Fibronectin and C4-binding protein are selectively bound by aggregated amyloid P component.

Serum amyloid P component (SAP) is a normal plasma protein, closely related to C-reactive protein, which is deposited together with amyloid fibrils in all forms of amyloidosis. It is also a normal constituent of human tissues, where it is found in vascular basement membranes and in association with the peripheral microfibrillar mantle of elastic fibres throughout the body. Very similar, highly conserved, homologous proteins are present in the sera of all vertebrates in which they have been sought, and in all cases these proteins display calcium-dependent binding affinity for agarose. The physiological function or pathogenetic significance of this reactivity are not known but we report here for the first time that under appropriate conditions human SAP can also bind certain serum glycoproteins. SAP, which had been aggregated either by direct conjugation to CNBr-activated Sepharose beads, or by complexing with anti-SAP antibodies immobilized on such beads, selectively took up fibronectin and C4-binding protein from whole normal human serum. The reaction was calcium dependent and the two ligands were bound independently of each other or of other serum constituents. Experiments with isolated fibronectin and SAP complexed by anti-SAP-Sepharose indicated that close association of pairs of SAP molecules was required for fibronectin to be bound and that each SAP dimer was capable of taking up a single molecule of fibronectin. There was no evidence that SAP in its native state in the serum was complexed with either fibronectin or C4-binding protein. The present findings significantly extend knowledge of the properties of SAP and open the way to characterisation of its physiological ligand(s) and thence to elucidation of its function.

The acute-phase response in (NZB X NZW)F1 and MRL/l MICE.

The acute-phase plasma protein response to disease activity in murine models of autoimmune lupus-like disease was investigated by measurement of the concentration of serum amyloid P component (SAP) in NZB X W and MRL/l mice. The levels of SAP, which is a major acute-phase protein in mice, did not rise at all in response to progression of disease in NZB X W mice between the ages of 1 and 9 mo. This resembles the behavior of acute-phase proteins such as C-reactive protein and serum amyloid A protein in human systemic lupus erythematosus, and just as in human lupus, where the occurrence of intercurrent microbial infection can stimulate an acute-phase response, so injection of bacterial lipopolysaccharide or casein into the NZB X W mice stimulated "normal" acute-phase SAP production. In marked contrast, MRL/l mice developed greatly increased levels of SAP, which correlated closely with progression of their pathology as they aged. The disease profile of the MRL/l strain includes rheumatoid factors and spontaneous polyarthritis and their SAP response resembles the behavior of acute phase proteins in human rheumatoid arthritis. Different patterns of acute-phase response in different autoimmune disorders may thus be a reflection of the genetic predisposition to particular diseases and/or contribute to their pathogenesis. The existence of animal counterparts for the various clinical patterns of human acute-phase protein production will assist in experimental investigation of the underlying mechanisms and of the biological role of the acute-phase response.

Binding specificity of serum amyloid P component for the pyruvate acetal of galactose.

Serum amyloid P component (SAP) is a normal plasma protein that is of interest because of its presence in amyloid deposits, its presence in normal human glomerular basement membrane, and its stable evolutionary conservation. It has calcium-dependent ligand-binding specificity for amyloid fibrils, fibronectin (Fn), C4-binding protein (C4bp), and agarose. Although the binding to agarose, a linear galactan hydrocolloid derived from some marine algae, is unlikely per se to be related to the physiological function of SAP, it does provide a model system in which to explore the precise ligand requirements of SAP. We report here that the amount of SAP from human, mouse, and plaice (Pleuronectes platessa L.) serum able to bind to agarose from different sources reflect precisely their pyruvate content. Methylation with diazomethane of the carboxyl groups in the pyruvate moiety of agarose completely abolishes SAP binding to agarose. The pyruvate in agarose exists as the 4,6-pyruvate acetal of beta-D-galactopyranose. We have therefore synthesized this galactoside, using a novel procedure, established its structure by analysis of its nuclear magnetic resonance spectra, and shown that it completely inhibits all known calcium-dependent binding reactions of SAP. The R isomer of the cyclic acetal, methyl 4,6-O-(1-carboxyethylidene)-beta-D-galactopyranoside (MO beta DG) was effective at millimolar concentration and was more potent than its noncyclic analogue, while pyruvate, D-galactose, and methyl beta-D-galactopyranoside were without effect. The autologous protein ligands of SAP presumably, therefore express a structural determinant(s) that stereochemically resembles MO beta DG. Availability of this specific, well-characterized, low molecular weight ligand for SAP should facilitate further investigation of the function of SAP and its role in physiological and pathophysiological processes.

