Serum amyloid P component prevents high-density lipoprotein-mediated neutralization of lipopolysaccharide

Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.     

Circulating serum amyloid P component is the precursor of amyloid P component in tissue amyloid deposits.

Intravenous administration of 125I-labelled isolated mouse serum amyloid P component (SAP) to mice with systemic amyloidosis was followed by specific deposition of the labelled protein in amyloidotic organs. Although only a small proportion of the total injected dose became localized in this way, the amount correlated with the quantity of amyloid present in different organs and was greatest in the spleen. No such localization was detected in the organs of control, untreated mice or animals which had received inflammatory stimuli but did not have amyloidosis. The labelled SAP was found by autoradiography to be present in the same distribution within the tissues as the Congophilic amyloid deposits. These observations establish directly, for the first time, that circulating SAP is the precursor of the amyloid P component (AP) in systemic amyloidosis. They were confirmed by the further finding that intravenous injection into amyloidotic mice of human SAP, either in whole human serum or in isolated pure form, was followed by appearance of the human SAP in the mouse amyloid deposits. In addition to elucidating one aspect of the pathogenesis of amyloid deposition and strengthening the homology of functional behaviour between SAP of different species, the present results suggest a means for selective targeting of diagnostic tracers and/or effector agents to amyloid deposits in vivo.     

Early B-lymphocyte precursors and their regulation by sex steroids

Summary: This review describes an improved characterization of early B-lymphocyte precursors in mice and the remarkable sensitivity of the same cells to hormones. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) was used as a marker to image and characterize bone marrow cells lacking all lineage-associated markers. Most early TdT⁺ precursors have a distinctive density of c-kit and express the interleukin-7Ra chain, as well as flt-3/flk2, but lack CD34. An understanding of those cell surface properties made it possible to obtain highly enriched, viable cells with the potential to give rise to CD19+ lymphocytes in culture. A series of other flow cytometry and culture experiments suggested a possible differentiation sequence for these early pro-B cells. This new model was used to advantage in our studies of sex steroids. It appears that early precursors represent a hormone-sensitive control point for determining numbers of new B lymphocytes that are produced within bone marrow. We also compare and contrast these findings with B lymphopoiesis in humans.     

Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation

Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.     

Quantitation of serum amyloid P component by an enzyme-linked immunoassay

A solid phase enzyme-linked immunoassay (ELISA) for the murine acute-phase reactant, serum amyloid P component (SAP), was developed. The assay is based on our finding of a calcium-dependent binding of SAP to trinitrophenyl-conjugated proteins. The wells of polystyrene microtiter plates are coated with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), then incubated with SAP-containing samples. The amount of SAP is determined by indirect ELISA, wells being sequentially incubated with rabbit anti-SAP antiserum and horseradish peroxidase-linked donkey anti-rabbit IgG conjugate. The limit of sensitivity of the assay is 0.6 ng/ml SAP. Comparison with data on sera obtained by rocket immunoelectrophoresis assay yielded a correlation coefficient of 0.89. The binding of SAP to TNP-KLH was inhibited by calcium chelators and low concentrations of non-ionic detergents. Chromatography of serum on TNP-Sepharose provided a efficient and simple way of purifying SAP. The assay was also adapted for the quantitation of human SAP. The use of the assay in studying the binding specifities of SAP and its physiological role is discussed.     

Measurement of murine serum amyloid P component by rate nephelometry

Murine serum amyloid P component (SAP) is an acute-phase protein that is increased 2–10-fold in concentration following appropriate inflammatory or infections stimuli. Previous studies of the acute-phase SAP response have employed quantitative immunoelectrophoresis or radioimmunoassay to measure SAP concentration. A rate nephelometric procedure has been developed which measures SAP concentration rapidly and with equivalent or greater precision than the previously applied techniques. This simple method will facilitate experimental and clinical studies of the acute-phase response.     

