CD59 (homologous restriction factor 20), a plasma membrane protein that protects against complement C5b-9 attack, in human atherosclerotic lesions.

Blood cells express a cell membrane protein, termed homologous restriction factor 20 (HRF20) and identical to CD59, that can inhibit complement C5b-9 insertion into their membranes. In this report, we investigated by immunohistochemistry whether CD59 was present on cells in human atherosclerotic lesions since membranous C5b-9(m) has been found in lesions. Using a monoclonal anti-CD59 antibody, a cellular CD59 staining pattern was apparent in nearly all lesion specimens. CD59 stain co-localised with macrophage (CD14), T lymphocyte (CD7), endothelial cell (anti-factor VIII related antigen) and smooth muscle cell cytoskeletal-specific antigens (anti-alpha actin and muscle myosin). Endothelial cells always exhibited a more intense stain than the other cell types. CD59 antigen was not localised to any one area of the lesions. Usually CD59-positive cells occurred in clusters rather than as randomly spaced individual cells. CD59 did not stain all cells of the lesion and in particular did not appear to stain all smooth muscle cells. Areas of CD59-negative cells were sometimes observed to exhibit a cellular C5b-9 staining pattern. C5b-9 deposits were also observed in CD59-positive regions. Normal saphenous vein stained strongly for CD59 at the endothelial lining and weakly in the media. Capillaries in atherosclerotic intima always stained strongly for CD59. We conclude that HRF20 is constitutively expressed on endothelium and is under regulatory control in smooth muscle cells. Cellular C5b-9 attack in atherosclerotic lesions is therefore most likely to occur on smooth muscle cells.

The apolipoprotein(a) moiety of lipoprotein(a) interacts with the complement activation fragment iC3b but does not functionally affect C3 activation or degradation.

A previous study has shown that complement component C3 binds to recombinant apolipoprotein(a) (r-apo(a)). In the present report we have investigated the interactions between lipoprotein(a) (Lp(a)), r-apo(a) and C3 in relation to complement activation and degradation. Neither Lp(a) nor r-apo(a) affected complement activation as indicated by sheep and rabbit red blood cell hemolytic assays, and by assessment of the amount of C3a generated in zymosan-activated human serum in the presence or absence of Lp(a). Crossed immunoelectrophoretic analyses indicated that Lp(a) retarded the migration of iC3b in complement-activated serum but had no effects on C3, C3b, C3c or C3dg. Recombinant apo(a) exhibited the same properties as intact Lp(a) indicating that it is the apo(a) portion of Lp(a) that mediates this effect and not the lipid moiety. Low density lipoprotein had no effect on the migration of C3 cleavage fragments. Treatment of Lp(a) or apo(a) with neuraminidase abolished their capacity to alter iC3b migration. SDS-PAGE immunoblotting analysis of C3 activation fragments generated in the presence of Lp(a) demonstrated the usual physiologic C3 cleavage fragments. Rocket intermediate gel immunoelectrophoresis of complement-activated serum demonstrated that Lp(a) did not hinder or accelerate the generation of C3c and C3dg breakdown fragments of iC3b. The results indicate that the apo(a) moiety of Lp(a) alters the migration of iC3b in an electric field but does not affect complement activation or degradation of activated C3. The sialic acid residues on apo(a) are necessary for the apo(a)-iC3b interaction.

Expression of decay-accelerating factor on synovial lining cells in inflammatory and degenerative arthritides.

The decay-accelerating factor (DAF) is a complement regulatory cell surface protein that protects cells from complement-mediated lysis. We analysed synovial tissue biopsies from patients with chronic arthritides for the presence of DAF using immunohistochemistry. DAF was expressed in the synovial lining cell layer both in rheumatoid arthritis (RA) and in osteoarthritis (OA). DAF was also on vascular endothelial cells of synovial tissue. A significant correlation was found between the expression of DAF and of HLA-DR in the lining layer, suggesting that DAF may be induced during a local inflammatory response. In addition, C5b-9 terminal complement complexes were found in several DAF-positive cases, suggesting that complement activation might, in itself, induce DAF expression. We propose that the occurrence of DAF may represent a physiological mechanism for local complement regulation in synovial tissue.