Amyloid P-component is a constituent of normal human glomerular basement membrane.

Glomerular and other vascular basement membranes were found to contain an antigen that was immunochemically indistinguishable from serum amyloid P-component. There was no immunological cross-reactivity between antisera to serum amyloid P-component and to collagen types I, III, IV, or V. The amyloid P-component antigen was confined to the endothelial aspect, the lamina rara interna, of glomerular basement membrane. It could not be eluted by high-ionic-strength saline, EDTA, dithiothreitol, or either polar or nonpolar detergents, but was released into solution when isolated glomerular basement membrane was digested by highly purified bacterial collagenase. Most of these P-component molecules and their constituent polypeptide chains were of higher molecular weight and lower isoelectric point than serum amyloid P-component. These findings indicate that, as well as being a normal plasma protein and a universal constituent of amyloid deposits, P-component is also a normal matrix glycoprotein of basement membrane in which it is covalently linked to collagen and/or other matrix proteins. This may be relevant both to the pathogenesis of amyloidosis and to other aspects of physiology and pathology of basement membranes.

Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins.

Immobilized rabbit and rat C-reactive protein (CRP) were found to selectively bind apolipoprotein B (apoB)-containing lipoproteins (low density lipoprotein, LDL and very low density lipoprotein, VLDL) from whole serum in a manner similar to that previously reported with human CRP. In acute phase human serum the CRP is in a free form, not complexed with lipoprotein or any other macromolecular ligand, and in acute phase serum from most rabbits fed on a normal diet the rabbit CRP was also free. However, in acute phase serum or heparinized plasma from hypercholesterolemic rabbits part or all of the CRP was found by gel filtration and immunoelectrophoretic techniques to be complexed with beta-VLDL, an abnormal apoB-containing plasma lipoprotein present in these animals. The presence of extent in different serum samples of CRP complexed with lipoprotein correlated closely with the serum apoB concentration. The formation of complexes between native, unaggregated rabbit CRP in solution and apoB-containing lipoproteins was readily demonstrable experimentally both with the isolated proteins and in whole serum. In all cases these interactions were calcium-dependent and inhibitable by free phosphoryl choline. The present findings extend earlier work in man and the rabbit and indicate that among the C-reactive proteins from different species, which are structurally highly conserved, the capacity for selective binding to apoB-containing plasma lipoproteins is also a constant feature. These interactions may therefore be related to the in vivo function of CRP in all species and this function may in turn be relevant to pathological conditions, such as atherosclerosis, in which lipoproteins are important.

Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein.