Serum amyloid P component and C-reactive protein opsonize apoptotic cells for phagocytosis through Fcgamma receptors

Serum amyloid P component (SAP) and C-reactive protein (CRP) are opsonins that react with nuclear autoantigens targeted in systemic autoimmunity. CRP and SAP bind to apoptotic and necrotic cells, which are potential sources of these autoantigens. We have recently determined that the receptors for CRP on phagocytic cells are Fcgamma receptors. The goal of this study was to determine whether CRP and SAP promote phagocytosis of apoptotic cells and to identify the receptors involved. Apoptosis was induced in human neutrophils (PMN) and the Jurkat T-cell line by UV-irradiation. SAP treatment of apoptotic human PMN increased ingestion by autologous macrophages. Both SAP and CRP increased ingestion of apoptotic, but not normal Jurkat cells by J-774 macrophages and mouse peritoneal macrophages. Neither SAP nor CRP increased ingestion of apoptotic Jurkat cells by macrophages from FcR gamma-chain deficient mice, which lack FcgammaRI and FcgammaRIII. Inhibition of FcgammaRIII-mediated uptake using mAb 2.4G2 eliminated opsonization by SAP, but not by CRP. These results indicate that pentraxins promote uptake of apoptotic cells through FcgammaRI and/or FcgammaRIII. Ingestion through these receptors is expected to alter the pattern of cytokine production and antigen presentation in response to apoptotic cells.     

Nonamyloidotic monoclonal immunoglobulin deposits lack amyloid P component

Deposits in tissues from 13 patients with amyloid, 8 with monoclonal light chain or light and heavy chain deposition disease, and 2 with both amyloid and nonamyloidotic light chain deposits of the same isotype were examined in parallel for the presence of amyloid P component by immunofluorescence and/or immunoperoxidase methods. Amyloid P component was detected in the amyloid but not the nonamyloid deposits, even in the 2 individuals in whom both types of deposits were present, indicating a specific relationship between the amyloid P component and the amyloid fibrils.     

Immunoradiometric assay for human serum amyloid P component

Human serum amyloid P component (SAP) is of increasing interest for its possible pathogenic role in amyloidosis and Alzheimer's disease, and as a therapeutic target in these conditions. We have developed and validated a robust and reproducible immunoradiometric assay (IRMA) for human SAP in serum, plasma and cerebrospinal fluid, and characterized the notable stability of human SAP immunoreactivity during storage of undiluted serum at 4°C and 37°C as well as frozen at -30°C. SAP values were also stable after repeated freeze thawing of highly diluted serum samples. The 100 fold dynamic range of the assay, 0.5-50 μg/L, encompassed all values seen in blood and cerebrospinal fluid, when tested at suitable dilutions, from both normal healthy individuals and patients, including subjects receiving the SAP-depleting drug, CPHPC. Furthermore by comparing the IRMA values in the presence and absence of calcium, the new assay revealed interference due to the binding of CPHPC by SAP, which was markedly enhanced in heparinized plasma. It is therefore essential that SAP assays in samples from patients on CPHPC be conducted in the absence of free calcium, in order to completely abrogate interference and determine the actual total SAP concentration. Estimates by the IRMA of SAP concentration in 49 serum samples from amyloidosis patients corresponded closely with those obtained by the established standard electro-immunoassay method and by a newly developed commercial ELISA kit (Hycult Biotechnology).     

血清淀粉样P成分的DNA结合特性

目的 检测血清淀粉样P成分(SAP)与不同DNA的结合活性并研究SAP与DNA的结合是否有利于此类抗原的清除，探讨它在SLE发生发展中的作用及意义。方法 用亲和层析和凝胶过滤方法自小鼠和人血清中纯化SAP，分别提取来自细菌、质粒、酵母、小鼠淋巴细胞等不同DNA，用DOT-EIA方法检测SAP与它们的结合活性，用巨噬细胞吞噬实验观察SAP与DNA结合后对DNA清除的影响。结果 人和小鼠SAP均与细菌DNA结合最强，其次为质粒、酵母DNA，与淋巴细胞DNA和小牛胸腺DNA的结合较弱，与单链λDNA结合最弱；与来源于活化状态小鼠淋巴细胞DNA的结合力高于非活化状态；SAP与活化淋巴细胞DNA结合后可促进巨噬细胞对DNA的吞噬。结论 SAP与不同DNA结合活性各异。     

Serum amyloid P component binding to Shiga toxin 2 requires both a subunit and B pentamer

Solid-phase binding, competitive binding, and cytotoxicity neutralization assays indicate that the B pentamer and A subunit both contribute to human serum amyloid P (HuSAP) component binding to Stx2. A polyvalent globotriaosyl-ceramide receptor analog, Daisy, did not competitively inhibit HuSAP binding, implying that the two ligands bind to different Stx2 domains.     