Bacterial lipopolysaccharides alter human endothelial cell morphology in vitro independent of cytokine secretion.

Lipopolysaccharides derived from six bacterial species were found to alter human endothelial cell morphology in vitro in a species-dependent and dose-dependent manner. Lipopolysaccharides derived from Salmonella enteritidis (SeLPS) induced the strongest response, whereas lipopolysaccharides from Vibrio cholerae produced no effect. Cell-shape changes induced by SeLPS (10 micrograms/ml) were noticeable by 24 hours and reached a maximum by 72 hours, thus paralleling the effects produced by the recombinant cytokines interferon gamma (IFN-gamma) (10(3) U/ml), tumor necrosis factor (10(2) U/ml), and interleukin-1 (5 to 10 U/ml). Pretreatment of human endothelial cells with IFN-gamma (10(3) U/ml) for 24 hours induced an accelerated morphologic response to subsequent SeLPS (10 micrograms/ml) stimulation and vice versa, suggesting independent pathways of action. Simultaneous treatment of human endothelial cells with SeLPS and IFN-gamma produced a more rapid onset of morphologic changes and a stronger overall effect. Culture medium containing polymyxin B inhibited cell-shape changes induced by lipopolysaccharides but not those induced by the cytokines. Conditioned medium generated over a 12-hour period following a 24-hour SeLPS/IFN-gamma stimulation did not contain measurable cytokine antigens, nor did it induce biologic responses associated with interleukin-1, IFN-gamma, and tumor necrosis factor alpha. The results indicate that lipopolysaccharides act directly on endothelial cells as well as synergistically with IFN-gamma to alter cell morphology and may, therefore, contribute to the vascular pathology of gram-negative infections.

Analysis of complement C3 activation products in human atherosclerotic lesions.

Cleavage of the complement C3 protein is essential for complement activation. Saline extracts of human atherosclerotic lesions were examined by various techniques for the presence of C3 cleavage fragments. Crossed intermediate gel immunoelectrophoresis revealed that native C3 was the predominate C3 protein in extracts and that the C3dg fragment was also detected. SDS-PAGE/Western blot analyses of lesion extracts employing monoclonal antibodies directed at C3c and C3dg fragment determinants demonstrated molecular weight bands corresponding to the known molecular weights of all the physiologic C3 cleavage fragments, except C3b which is known to have a short half-life. After C3, the two most common fragments observed were C3c and C3dg. No bands other than those corresponding to known C3 cleavage fragments were observed and control antibody stains were always negative. In some blots bands with a greater molecular mass than C3 were evident, indicating that some of the C3 in lesions may be covalently bound to an activator. We have previously identified a large (100-500 nm) nonapoprotein containing lipid particle (LCA) as a major complement activating structure in human atherosclerotic lesions. Fractionation of lesion extracts by molecular sieve chromatography and sucrose density gradient centrifugation failed to reveal a concordance between LCA and C3 antigens. The results indicate that complement activation, i.e. C3 convertase formation, takes place in human atherosclerotic lesions and that activated C3 is degraded according to normal complement regulatory mechanisms.

Immunohistochemical detection of macrophages and T lymphocytes in atherosclerotic lesions of cholesterol-fed rabbits.

