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Substance P primes human neutrophil activation: a mechanism for neurological regulation of inflammation.

The neuropeptide substance P (SP), a member of the tachykinin family, has stimulatory effects on various cell types at nanomolar concentrations. SP has also direct effects on polymorphonuclear leukocytes (PMNs). However, unlike other cells, stimulation of PMNs requires extremely high concentrations of the peptide (greater than 10 microM), suggesting that direct PMN activation by SP is not physiologically relevant. By measuring primed stimulation of PMNs, we now demonstrate potent synergistic effects of nanomolar doses of SP on the migratory and cytotoxic functions of human PMNs stimulated by fMLP and C5a. This synergism between SP and chemotactic peptides reveals a new regulatory activity of SP and suggests that neurogenic stimuli may prepare neutrophils for an exaggerated inflammatory response to other phlogistic mediators.

Cell Movement↗

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

Antibodies, Monoclonal↗

Acute inflammation induced by immune complexes in the microcirculation.

The aims of the studies presented in this publication were to elucidate the morphology and quantitate the kinetics of an inflammatory reaction elicited by immune complexes and to ascertain the role of complement in the reaction. The hallmark of both the direct active (DAA) and reversed passive (RPA) Arthus reactions was the accumulation of immune precipitates and polymorphonuclear leukocytes (PMNs) in and around vessels. Using fluoresceinated antigen as a tracer, immune complexes localized in the lumina and walls of venules and small veins in the DAA and in the wall of vessels and perivascularly in RPA. PMNs accumulated at these same sites, phagocytosed the fluoresceinated complexes and became degranulated. The precise localization of immune complexes was achieved by examining the same tissue sections first by fluorescence microscopy, followed by conventional staining and examination by light microscopy. Marked stasis of the microcirculation was observed, particularly in DAA, in which a few immune complex-containing PMNs were entrapped in a mass of densely packed red blood cells. Some edema was observed in early lesions and definitive separation of collagen fibers was noted in lesions older than 2 hr. Hemorrhage became the dominant characteristic of both types of reactions from 2 hr onward. By administering radiolabeled cells, proteins, and microspheres as a "pulse," given at various times before sacrifice, the quantitation and kinetics of the inflammatory lesions elicited by immune complexes could be elucidated. In RPA all parameters quantitated reached a peak soon after elicitation of the reaction (2-4 hr), which is in keeping with other forms of acute inflammation. In DAA there was some difficulty in assessing the quantitation because of interanimal variations and because of progression of the inflammatory lesions, as the antigen diffused peripherally from the site of its injection. Peak activities occurred in 4- to 8-hr-old lesions. These observations and a comparison of the center and periphery of the lesions, strengthen the contention that the RPA and DAA have common features and features which differ. In common are immunological mechanisms (antigen-antibody interaction and complement activation) and cellular events (polymorphonuclear leukocyte chemotaxis, phagocytosis, and release of lysosomal contents). Different features are the site of immune complex formation and its sequelae. In RPA they form primarily in the wall of venules and small veins and hence have a marked effect on increase in vessel permeability. In the DAA most of the complexes form and the leukocytes accumulate in the lumen of the same vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Complement C5a (desArg) generation in serum exposed to damaged aortic endothelium.

Complement activation by injured endothelial cells was investigated using ex vivo rabbit thoracic aortas as a source of endothelium and a neutrophil aggregation (NA) bioassay to detect the complement cleavage product C5a (desArg). Endothelium, oxygen-starved by incubating aortas 30 min with Tyrode's buffer, activated complement as demonstrated by a 57% increase in the NA response induced by serum from buffer-treated aortas as compared to serum from untreated control aortas. Incubation of MgEGTA serum in injured aortas resulted in a 27% (P less than 0.025) weaker NA response than normal serum, indicating participation by both classical and alternative pathways of complement activation. Serum from aortas incubated 30 min with 100 micrograms/ml cholestane-3 beta, 5 alpha, 6 beta-triol, a cytotoxic cholesterol oxidation derivative, induced a NA response comparable to that from serum from aortas treated 30 min with Tyrode's buffer. Heat inactivation of serum prior to aortic incubation abolished NA activity and serum incubated in deendothelialized aortas lacked NA activity. Fractionation of serum samples from these experiments on Sephadex G-100 revealed a single peak of NA activity corresponding to the molecular weight of C5a (desArg). Endothelial cell injury was demonstrated by the inability to exclude Trypan blue dye and by scanning electron microscopy. These data demonstrate that damaged arterial endothelium can effectively activate the complement system, resulting in the production of an anaphylatoxic inflammatory mediator.

