histamine dosage was increased every 5 minutes by 0.1 to 0.2 microgram/kg/min until mean BP fell greater than 15 mm Hg or a dosage of 1.6 micrograms/kg/min was reached. Plasma catecholamine samples were taken between the fourth and fifth minute of each histamine dosage. Identical measurements were made during nitroglycerin-induced hypotension in these subjects. Histamine produced threefold greater increases in heart rate and plasma norepinephrine (NE) levels than did nitroglycerin for comparable decreases in BP. Although NE levels increased twofold to fivefold from baseline with histamine infusion, epinephrine levels increased minimally at the highest doses or not at all. Our data demonstrate that histamine selectively releases NE from adrenergic nerve terminals without significant adrenal catecholamine release. We suggest that neural NE release plays an important role in the cardiac effects of histamine.     

Cardiovascular responses to tilting in tetraplegic man

1. A study has been made of the effect of head-up tilt on blood pressure, heart rate, forearm blood flow and occluded vein pressure in the hand and foot in non-bedridden patients with chronic, closed, complete, localized traumatic transection of the cervical spinal cord.2. In typical responses the blood pressure fell and the heart rate rose progressively for about 2 min, tending to plateau thereafter. The average falls in mean blood pressure for tilts of +30, +45 and +60 degrees were from 68 to 44, 74 to 36 and 70 to 44 mm Hg respectively, and the corresponding heart rate increases were from 67 to 85, 62 to 97 and 62 to 103 beats/min respectively. One patient lost consciousness when his blood pressure was 29/19 mm Hg and one other patient experienced symptoms suggesting cerebral ischaemia.3. Following the return to horizontal, the blood pressure and heart rate usually returned to their previous values within 1 min and the blood pressure tended to overshoot slightly in the following few minutes. No immediate blood pressure overshoot occurred after the tilt except in association with a spasm of skeletal muscles.4. There was consistently a decrease in forearm blood flow, sometimes to unrecordably low levels and a venoconstriction with postural change and it is argued that these changes are the result of spinal cardiovascular reflexes to peripheral vessels. Following the return to the horizontal position, there was occasionally a large immediate increase in forearm blood flow.5. Following the intravenous administration of 3 mg atropine on two occasions, head-up tilting produced further small heart rate increases.6. Spasms of skeletal muscle occur frequently in these patients and greatly modify the effects of postural stimuli. Bladder percussion also modifies the normal response.     

Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.

Interleukin-8 (IL-8) is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonuclear neutrophils (PMNs). T lymphocytes, and basophils. Cellular sources of IL-8 include monocytes, PMNs, endothelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccharide, IL-1, and tumor necrosis factor-alpha. This report demonstrates that C5a, in addition to being a direct mediator of inflammation, can induce both IL-8 synthesis and high levels of release from monocytes. Natural human C5a and a synthetic C-terminal analogue peptide of C5a each induced IL-8 synthesis and release from CD14+ human peripheral blood mononuclear cells. Antigenic reactivity based on enzyme-linked immunosorbent assay gave evidence that IL-8 was present in the culture supernatants of stimulated peripheral blood mononuclear cells. Proof that supernatant levels of IL-8 attain biologically significant quantities was provided by human PMN chemotaxis assays. The quantity of IL-8 recovered from C5a-activated monocytes in peripheral blood mononuclear cells is up to 1,000-fold greater than that released from comparable numbers of PMNs under similar conditions. Therefore, IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at sites of infection, inflammation, or tissue injury. This observation suggests an important humoral amplification loop for inflammatory events involving both complement activation and cytokine release.     

Cardiovascular responses to partial and total immersion in man

1. Short-term cardiovascular effects of partial and total immersion of eighteen human subjects in the horizontal plane have been examined. Brachial arterial pressure, heart rate, forearm blood flow and respiratory movements were monitored simultaneously throughout the experiments. Forearm vascular resistance was calculated from the mean blood pressure and mean flow.2. Total immersion, including the face, with breath-holding resulted in a 61 +/- 43% increase in forearm vascular resistance with an associated 29 +/- 15% reduction in forearm blood flow. The concurrent bradycardia was significantly different from the heart rate changes during breath-holding with the torso only immersed, or during total immersion with snorkel-breathing. Neither breath-holding in air or with only the torso immersed, nor total immersion with snorkel-breathing produced such a diving response.3. Breath-holding, after several minutes of total immersion and snorkel-breathing, produced an attenuated diving response. It therefore appears that a full diving response can be obtained only when the apnoea commences at the moment of face immersion.4. The present investigation supports the concept that in man face immersion is an essential predisposing factor for the diving response, and cortical inhibition of the respiratory centre is important for its initiation and maintenance.     

Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum

There is now strong evidence for non-immune or inflammatory functions of complement, notably in the central nervous system. In particular, it has been recently reported that the anaphylatoxin receptors C3aR and C5aR are transiently expressed in the cerebellar cortex of newborn rat, suggesting that anaphylatoxins are involved in the histogenesis of the cerebellum. In the present study, we have investigated the effects of C3aR and C5aR agonists and antagonists on the development of the cerebellum of 11-12-day-old rats in vivo and in vitro. Sub-dural injection of C3aR and C5aR agonists at the surface of the cerebellum transiently modified the thickness of the cortical layers. The C5aR agonist provoked an enlargement of the external granule cell layer (EGL) that was due to increased proliferation of immature granule neurons. Conversely, the C3aR agonist decreased the thickness of the EGL and increased the thickness of the internal granule cell layer (IGL), suggesting that C3a accelerates the migration process of granule cells from the EGL to the IGL. Video-microscopy examination of cultured granule neurons confirmed the role of C3aR in cell motility. These results provide clear evidence for the involvement of anaphylatoxin receptors in the histogenesis of the cerebellar cortex.