Human atherosclerotic plaques contain significant numbers of T lymphocytes and monocyte-derived macrophages. Cytokines released from activated T lymphocytes induce aberrant expression of major histocompatibility complex class II (Ia) antigens by vascular smooth muscle cells and may also regulate cell proliferation and metabolism in the vessel wall. We have analyzed the arteries of cholesterol-fed rabbits to study the sequence of lymphocyte and monocyte entry into the forming atherosclerotic lesion. Rabbits were fed 0.3% cholesterol for 1-10 weeks, and monoclonal antibodies to rabbit leukocyte differentiation antigens and Ia antigen were applied to sections of the aorta. Monocytes were already observed 1 week after initiation of cholesterol feeding, and they accumulated in the intima, where they formed the bulk of the foam cell-rich lesion. T lymphocytes also adhered to the aortic surface from 1 week onward, and also accumulated in the lesion, although in lower proportions than did monocytes. In 10-week lesions, approximately 6% of cells expressed the T-lymphocyte marker L11/135. Ia antigen expression was frequent throughout the lesion in all phases of its development, and most of the Ia-expressing cells could be identified as monocyte-derived macrophages. These data indicate that the cholesterol-fed rabbit is a useful model for studying the role of monocytes and T lymphocytes in atherosclerosis.

[Paroxysmal nocturnal hemoglobinuria, a cell surface molecular defect]. / Paroxysmal nokturn hemoglobinuri en molekyldefekt på cellytan.

Paroxysmal nocturnal haemoglobinuria is an acquired haemolytic anaemia that may develop into aplastic anaemia or myeloid leukaemia. It has recently been shown that paroxysmal nocturnal haemoglobinuria is due to a defective coupling of specific proteins to glycolipids on the cell surface of haematopoietic cells. One of these proteins is decay-accelerating factor (DAF), and the absence of DAF on the surfaces of blood cells leads to the haemolytic symptoms. The molecular biology of DAF and its relationship to paroxysmal nocturnal haemoglobinuria symptoms is described in this brief review. The molecular defect of paroxysmal nocturnal haemoglobinuria is illustrated in a case report.

Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation.

Complement receptors and regulatory proteins in human atherosclerotic lesions.

Complement activation in human atherosclerotic lesions is indicated by the presence of C5b-9 terminal complexes. By using monoclonal antibodies to the complement C3b receptor (CR1) and the iC3b receptor (CR3), it was observed that approximately 20% of the cells in complicated human carotid lesions express CR1 and CR3 antigens. One to five percent of complement receptor-positive cells stained for smooth muscle cell-specific myosin, and the remainder were determined to be predominantly macrophages, based on their reactivity to anti-LeuM3 (CD14) monoclonal antibody. No C3dg receptor (CR2)-positive cells were observed in any of the eight lesions examined. The complement regulatory glycoprotein decay accelerating factor (DAF) was widely distributed extracellularly, in addition to being present on 20% to 60% of the total cell population. Factor H, a plasma protein that regulates alternative pathway C3 convertase formation, was observed extracellularly in 70% of the lesions examined. C1 inhibitor was present in a few plaque specimens, was relatively sparse, and appeared largely cell associated. Terminal C5b-9 complement complexes were pervasive in all lesions. Both the complement regulatory proteins and the activation products were limited to the area of lesion involvement and were absent from normal arterial wall. The results demonstrate that molecules involved in complement regulation and complement ligand binding are present in atherosclerotic lesions, where they may function to modulate the activities of complement.

Immune mechanisms in atherosclerosis.

