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PubMed1986

Microvascular deposition of complement membrane attack complex in dermatomyositis.

We examined the role of the complement system in the pathogenesis of dermatomyositis. Using an antibody against the neoantigens of the terminal C5b-9 membrane attack complex, we performed immunocytochemical studies that localized this complex to the intramuscular microvasculature (arterioles and capillaries) of muscle biopsy specimens from 10 of 12 patients (83 percent) with childhood dermatomyositis and 5 of 19 patients (26 percent) with adult dermatomyositis. Fifty-two control specimens, including 14 from patients with polymyositis and 12 from patients with denervation atrophy (a condition known to be associated with necrotic capillaries), showed no deposition of membrane attack complex in the microvasculature. These findings indicate that the complement system is deposited, bound, and activated to completion within the intramuscular microvasculature of patients with dermatomyositis. In addition to providing further evidence for the presence of vasculopathy in dermatomyositis, these findings suggest a primary role for complement in mediating vessel injury in the disease, particularly in its childhood form.

Adult↗
PubMed2001

In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis.

BACKGROUND: Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism(s) of action. METHODS: Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined by SC5b-9 enzyme-linked immunoassay. RESULTS: SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [Na+]i without affecting [K(+)](i) in fast-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+). CONCLUSIONS: Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.

Animals↗
PubMed1973

Lambda-chain production in human lymphoblast-mouse fibroblast hybrids.

Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity were hybridized with thymidine kinase (EC 2.7.1.21)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (lambda) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human lambda chain, and continued to do so after prolonged culture. As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or alpha(1)-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive lambda-chain reactivity, suggesting that the kinase and lambda-chain loci are not linked. The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system.

Animals↗
PubMed1976

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.

Cell Membrane Permeability↗
PubMed1978

Electron spin resonance studies on interaction of complement proteins with erythrocyte membranes.

Sheep erythrocytes have been spin labeled with 5-, 12-, and 16-nitroxystearic acid in order to investigate complement-induced changes in the physical state of the lipid bilayer. Formation of osmotic lesions in the membrane causes an increase in the fluidity of the membrane which overcomes the decrease in membrane fluidity caused by the interaction of the complement proteins. A decrease in membrane fluidity is observed only when complement-lysed membranes are resealed or when complement proteins react with isosmolar ghosts that do not undergo osmotic lysis. The decrease in bulk fluidity of the membrane is first observed when C8 binds to the membranes bearing C5b67 and is enhanced upon the subsequent binding of C9. The decrease in membrane fluidity shown by the electron spin resonance spectra of spin-labeled fatty acids suggests that certain of the complement proteins penetrate the membrane and interact with hydrophobic regions of the lipid bilayer.

Animals↗
PubMed1978

Steady-state analysis of tracer exchange across the C5b-9 complement lesion in a biological membrane.

Resealed erythrocyte ghosts have been used to define the kinetics of tracer exchange across the membrane-bound terminal complex of the complement cascade (C5b-9). Under steady-state conditions and at net chemical equilibrium, C5b-9 ghosts showed no significant lysis above control levels as measured by hemoglobin efflux. In 1 mM sucrose at 37 degrees C, [14C]sucrose isotopic exchange diffusion into C5b-9 ghosts occurred at 4.8 (+/- 0.5, SEM) X 10(-20) mol sec-1 per functional lesion, equivalent to an apparent permeability coefficient of 4.8 X 10(-14) cm3 sec-1 for the single C5b-9 lesion. No significant uptake of [14C]sucrose above control levels was observed in C5b67 ghosts. The apparent rate of tracer permeation through the complement lesion is one to two orders of magnitude slower than predicted by a model of a transmembrane channel of dimensions permitting free diffusion of sucrose. The data support earlier assertions from this laboratory that diffusion of small molecules across the complement lesion in biological membranes is significantly restricted.

Biological Transport↗
PubMed1979

Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

Binding Sites↗
PubMed1981

Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

To evaluate the effect of membrane lipid acyl-chain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4, or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles.

Complement C5↗
PubMed1981

Platelet factor 4 is chemotactic for neutrophils and monocytes.

Platelet factor 4 is shown to be a chemotactic protein for human polymorphonuclear leukocytes and monocytes at concentrations found in human serum and reached locally in injured tissue. The maximum chemotactic response to platelet factor 4 nearly equals that achieved with saturating concentrations of the chemotactic activity derived from the fifth component of human complement, C5. Cells desensitized to C5 chemotactic activity retain chemotactic responsiveness to platelet factor 4. Serum contains inhibitory capacity against the chemotactic activity associated with platelet factor 4. Our results suggest that the local release of platelet factor 4 may be an important stimulus attracting inflammatory cells to sites of blood vessel injury.

Blood Coagulation Factors↗
PubMed1981

Homologous species restriction in lysis of erythrocytes by terminal complement proteins.

The cytolytic efficiency of the terminal complement protein complex, C5b-9, varies with the species of origin of C8 and C9. In the present study, we explored the susceptibility of erythrocytes from various species to lysis by C5b6,7 plus C8 and C9 from different species. EC5b6,7 intermediates were prepared on human, guinea pig, rabbit, mouse, and rat erythrocytes with human C5b6 and guinea pig C7. The degree of lysis of these intermediates by C8 and C9 was found to vary widely depending on the species of the proteins and the target cells. In all cases, lysis was least efficient when C8 and C9 were homologous with respect to the target cell species. This effect was mostly attributable to C9. The inefficient lysis in a homologous system is not due to a failure of C9 binding. Rather, the poor lysis in the homologous system may be attributable to inefficient insertion or channel formation.

Animals↗
PubMed1982

Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.