The acute phase response among plasma proteins is a normal response to tissue injury and is therefore a fundamental aspect of many diverse disease processes. It probably usually has a beneficial net function in limiting damage and promoting repair but in some circumstances it may have pathological consequences. Sustained high levels of acute phase proteins and especially SAA are associated with the development of amyloidosis in some individuals. Increased concentrations of CRP may, by activating the complement system, contribute to inflammation and enhance tissue damage. Failure of the normal or appropriate CRP response may also possibly have deleterious effects. SAA is a polymorphic protein which is normally present only in trace amounts but which, during the acute phase response, becomes one of the major apolipoproteins associated with high-density lipoprotein particles. The function of apoSAA is not known but it must have considerable physiological significance apart from its role as the putative precursor of amyloid A protein fibrils. CRP and SAP have been very stably conserved throughout vertebrate evolution and homologous proteins are apparently present even in vertebrates. This strongly suggests that they have important functions although these have not yet been precisely delineated. The main role of CRP may be to provide for enhanced clearance of inappropriate materials from the plasma whether these are of extrinsic origin, such as microorganisms and their products, or the autologous products of cell damage and death. The interaction between aggregated CRP and plasma low-density lipoprotein may play a significant part in the normal function of CRP and may also have a role in lipoprotein metabolism, clearance, and deposition. SAP is a normal tissue protein as well as being a plasma protein. Aggregated SAP selectively binds fibronectin and this may represent an aspect of the normal function of SAP. The deposition of SAP in amyloid is evidently not a normal function but it is not known whether this deposition is involved in the pathogenesis of amyloid or whether it is merely an epiphenomenon. In any case immunohistochemical staining for SAP is useful in the diagnosis of amyloid, in investigation of glomerulonephritis, and in studying disorders of elastic tissue. Regardless of its physiological or pathophysiological functions, the assay of serum CRP is a valuable aid to clinical management in a number of different situations and in different diseases provided results are interpreted in the light of full clinical information.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-dependent aggregation of human serum amyloid P component.

Normal human serum or isolated human amyloid P component was ultracentrifuged on density gradients containing either 10 mM EDTA or different concentrations of Ca2+ between 0.15 and 2.15 mM. In the presence of Ca2+ concentrations of 1 mM or more human P component sedimented more rapidly than it did in the presence of lower Ca2+ levels or of EDTA. This phenomenon was due to Ca2+-dependent aggregation of P component molecules and did not require the presence of any other serum constituents. It was completely inhibited by incorporating a physiological concentration (40 mg/ml) of serum albumin in the gradients, suggesting that free ionized Ca2+ is required to promote aggregation of the P component. P component from the mouse and the plaice (Pleuronectes platessa L.), a marine teleost, did not undergo the same Ca2+-dependent aggregation as human P component. These observations resolve a discrepancy existing in the literature concerning the sedimentation rate of human P component in density gradient ultracentrifugation and shed new light on its behaviour with respect to Ca2+ which may be relevant to the deposition of P component in amyloidosis.

Isolation and characterisation of goat C-reactive protein.

A pentraxin was isolated from acute phase goat serum by its calcium-dependent affinity for agarose, and although it did not bind to phosphorylcholine immobilised on Sepharose, its binding to agarose was reversed by exposure to fluid phase phosphorylcholine. It was identified as goat C-reactive protein on the basis of its immunochemical cross-reactivity with human and bovine C-reactive protein. The molecule was composed of five identical, glycosylated, non-covalently associated subunits, each of molecular weight approx. 24,000. Acute phase serum levels in a small number of samples were not significantly different from normal levels (means 72 and 55 micrograms/ml, respectively), suggesting that goat C-reactive protein is not a major acute phase reactant. No other pentraxin was detected in goat serum.

Epileptic discharges produced in monkeys by injection of spleen cells from rabbits immunised with monkey brain.

Cell suspension from the spleen of rabbits immunised with monkey brain gave rise to epileptic discharges in the monkey when injected intracortically, except in the case of one injection from a rabbit not recently given a booster treatment. In contrast, cell suspensions from the spleen of unimmunised rabbits, or of a rabbit immunised with a non-brain material, in all cases failed to be effective. The epileptic discharges began about 2 weeks after injection, were variable in frequency, and lasted throughout the period of recording.

Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response.

A sensitive and precise immunoassay for equine serum amyloid A protein (SAA) was established and used to determine, for the first time, the circulating concentration of this protein in health and disease. As in other species, equine SAA was present only at trace levels in healthy animals but behaved as an extremely sensitive and rapidly responding acute phase reactant following most forms of tissue injury, infection and inflammation, objectively reflecting the extent and activity of disease. Measurements of SAA should make a significant contribution to diagnosis and management of viral and bacterial infection in horses, and routine serial assays could provide an objective criterion for monitoring prospectively the general health of horses in training and racing.

C-reactive protein in dogs.