Age-related deposition of amyloid P component in normal human testis

The distribution of amyloid P component in normal human testes from fetal life to old age was studied by a direct immunofluorescent technique on frozen sections. Amyloid P is readily and invariably detected in association with elastic fibres around seminiferous tubules and in blood vessels from the age of 30 years upwards. The same is true for most cases during the twenties, but in no case below the age of 18 was its presence demonstrable.     

Effects of serum amyloid P component on human lymphocytes

The P component of amyloid is a normal serum protein designated SAP. SAP has substantial homology with C-reactive protein (CRP). Recent studies have established the structure, tissue distribution and binding reactivities of SAP; however, as yet, very minimal insight into its function has been achieved. Recent studies, though somewhat controversial, have suggested a regulatory role for CRP on the immune response. In view of these studies, we wanted to evaluate the in vitro effects of SAP on several immunological properties of human lymphocytes. We found that SAP had a marked inhibitory effect on the proliferative response of lymphocytes to a variety of T-dependent mitogens. In addition, SAP markedly enhanced the formation of active E rosettes, a marker of activated T cells.     

Evaluation of systemic amyloidosis by scintigraphy with 123I-labeled serum amyloid P component

BACKGROUND. In systemic amyloidosis the distribution and progression of disease have been difficult to monitor, because they can be demonstrated only by biopsy. Serum amyloid P component (SAP) is a normal circulating plasma protein that is deposited on amyloid fibrils because of its specific binding affinity for them. We investigated whether labeled SAP could be used to locate amyloid deposits. METHODS. Purified human SAP labeled with iodine-123 was given intravenously to 50 patients with biopsy-proved systemic amyloidosis--25 with the AL (primary) type and 25 with the AA (secondary) type--and to 26 control patients with disease and 10 healthy subjects. Whole-body images and regional views were obtained after 24 hours and read in a blinded fashion. RESULTS. In the patients with amyloidosis the 123I-SAP was localized rapidly and specifically in amyloid deposits. The scintigraphic images obtained were characteristic and appeared to identify the extent of amyloid deposition in all 50 patients. There was no uptake of the 123I-SAP by the control patients and the healthy subjects. In all patients with AA amyloidosis the spleen was affected, whereas the scans showed uptake in the heart, skin, carpal region, and bone marrow only in patients with the AL type. Positive images were seen in six patients in whom biopsies had been negative or unsuccessful; in all six, amyloid was subsequently found on biopsy or at autopsy. Progressive amyloid deposition was observed in 9 of 11 patients studied serially. CONCLUSIONS. Scintigraphy after the injection of 123I-SAP can be used for diagnosing, locating, and monitoring the extent of systemic amyloidosis.     

Human serum amyloid P component binds to peripheral blood monocytes

The binding of human serum amyloid P component (SAP) to blood cells and monocyte-derived dendritic cells was investigated by flow cytometry. Monocytes bound biotinylated SAP with avidity in a dose-dependent and saturable manner. By contrast, the binding of SAP to monocyte-derived dendritic cells was weak. No binding to erythrocytes, NK cells, T lymphocytes or B lymphocytes could be detected. The SAP-monocyte interaction was calcium-independent and readily inhibited by C1q. SAP was nonmitogenic for human mononuclear cells and had no apparent influence on lymphocyte proliferation induced by mitogenic lectins. We speculate that binding of SAP by monocytes could be of physiological relevance at extravascular sites by influencing complement regulation.     

Isolation and characterization of guinea-pig serum amyloid P component.