Animals↗
PubMed1983

Arachidonate metabolism by human polymorphonuclear leukocytes stimulated by N-formyl-Met-Leu-Phe or complement component C5a is independent of phospholipase activation.

Release of arachidonic acid by the membrane phospholipase and metabolism by the 5-lipoxygenase pathway was examined in human polymorphonuclear leukocytes (PMNs). The 5-lipoxygenase pathway is activated when PMNs are given arachidonic acid in ethanol and there is extensive metabolism to 5-hydroxyicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). This activation event was shown to be altered by the ethanol because resting PMNs given arachidonic acid with bovine serum albumin fail to metabolize arachidonic acid. However, cells activated by the inflammatory agents N-formyl-Met-Leu-Phe (fMLF) or complement component C5a recruit the 5-lipoxygenase to metabolize exogenous arachidonic acid to 5-HETE and LTB4. When PMNs were incubated with arachidonic acid-bovine serum albumin and challenged with fMLF or C5a (des-Arg-C5a) they produced 49-75 pmol of LTB4 and 310-440 pmol of 5-HETE per 10(7) cells. PMNs stimulated by fMLF or C5a (des-Arg-C5a) do not induce membrane phospholipases to mobilize endogenous arachidonic acid and neither 5-HETE nor LTB4 is formed. In contrast, PMN stimulation by the ionophore A23187 activates both the membrane phospholipase and the 5-lipoxygenase to produce 5-HETE and LTB4 from endogenous arachidonic acid. Our results indicate that the lipoxygenase pathway is inoperative in resting PMNs but can be recruited by chemotactic factors to act on arachidonate from extracellular sources. It was previously believed that formation of 5-HETE and LTB4 by the PMN depends solely on phospholipase to mobilize endogenous arachidonic acid. The results reported here refute this concept and indicate that the role of phospholipase activation in PMN may be overestimated. Therefore, subsequent involvement of lipoxygenase products in mediating stimulation of PMN by inflammatory factors (e.g., as in aggregation and chemotaxis) remains in question unless an exogenous source of arachidonate can be identified.

Arachidonic Acid↗
PubMed1984

Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.

The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximately equal to 20 nM), with both C5a and ionomycin, cytosolic Ca2+ increases to less than 200 nM, and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O-2 when exposed to ionomycin but not to C5a. These results and other considerations suggest that (i) activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM; (ii) C5a receptor-mediated activation of Ca2+ influx may require cooperation between the neutrophil surface and some cytoplasmic organelle and/or redistribution of the C5a-receptor complexes on the cell surface; and (iii) the phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.

Animals↗
PubMed1985

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

Animals↗
PubMed1988

Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations.

Two-dimensional 1H NMR investigations were used to locate elements of regular secondary structure in the human complement protein C3a (the des-Arg77 derivative) in solution. The results were compared to a refined crystal structure based on the 3.2-A resolution structure of des-Arg77-C3a [Huber, R., Scholze, H., Paques, E. P. & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399]. In excellent agreement with the x-ray data, helices occur in the regions of residues 17-28 and 36-43 in solution. In contrast to the x-ray data, where a third long helix was found from residue 47 to residue 73, the solution data show a shorter helix in the region from residue 47 to residue 66, followed by a transition range at positions 67-70, leading into a six-residue carboxyl-terminal peptide in dynamic random coil conformation. At the amino terminus, a well-defined helix is observed in solution for the residues 8-15 region, which, like the carboxyl terminus, gradually changes to dynamic random coil toward the end of the polypeptide chain. This is at variance with the x-ray data as well, in which residues 13-15 are nonhelical and no electron density could be assigned to the first 12 residues due to disorder.

Anaphylatoxins↗
PubMed1994

Mapping of the C5a receptor signal transduction network in human neutrophils.

Human neutrophils respond to chemoattractants, resulting in their accumulation at an inflammatory site. Chemoattractants such as the C5a peptide, derived from the C5 complement factor, bind to inhibitory guanine nucleotide binding protein (Gi)-coupled seven membrane-spanning receptors expressed in neutrophils. C5a receptor activation results in the Gi-dependent activation of the mitogen-activated protein (MAP) kinase pathway in human neutrophils. C5a receptor ligation activates both B-Raf and Raf-1, with B-Raf activation overlapping but temporally distinct from that of Raf-1. B-Raf and Raf-1 both efficiently phosphorylate MAP kinase kinase (MEK-1). C5a also stimulates guanine nucleotide exchange and activation of Ras. Ras and Raf activation in response to C5a involves protein kinase C-dependent and -independent pathways. Activation of both Raf-1 and B-Raf was inhibited by protein kinase A stimulation, consistent with the inhibitory effects of increased cAMP levels on neutrophil function. The findings define a functional signal transduction pathway linking the neutrophil C5a chemoattractant receptor to the regulation of Ras, B-Raf, Raf-1, and MAP kinase.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMedDate not supplied

Effects of cigarette smoking on blood levels of leukotrienes and plasma levels of complements C3a and C5a in healthy volunteers.

We reported previously that cigarette smoking induces bronchoconstriction. In the present investigation we measured the level of peripheral venous blood leukotrienes, which are known to cause marked bronchoconstriction, and plasma complement C3a and C5a, which are known to have chemotactic and anaphylatoxic activities. All of these showed a significant increase after cigarette smoking. These data may suggest that the activation of complement induces the release of leukotrienes, and that these leukotrienes cause bronchoconstriction. It appears likely, therefore, that cigarette smoking induces an increase in peripheral venous blood leukotrienes and complement C3a and C5a, and that such an increase may elicit bronchoconstriction.

Adult↗