Animals↗

A proton nuclear magnetic resonance study of the conformation of bovine anaphylatoxin C5a in solution.

The solution conformation of bovine anaphylatoxin C5a has been investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H-NMR spectrum is assigned in a sequential manner using a variety of two-dimensional NMR techniques. A qualitative interpretation of the short range nuclear Overhauser enhancement data involving the NH, C alpha H and C beta H protons suggests that C5a has four helices comprising residues 5-11, 15-25, 33-39 and 46-61, and is composed of a globular head (residues 5-61) and a C-terminal tail. The polypeptide fold was determined by hybrid distance geometry-dynamical simulated annealing calculations on the basis of 203 approximate interproton distance restraints, 22 distance restraints for 11 intrahelical hydrogen bonds (identified on the basis of the pattern of short range NOEs and slowly exchanging backbone amide protons) and restraints for the 3 disulfide bridges. The overall polypeptide fold is similar to that of the sequence related human recombinant anaphylatoxin C5a [(1988) Proteins 3, 139-145].

Amino Acid Sequence↗

Different sensitivities of the responses of human neutrophils stimulated with immune complex and C5a anaphylatoxin to pertussis toxin.

When stimulated with immune complex or C5a anaphylatoxin, human neutrophils undergo an increase in the concentration of intracellular Ca2+ [( Ca2+]i) that precedes the onset of superoxide (O2-) production. The extracellular Ca2+ is required for the O2- production of neutrophils stimulated by C5a, but is only partially required for those by immune complex. The addition of pertussis toxin to neutrophils does not inhibit the rise in [Ca2+]i and O2- production induced by immune complex but does inhibit those induced by C5a. These results suggest that a different sequence of reaction is involved by different stimulants, the anaphylatoxin and immune complex.

Antigen-Antibody Complex↗

Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver.

The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.

Anaphylatoxins↗

Role of complement in thrombogenesis. I. Complement-dependent coagulation in a model system.

Chondroitin sulphate was used to isolate from plasma a system that clotted with Russell's viper venom, brain extract and activated contact factors. Clotting appeared to depend on concomitant change in C4, C3 and C1s in the system. Brain extract additionally reacted with C9. Reconstitution of specifically defective plasmas suggested a specific role for each of these complement components in clotting.

Antigen-Antibody Reactions↗

Detection of the terminal complement complex in patient plasma following acute myocardial infarction.

The mechanisms of inflammation responsible for the myocardial tissue damage seen after an acute myocardial infarction (AMI) have not been clearly identified. Recent lines of evidence, demonstrating depressed sera levels of individual complement components in patients after myocardial infarction, have suggested involvement of the complement (C) system in micro- and macrovascular injury subsequent to AMI. The present study assessed the role of complement as a mediator of myocardial inflammation by quantifying products of complement activation including, the terminal complement complex (TCC) the cytolytic component of the complement system, C1rC1s-C1 inhibitor complex and C3bBbP complex, formed following activation of the classical and alternative pathway, respectively, and anaphylatoxins C3a and C5a in 41 patients following AMI. Plasma TCC and C1rC1s-C1 inhibitor complex concentrations increased up to 32-fold (P less than 0.001) and 8-fold (P less than 0.001), respectively, while the C3bBbP complex, C3a des-Arg and C5a des-Arg each increased over 2-fold (P less than 0.001) 16 h after AMI, and were only minimally detectable during non-inflammatory myocardial conditions. Furthermore, TCC concentrations increased over 150% (P less than 0.001) one day after patients reinfarcted, subsequent to hospitalization for a primary AMI. These results demonstrate activation of complement after AMI and suggest that inflammatory mediators of the complement system may contribute to myocardial tissue damage during the infarction process.

Adult↗

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗

Purification of human C5a des arg by immunoadsorbent and molecular sieve chromatography.

Human C5a des arg was isolated from complement-activated serum by immunoadsorption followed by Sephadex G-75 chromatography. C5a des arg obtained by this 2-step procedure was shown to be immunologically identical to C5a des arg purified by a conventional multi-step method, homogeneous on SDS-polyacrylamide gels, and biologically active. Although this technique yields approximately the same amount of C5a des arg/liter of activated serum as that obtained by conventional methods, its simplicity and relative rapidity make it a practical alternative.

Animals↗

Electroimmunoassay-immunoblotting (EIA-IB) for the utilization of monoclonal antibodies in quantitative immunoelectrophoresis: the method and its applications.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis-immunoblotting.

Animals↗