To summarize, it is possible that T cell activation in the plaque has four different effects: a direct inhibition of smooth muscle proliferation mediated by IFN-gamma, an indirect stimulation of smooth muscle proliferation via IFN-induced macrophage activation, an induction of responsiveness to PDGF by induction of PDGF receptor expression, and finally, an up-regulation of HDL receptors. The net effect of T cell activation during the vascular response to injury may, therefore, depend on the balance between these mechanisms in any given situation during lesion development. T cell activation may itself be regulated by apolipoprotein E-containing LDL, which thus could form a direct link between lipoprotein accumulation and immune activation. We have recently tried to assess the effect of T cell activation during the response to experimental arterial injury with the use of a drug model. Cyclosporin A is a drug that specifically inhibits T cell activation. Rats treated with cyclosporin A for a short period had significantly smaller intimal lesions than did controls after balloon injury. This could be due to an inhibition of T cell activation, resulting in an inhibition of monocyte-macrophage activation and thereby loss of an important stimulus for intimal cell proliferation. When interpreting these results, one must, however, bear in mind that cyclosporin A could exert as yet unknown nonimmune vascular effects. It is also worth stressing that cell proliferation in the human atherosclerotic plaque may not be as high as in experimental animal lesions. In fact, cell replication may be a very rare event in the average advanced atherosclerotic plaque. Cell proliferation may, however, be associated with an episodic growth of lesions, and growth factor-mediated responses could, therefore, be important for the eventual clinical outcome in the individual patient. In conclusion, cytokines produced during the immune response affect growth and differentiation of vascular cells and could modulate both the response to injury and the local lipid metabolism in an atherosclerotic plaque There is indirect support for paracrine secretion of several of these factors in the atherosclerotic plaque, and activated T lymphocytes and macrophages are abundant in the plaque. This points to the possibility that specific immune responses are associated with the development of atherosclerosis. It is unknown, however, to what extent such immune responses occur or which antigens may elicit these responses.

Decay-accelerating factor is expressed on vascular smooth muscle cells in human atherosclerotic lesions.

Decay-accelerating factor (DAF) is a constitutively expressed plasma membrane glycoprotein on blood cells and endothelium that inhibits cell surface C3/C5 convertase formation, thus inhibiting complement activation and protecting cells from lysis by the terminal complement components. Using monoclonal anti-DAF antibodies in conjunction with anti-smooth muscle cell (SMC)-specific myosin antibodies, it was found by immunohistochemistry that vascular SMC in advanced human carotid atherosclerotic lesions express DAF antigen. The percentage of DAF-positive SMC ranged from 20 to 60% between different patient samples and SMC DAF expression was limited to SMC in the lesion proper. Normal arterial wall SMC exhibited no DAF-specific immunostaining. Essentially 100% of passaged cultured vascular SMC derived from normal human uterine artery, or from umbilical vein, expressed DAF as assessed by immunocytochemistry. A 68-kD band was observed on SDS-PAGE autoradiograms of DAF-immunoprecipitated radiolabeled cultured SMC extracts. Sensitization of rabbit erythrocytes with DAF-containing SMC extracts conferred protection against complement-mediated hemolysis in normal human serum and the protective effect could be reversed by treatment with anti-DAF antibodies. We conclude that DAF is induced on vascular SMC during atherogenesis and in culture.

Prelesional complement activation in experimental atherosclerosis. Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits.

Cholesterol accumulation in the tunica intima of large and medium sized arteries is a characteristic of atherosclerotic lesion development. Cholesterol activates the complement system in vitro generating C5b-9 complexes, and C5b-9 antigens have been observed in atherosclerotic lesions. We therefore investigated whether complement activation correlates temporally with accumulation of intimal cholesterol in vivo. Frozen sections of aortic tissue from rabbits fed a 0.3% cholesterol diet for 0, 2, 6, and 10 weeks were stained with antibodies directed against neoantigens of the C5b-9 complex. Lipids were detected with oil red O, filipin, and antibodies against apoprotein B. C5b-9 complexes were observed to colocalize with extracellular lipid in the subendothelial space beginning at 2 weeks on the diet and extending past 10 weeks. Quantitation of aorta extracted C5b-9 complexes by an enzyme-linked immunosorbent assay revealed a progressive increase in aortic C5b-9 content as a function of time on the cholesterol diet. An approximately 30-fold increase in aortic C5b-9 occurred between 0 and 10 weeks on the diet, whereas plasma C5b-9 remained at normal levels throughout this time. Aortic cholesterol and triglyceride levels approximately doubled between 0 and 10 weeks on the diet and plasma cholesterol increased nearly 50-fold. Intimal C5b-9 deposition preceded monocyte infiltration and foam cell development. The data indicates that early cholesterol accumulation in the aortic intima results in complement activation. Since complement activation products are chemotactic for monocytes and promote their adhesion to the endothelium, complement may provide a mechanism by which monocytes are attracted to arterial regions of lipid accumulation.

