Search

Concentration and preferential retention of immunoglobulins and complement components were studied in comparison with other plasma proteins in 42 human aortae with atherosclerosis. Saline and acid extracted IgG, IgA, IgM, C1q, C3c, C4, C9, C3A, C-reactive protein, alpha 1-antitrypsin, alpha 2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined using the radial immunodiffusion. The fibrous plaques and their adjacent areas contained higher levels of each protein than intima with only fatty streaks. No significant differences were found between the fibrous plaques and their adjacent areas presenting intimal thickenings. Saline eluted IgG and IgA were significantly higher in the fibrous plaque intima than in intimal samples with fatty streaks and were the only proteins detected in the acid eluates. The complement components were present in all saline eluates, while C-reactive protein was found in 23 samples. Crossed immunoelectrophoretic studies showed the activation of saline C3 and C4. In 8 cases serum levels of the studied proteins were compared with their concentration in saline eluates obtained from intima and media. The immunoglobulins and complement components presented higher intima/serum and lower media/intima retention ratios than the other studied proteins suggesting their preferential retention in the intima. The presence of immune related proteins in the atherosclerotic intima and their preferential retention might be explained not only by an altered permeability but also in relation to their function.

The co-localization of terminal C5b-9 complement complexes and macrophages was investigated in human arteries with atherosclerosis using a double-labeling immunohistochemical technique. Macrophages were found in all the atherosclerotic arteries, with the accumulation correlating positively with the degree of atherosclerosis. This accumulation was associated with an increase of C5b-9 deposits, as well as with an increase in the number of deposits containing both complement components and macrophages ('co-localization'). This co-localization was found to pertain both to intact macrophages and to macrophage remnants. These data suggest that C5b-9 complement complex might be formed on macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.

The relationship between macrophages and the terminal C5b-9 complement complexes was investigated in human arteries affected with atherosclerosis by using monoclonal antibodies and indirect immunoperoxidase, immunogold silver staining, and double-labeling immunohistochemical techniques. Macrophages were found in all the atherosclerotic arteries as immunoreactive deposits with a nucleus, considered as intact cells, or without a nucleus, considered as cell remnants. The double-labeling technique shows C5b-9 deposits partially colocalized on the intact macrophages or on the cell debris of macrophage origin. These data suggest that C5b-9 complement complex may be formed on activated or dying macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.

The terminal C5b-9 complement complex was investigated in 15 aortic, 2 femoral fibrous plaques and 5 fatty streaks aortic intimae using indirect immunofluorescence and immunoperoxidase. All the fibrous plaques presented C5b-9 deposit-like threads in the fibrous cap and masses in the amorphous areas of the plaque. The deposits were frequently associated with other immune-related proteins such as: IgG, IgA, IgM, Clq, C3c and C4 which were simultaneously investigated. Fatty streaks intimae presented no C5b-9 and complement component deposits. Whereas the demonstration of the complement components could merely reflect a non-specific trapping, the presence of assembled C5b-9 in the damaged tissue is more indicative of the involvement of complement activation in the progression of atherosclerotic lesions.

The terminal C5b-9 neoantigens of the complement complex, S-protein (Vitronectin), C3c, C3d and apolipoprotein B were localized on 16 aortic fibrous plaques, 8 aortic intimal thickenings, 4 fatty streaks intimae, 12 coronary fibrous plaques, 3 coronary intimal thickenings, 6 femoral and 5 basilar fibrous plaques, using an indirect and double-staining immunoperoxidase method. The granular specific deposits were localized in the fibrous cap and deeper parts of the plaque or in the deeper intima and inner-third media of intimal thickenings and fatty streaks intimae, in relation to the degree of atherosclerotic involvement. The different localization of C5b-9 and S-protein demonstrated by the double-staining technique is more suggestive for the assembly of the complex into the arterial wall and not for its preformed passage from circulation. The relation of these immune deposits to the degree of fibrosis and necrosis and their presence from the initial stages through to the advanced lesions could ascribe a role to the complement system in atherosclerosis.

The concentrations of IgG, IgA, IgM, Clq, C3c, C4, C9, C3A, Albumin, Transferrin, Alpha-1-antitrypsin, Alpha-2-macroglobulin were determined in the serum, aortic atherosclerotic intima and media of 8 patients. The protein levels in the intima were dependent on their serum concentration. The passage of proteins from serum to media was investigated using the ratios between their intima/serum and media/intima concentrations. Immunoglobulins and complement components displayed higher intima/serum and lower media/intima ratios than the other proteins suggesting a preferential retention of the immune related proteins in the intima. This trapping into the intima seems to be related to their function too, suggesting a certain involvement in the progression of the atherosclerotic lesions.

Saline and acid eluates of intima with only fatty streaks, fibrous plaque and intima surrounding the fibrous plaque were obtained from 42 human aortae. IgG, IgA, IgM, C1q, C3c, C4, C9, C3A, C-reactive protein, alpha-1-antitrypsin, alpha-2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined in these eluates using the radial immunodiffusion technique. Saline extracted IgG and IgA were significantly higher in the fibrous plaque and adjacent tissue than in the fatty streak intima, where IgM was the highest. IgG and IgA were only present in acid eluates. The complement components were present in all saline eluates, but only traces of C3c and C1q in some acid eluates. Statistically significant differences were found for C1q and C9. C-reactive protein was present in 23 samples. The results could suggest an involvement of immune mechanisms in the progression of the atherosclerotic lesion.