Serum concentrations of C-reactive protein (CRP) in dogs with various diseases or undergoing various procedures were measured by specific immunoassay. In 20 healthy dogs from various sources, values were all less than 5 mg/L, but in 22 healthy dogs from a single source, values ranged from less than 5 mg/L in 14 dogs and from 8 to 67 mg/L in 8 dogs. Increased concentrations of serum CRP were attained 24 hours after injection of casein (n = 9; median 188 mg/L), ovariohysterectomy (n = 11; median, 144 mg/L), or elective, nonacute orthopedic surgery (n = 10; median, 83 mg/L). After inoculation of Leptospira interrogans serovar canicola (n = 5), the behavior of serum CRP as an acute-phase reactant provided a sensitive and precise objective reflection of in vivo response. The CRP concentration in random single-serum samples from 73 dogs with other inflammatory and noninflammatory disorders ranged from normal (less than 5 mg/L) to 246 mg/L and generally correlated with the extent and activity of disease.

Studies of the in vivo synthesis and catabolism of serum amyloid P component (SAP) in the mouse.

The production of mouse serum amyloid P component (SAP), a major acute phase protein of liver origin, was studied immunocytochemically using the peroxidase staining technique. SAP was not detectable in the cytoplasm of hepatocytes from normal, unstimulated mice, nor was it observed before 24 h after an acute phase stimulus. 125I-labelled mouse SAP was cleared from the plasma in vivo with a half-life of 7.0-8.25 h in all animals studied including: normal mice of different strains with different genetically determined plasma SAP concentrations; mice undergoing acute phase responses with greatly elevated plasma SAP levels and mice with casein-induced amyloidosis. The circulating level of SAP is thus independent of its rate of clearance and catabolism and is determined by the rate of synthesis and/or secretion of SAP.

In vivo turnover studies of C-reactive protein.

The in vivo plasma clearance rate of the acute phase reactant C-reactive protein (CRP) was studied in mice and rats. The clearance rate of 125I-human CRP in mice and 125I-rat CRP in rats showed a T1/2 of approximately 4 h. The T1/2 was independent of circulating levels of CRP and was not affected by the presence of C-polysaccharide (CPS), a ligand to which CRP binds. However, in mice receiving sufficient CPS, more radioactivity localized to the spleen compared to mice receiving 125I-CRP only. 125I-CPS was rapidly cleared at the same rate by normal mice and by mice undergoing an acute phase response while rats cleared 125I-CPS more slowly despite having high circulating CRP concentrations. These findings suggest that CRP does not provide a mechanism for extremely rapid clearance of its ligands from the circulation, although the handling and subsequent fate of these ligands may be affected.

Amyloid P-component in mice injected with casein: identification in amyloid deposits and in the cytoplasm of hepatocytes.

Amyloid P-component was sought by immunofluorescence in the tissues of CBA mice receiving repeated subcutaneous injections of casein. P-component appeared in a perifollicular distribution in the spleen after about twenty casein injections, and correlated precisely with amyloid deposits identified by staining with Congo red. In mice which had received at least fourteen injections of casein, P-component also became detectable within the cytoplasm of periportal hepatocytes. This is the first demonstration of P-component in murine amyloid and of a possible site for in vivo synthesis of P-component.

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.

'Amyloid degrading activity' of human serum, an in vitro clearing effect which does not involve degradation of amyloid fibrils.

Clearing of turbid amyloid A fibril containing agarose gels by human serum has been ascribed to 'amyloid degrading activity'. We report here that this optical phenomenon is not due to an enzymatic reaction, does not involve proteolysis of the fibril subunits and is not inhibited by sera of patients with AA amyloidosis. The extent of clearing correlates closely with the serum albumin concentration and, as previously reported by others, serum albumin itself causes clearing comparable to that of whole serum. Furthermore addition of albumin solutions to turbid aqueous suspensions of AA amyloid fibrils causes immediate clearing. Serum albumin is known to clarify turbid non-amyloid fibril containing gels and is used commercially to improve the optical properties of radial immunodiffusion plates. We therefore propose that this property of albumin, the mechanism of which is not yet understood, underlies the so called 'amyloid degrading activity' of human serum and the latter is not therefore likely to be of in vivo biological or clinical significance.