Immunologic control of vascular cell growth and differentiation.

Several recent studies demonstrate that T lymphocytes and monocytes enter the vessel wall during pathologic conditions and accumulate both in atherosclerotic and arteritic lesions. This brief review summarizes our phenotypic analysis of such T lymphocytes. Effects of T lymphocytes and their secretory products (lymphokines) on vascular cells are discussed on the basis of both cell culture studies and animal experiments. Finally, the role of complement activation for recruitment of blood-borne cells is evaluated.

The complement system in atherosclerosis.

Complement is a term referring to a collection of plasma proteins, specific cellular receptors and cell surface regulatory molecules. Activation of the complement system to completion results in the formation of C5b-9 terminal complexes. These complexes have been observed in human atherosclerotic lesions by immunohistochemistry. Although the structure(s) which activate complement in lesions have not been defined, cholesterol and oxysterols exhibit this property in vitro. Endothelial cell damage leads to complement activation and endothelial cells overlying atherosclerotic lesions have been observed to contain C3 and C5b-9 antigens. Cardiac myocytes stain for complement proteins (C3, C4 and C5b-9) following myocardial infarction. Infarct size and extent of inflammatory cell infiltrates are diminished by decomplementation prior to experimentally-induced myocardial ischemia. Following myocardial infarction and ulceration of atherosclerotic lesions in human patients there is an increase in circulating complement activation products and a decrease in the level of native C1 through C4 proteins. Thus, it appears that complement plays a role in atherogenesis and its sequelae. Little is known however, about the pathophysiological effects complement activation products exert on lesion development, for example through modulation of macrophage functions, or how complement activation is regulated in lesions. Implications for complement in atherogenesis are discussed.

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.

Generation of complement anaphylatoxins and C5b-9 by crystalline cholesterol oxidation derivatives depends on hydroxyl group number and position.

Cholesterol crystals activate the human alternative complement pathway. Loss of Factor B hemolytic activity in C2-deficient serum was comparable to that in normal human serum after incubation with cholesterol crystals. Consumption of Factor B hemolytic activity in normal serum incubated with cholesterol occurred in a time- and dose-dependent manner. The reduced capacity of crystals-absorbed serum to activate C2, but not Factor B, on fresh crystals, indicated that cholesterol mediates antibody-dependent classical pathway activation in addition to alternative pathway activation in whole serum. Cholestane triol, an oxidation derivative of cholesterol which bears three hydroxyl groups, cleaved 5-fold more C3 than cholesterol in normal human serum. Three cholesterol derivatives, each bearing two hydroxyl groups, were intermediate activators between cholesterol and cholestane triol. The compounds differed, however, in their complement-activating ability, indicating that hydroxyl position as well as number exerts an influence on complement activation. Measurements of C3adesArg and C5adesArg antigens in cholesterol crystal treated serum revealed that approx. 10% of total serum C3 was cleaved and that, on a molar basis, only 3% C5 cleavage occurred relative to C3 cleavage. For 1 mole of C5a generated, 0.1 moles of fluid-phase C5b-9 was detected. Although the extent of C3 cleavage varied with each cholesterol derivative depending on the position and number of hydroxyl groups, the relative coupling efficiency of C3 and C5 cleavage and C5a and C5b-9 generation was similar for all compounds. The ability of cholesterol and its oxidation products to generate anaphylatoxins and C5b-9 complexes may be of importance in mediating inflammatory processes involved in atherogenesis.

Complement activation after aortic endothelial injury.

Ischemic endothelial cell injury results in C activation that precedes partially via the classic pathway. This may be relevant to clinical situations of ischemia such as occur during embolization or thrombus formation. The generation of C activation products (such as C3a and C5a) may mediate many of the inflammatory changes observed in infarcted tissues.