A pentraxin was isolated from acute-phase guinea-pig serum by calcium-dependent affinity chromatography on agarose. It was immunochemically identical to guinea-pig amyloid P component and therefore has been called guinea-pig serum amyloid P component (SAP). Guinea-pig SAP has an apparent MW of between 265,000 and 300,000 by different techniques, and is composed of 10 noncovalently associated subunits arranged in two pentameric annular discs interacting face-to-face. It is apparently composed of two types of subunit, which run as a closely spaced doublet on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least one type of subunit is glycosylated. The serum concentration was 16 +/- 4 mg/l in outbred animals, rising to 25 +/- 4 mg/l in an acute-phase response. Binding to agarose correlated with the agarose pyruvate content and was completely abolished by diazomethane treatment of the agarose, which methylates the pyruvate carboxylic moiety. Binding was also inhibited in the presence of free methyl 4,6-o-(carboxyethylidine)-beta-D-galactopyranoside. No protein resembling C-reactive protein (CRP) was obtained by calcium-dependent affinity chromatography of acute-phase guinea-pig serum on phosphorylcholine (PC)-Sepharose, and it not clear whether a counterpart of CRP exists in this species.     

Isolation of human C-reactive protein and serum amyloid P component

Procedures are described for the isolation in high yield of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP). CRP was obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled to cyanogen bromide-activated Sepharose. It was then gel filtered on Ultrogel AcA44 (acrylamideagarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity for agarose. Residual trace contaminants were removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and/or by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300 35–44% of the initial CRP was recovered in pure from according to biochemical and immunochemical criteria. SAP was isolated from normal serum by calcium-dependent affinity chromatography on unsubstituted Sepharose beads, followed by solid-phase immunoabsorption of contaminants and finally gel filtration on Sephacryl S-300. At least 50% of the SAP in the starting material was recovered in pure form according to biochemical and immuunochemical criteria. Ready availability of such preparations facilitates biochemical, biophysical and particularly biological studies of these plasma proteins.     

Serum amyloid P component and autoimmune parameters in the assessment of arthritis activity in MRL/lpr/lpr mice.

The concentration of immunoglobulins, anti-ssDNA, anti-dsDNA, anti-TNP antibodies, IgM rheumatoid factor, C3, immune complexes and serum amyloid P component (SAP), in the serum were measured in 68 male and female MRL/lpr/lpr mice, a strain affected with a systemic autoimmune disease. The degree of lymphoproliferation was assessed by the spleen weight. Spontaneous secretion of immunoglobulins and anti dsDNA antibodies were measured in spleen cell cultures. All mice presented age related increases or decreases (C3) in the level of measured parameters. Inflammatory lesions were detected, by light microscopy in the joints of all mice. There was a significant correlation, in both sexes between the serum level of SAP and the severity of the polyarthritis, as assessed by light microscopy. In female mice the levels of anti-dsDNA antibodies, immunoglobulins, either measured in serum or in the spleen compartment, and circulating immune complexes also showed correlation with the activity of the arthritis, but neither of these variables correlated as closely with arthritis scores as did serum SAP.     

Endometriosis: Secretion of interleukin-6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids

Endometriosis is generally associated with an immuno-inflammatoryprocess that takes place in the peritoneal cavity of patients.Interleukin (IL)-6, a multifunctional cytokine involved in numerousimmunological and prolifer-ative processes, has been found athigh concentrations in the peritoneal fluid of endometriosispatients. The purpose of this study was to investigate the abilityof endometriotic cells to produce IL-6 and to assess the regulationof its secretion by proinflammatory cytokines and sex steroids.Cultures of human endometriotic cells were exposed to differentconcentrations of cytokines and sex steroid hormones for varyingperiods of time. IL-6 secretion was measured using an enzyme-linkedimmunosorbent assay. Endometriotic cells spontaneously releasedIL-6 in culture. IL-1 and tumour necrosis factor (TNF)- (0.1–100.0ng/ ml) potentiated IL-6 secretion in a time- and dose-dependentmanner. Interferon- (0.4–400 ng/ml) induced a dose-relatedincrease in IL-6 secretion and showed a synergjstic effect onthat secretion in combination with TNF- (10 ng/ ml). Eitherspontaneous or cytokine-induced IL-6 secretion was inhibitedby progesterone (10^–8–10^–5 M) and danazol(10^–6 M), whereas oestradiol (10^–8–10^–5M) had a limited inhibitory effect. The antiprogestin RU486(l0^–8–10^–4 M) antagonized the inhibitory effectsof progesterone and danazol, but showed agonist action whenused alone. These findings indicate that endometriotic tissuemay actively contribute to the biological changes observed inthe peritoneal fluid of endometriosis patients. They also providenew insights into the mechanisms of action of progesterone andthose of danazol and RU486 used in the treatment of endometriosis.