The terminal C5b-9 complex of the complement system was localized in 26 aortic, 3 iliac and 4 femoral human fibrous plaques using indirect immunofluorescence and immunoperoxidase. IgG, IgA, IgM, Clq, C3c, C4, C9 and fibrinogen were investigated simultaneously. All the fibrous plaques presented C5b-9 deposits appearing like thin threads in the fibrous cap and masses and spots in the amorphous areas. The extent and intensity were in agreement with the size of the fibrous plaques. The intimal thickenings presented less intense deposits which were absent in atherosclerosis-free samples. The C5b-9 deposits were frequently associated with immunoglobulins and complement components in the same areas. Whereas the demonstration of complement components reflected only a nonspecific trapping, the presence of assembled C5b-9 in the damaged tissues is more indicative of the involvement of complement activation in the tissue injury. The absence of C5b-9 in the atherosclerosis-free intima and its presence at lower intensity in the intimal thickenings than in the fibrous plaques suggest a pathogenic involvement in the chronic progression of the atherosclerotic lesion.

The assembly of the terminal C5b-9 complement complex is a prime mechanism of complement-induced membrane damage followed by inflammatory response mediation and subsequent extensive tissue damage. In the assembly process the terminal complement components expose neoantigenic determinants which can be recognized by specific antibodies. Using such a specific antibody, affinity-purified rabbit IgG and by means of immunoelectron microscopy, the C5b-9 neoantigens were localized on the structures of the human fibrous plaque from 3 iliac and 3 femoral arteries obtained at surgery. The immunoelectron-dense deposits were localized on the cell debris, enmeshed in the connective tissue matrix, consisting of irregular particles that frequently had the shape and size of intracellular organelles or vesicles with concentric osmiophilic lamellae. No deposits could be found on the intact cells, on the connective tissue matrix or on cholesterol and lipid deposits. The presence of C5b-9 neoantigens deposits in the fibrous plaques frequently associated with other immune-related proteins indicates that complement activation has occurred in situ and could be related to the chronic progression of the atherosclerotic lesion.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

The indirect and double-labelling immunoperoxidase techniques were used to localize the C5b-9 neoantigens, S-protein and macrophage immunoreactive deposits in myocardial areas with necrosis and sclerosis. Granular masses of C5b-9 neoantigens, diffuse areas of S-protein and nucleated and anucleated immunoreactive deposits of macrophage origin were localized in damaged myocardial areas but not in areas free of lesion. The presence of C5b-9 neoantigens associated to S-protein and macrophage deposits is suggestive for a local complement activation in the complex heterolytic events of myocardial injury.

A quantitative ELISA using monoclonal and polyclonal antibodies against neoantigens of the terminal C5b-9 complement complex was used to evaluate the presence of terminal complexes in 68 human arterial samples with or without atherosclerotic involvement. Plasma levels of SC5b-9 were directly compared with the corresponding levels eluted from the femoral arteries in six patients undergoing surgical procedures. The plasma concentration of SC5b-9 in these donors was in the range of 30-90 arbitrary units (AU)/ml, equivalent to 100-300 ng/ml SC5b-9 or 45-130 AU/100 mg plasma protein. All the arterial samples contained detectable amounts of C5b-9. The aortic normal and fatty streaks intimae presented a minimum mean value of 65 +/- 12 AU/100 mg total protein, in the range of normal plasma SC5b-9 levels. The corresponding media contained significantly higher amounts of terminal complexes (115 +/- 30 AU/100 mg protein). Markedly increased levels of C5b-9 were eluted from aortic intimal thickenings (350 +/- 100 AU/100 mg protein) and the corresponding media (300 +/- 53 AU/100 mg protein). Similar concentrations were found in aortic fibrous plaques (340 +/- 80 AU/100 mg protein). The observed correlation between C5b-9 levels and atherosclerotic alterations in arterial walls is suggestive of chronic complement activation with involvement of the terminal complement sequence at these sites. These processes may contribute to progression of the arteriosclerotic lesions.

Myocardial fragments with acute infarction (10 cases), scars after chronic infarction (6 cases), areas with focal sclerosis and necrosis (8 cases) compared with normal myocardial areas (8 cases), were processed for indirect and double-labelling immunoperoxidase techniques to localize C5b-9 neoantigens, S-protein, C3d and apolipoprotein B. Granular masses of C5b-9 and C3d and diffuse areas of S-protein and apolipoprotein B were localized in the acute or chronically damaged areas but not in areas free of lesion. Double-labelling data revealed similarly damaged areas of localization for C5b-9 and S-protein, and for C3d and apolipoprotein B, respectively, on rather different than usual tissue structures. C5b-9 determination by ELISA from myocardial eluates revealed lower levels of neoantigens in normal areas (2.3 +/- 0.3 micrograms/g dried tissue), higher levels in areas with sclerosis (7.9 +/- 0.7 micrograms/g dried tissue) and the highest amounts in areas with acute infarction (11.1 +/- 1.2 micrograms/g dried tissue). The presence of C5b-9 neoantigens in damaged myocardial areas with a different localization than S-protein is suggestive of local complement activation.