Complement activation mediated by mannan-binding lectin in plasma from healthy individuals and from patients with SLE, Crohn's disease and colorectal cancer. Suppressed activation by SLE plasma.

We have developed a method for quantitating mannan-binding lectin (MBL)-induced activation of the complement system (MBL-C4-AC) in human plasma. This method and an assay for MBL concentration were applied to plasma samples from healthy individuals and patients with systemic lupus erythematosus (SLE), Crohn's disease (CD) and colorectal cancer (CRC). The MBL concentration was measured by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-MBL-antibodies and MBL-C4-AC by an ELISA using solid-phase mannan, incubating with plasma samples and quantitating the complement (C) activation by the use of antibodies against the C split-products C4b/C4c. The MBL concentration was nonsignificantly elevated in plasma from SLE-patients, whereas MBL-C4-AC was suppressed (P < 0.04). There was no correlation between MBL concentration and MBL-C4-AC in plasma from SLE-patients. In contrast, a significant correlation was found between the MBL concentration and MBL-C4-AC in plasma from healthy individuals. The C4 concentration was significantly reduced (P < 0.002) in plasma from the SLE patients and showed a significant correlation to MBL-C4-AC. The MBL-C4-AC assay was highly effective in discriminating the SLE patients from the other patient groups and healthy individuals.

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies.

Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro. These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating antibodies against SAP were not detected. Preincubation of virus (A/Japan/57) with SAP prevented primary infection of mice and development of antiviral antibodies. After a single intranasal administration of SAP (40 microg) 1 h before primary infection with virus (2LD(50)), nine out of 10 mice survived on day 10 and these mice approached normal body weight, whereas control mice (one out of five surviving on day 10) died. The data provide evidence of the potential of intranasally administered SAP for prophylactic treatment of influenza A virus infections in humans.

ELISA for evaluating the incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes.

An ELISA that measures plasma derived complement (C) split-products C3b/iC3b deposited on solid-phase immune complexes during C activation is described. Plates are coated with BSA, anti-BSA and plasma is added. Deposited C3b/iC3b is then detected by biotinylated anti-C3c-antibodies, avidin-alkaline phosphatase and para-nitrophenylphosphate. A novel feature is that the assay measures residual C activation capacity rather than in vivo generated C activation products. The assay was applied to plasma from 250 healthy blood donors. No difference in activation capacity of either the alternative (AP) or classical pathway (CP) with regard to age or gender was demonstrated. The total coefficient of variation was <5.7%. The ELISA procedure was compared to a standard hemolytic complement CH(50) assay using plasma from 23 out-patients with systemic lupus erythematosus (SLE). There was a weak correlation between the two assays for both C pathways, but neither the ELISA nor the CH(50) assay showed any correlation with the diagnostic ACR-criteria for SLE. However, the capacity of the CP was significantly reduced in SLE out-patients compared to healthy blood donors (P<0.0001).

Localization of human serum amyloid P component and heparan sulfate proteoglycan in in vitro-formed Abeta fibrils.

Ultrastructural studies of the localization of serum amyloid P component (SAP) in amyloid fibrils have given divergent results. We here report for the first time that electron microscopy of SAP coincubated with Abeta1-42 peptides or with mature Abeta1-42 fibrils, revealed SAP molecules coating the surface of the mature fibrils and that protofibrils of Abeta1-42 did not bind SAP. Also when incubated with extracted amyloid light chain (AL)-fibrils the SAP molecules aligned on the fibril surface. Heparan sulfate proteoglycan bound to the surface of the Abeta fibrils with a spacing of about 50 nm. We conclude that SAP does not bind to protofibrils but to the surface of mature Abeta fibrils and that it may stabilize and protect the fibrils.

Complexes of serum amyloid P component and DNA in serum from healthy individuals and systemic lupus erythematosus patients.

Serum amyloid P component (SAP) binds in vitro to DNA; based on findings in SAP-deficient mice it was proposed that SAP's role is to handle chromatin and DNA, thereby preventing formation of anti-DNA antibodies. For the first time we have shown the presence of Ca2+-dependent SAP-DNA complexes, measured by ELISA, in sera from both healthy volunteers and systemic lupus erythematosus patients (SLE). The concentration of SAP-DNA complexes in SLE sera was significantly lower than in normal sera and particularly low in sera from patients with anti-DNA titers exceeding 50. The complexes were dissociated by the SAP ligand heparin and were not demonstrable in EDTA plasma. Normal sera showed similar capacity to form SAP-DNA complexes with both thymus and Escherichia coli DNA, whereas significantly lower amounts of complexes, in particular with E. coli DNA, were formed in SLE sera. SLE patients with moderate to high anti-DNA titers showed a significant negative correlation between serum SAP's binding of E. coli DNA and the anti-DNA titer.

Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin.

Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing soluble protein, protofilaments and some fibrils or only fibrils in a solid phase system (ELISA) was modest with a slow kinetics compared to the positive delta IgG control. Soluble A beta induced no detectable CA in a liquid phase system (complement consumption assay) while fibrillar A beta caused CA at 200 mg/ml and higher concentrations. Soluble beta 2-microglobulin (beta 2M) purified from peritoneal dialysates was found to be as potent a complement activator as A beta in both solid and liquid phase systems while beta 2M purified from urine exhibited lower activity, a difference which may be explained by differences observed in SDS-resistant oligomers and isoforms. Soluble Amyloid A-protein caused no significant CA. A beta and beta 2M activated complement via the classical pathway. The modifying influence by amyloid-associated molecules on A beta-induced CA was also investigated, but neither serum amyloid P component nor heparan sulfate did significantly alter the A beta-induced CA. The results indicate that not only fibrillar A beta but also oligomers of, in particular, beta 2M from patients with dialysis-associated amyloidosis are capable of inducing CA at supra-physiological concentrations.

An ultrastructural study of amyloid intermediates in A beta1-42 fibrillogenesis.

Many attempts have been made to define early stages and intermediates in amyloid fibrillogenesis that may be susceptible to inhibition. We have developed an in vitro system, based on the use of A beta1-42 peptides, in which the development of prestages of protofilaments and protofilament and fibril formation could, for the first time, be followed by electron microscopy, supported by fluorescence spectrometry. The first recognizable ultrastructures after incubation of A beta1-42 peptides at 37 degrees C were globular subunits (4-5 nm in diameter) that gradually became organized into short protofilaments (30-100 nm), which in turn formed fibrils mainly by lateral association. At this stage, part of the protofilaments were seen first as collaterals protruding from the fibrils and then, as they were gradually incorporated, as buds on the fibril surface. A continuous growth of A beta1-42 fibrils was observed, seemingly originating from a nucleus, which appeared to consist of aggregates of amyloid intermediates. That protofilaments are intermediates also in the in vivo formation of amyloid was supported by the finding that AL fibrils isolated from amyloid tissues also exhibited radiating protofilaments. The demonstrated globular subunits and early formed protofilaments may be attractive targets for inhibition of fibril formation.

Isoforms of murine and human serum amyloid P component.

Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H/HeN mice showed one more acidic isoform and an increase in the concentration of the most basic isoform. C57BL/6 J mice exhibited two to three new isoforms during the acute-phase response. This appears to be the first demonstration of the physiological existence of SAP isoforms. In contrast, demonstration of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.

Binding of properdin to solid-phase immune complexes: critical role of the classical activation pathway of complement.

The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme-linked immunosorbent assay using solid-phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake was hardly detectable. Alternative pathway-mediated deposition of C3 with slow kinetics was demonstrated in C2-deficient serum and in NHS depleted of C1q, factor D and properdin (C1qDP-depleted serum) after reconstitution with factor D and properdin. Efficient uptake of properdin required a functional classical pathway, in the presence of which C3 and properdin were rapidly deposited onto the IC. Judging from findings in C3-deficient serum, factor I-deficient serum, and C1qDPB-depleted serum, the uptake of properdin was strictly C3-dependent, and did not require the presence of factors B and D. Thus, C3b fixed to IC was the principal ligand for properdin in the assay. The findings could have biological implications relating to complement-mediated modification of immune complexes in disease.

Increased plasma concentration of serum amyloid P component in centenarians with impaired cognitive performance.

Serum amyloid P component (SAP) binds to all amyloid fibrils including those in the plaques and tangles of Alzheimer patients. To investigate whether the plasma SAP concentration correlated to cognitive impairment, we measured SAP levels in blood samples from 41 centenarians and compared these to the cognitive performance evaluated by Mini Mental State Examination (MMSE). We observed a significantly (p < 0.001) increased SAP concentration (48.3+/-16.9 microg/ml; mean +/- SD) in the centenarians compared to gender-matched controls (32.8+/-11.4 microg/ml). Six severely demented centenarians had an even higher SAP concentration (60.2 microg/ml), while the subgroup of cognitive intact centenarians (MMSE score >24) showed a normal SAP concentration (38.4+/-9.3 microg/ml). No dehydration or hepatic dysfunction was demonstrable in the centenarians. We conclude that the centenarians with impaired cognitive performance had significantly increased plasma concentrations of SAP, while the values for cognitive intact centenarians were within the normal range.

Serum amyloid P component binds to influenza A virus haemagglutinin and inhibits the virus infection in vitro.

Serum amyloid P component (SAP) is a member of the phylogenetically conserved and structurally related group of proteins called pentraxins. SAP exhibits multispecific calcium-dependent binding to oligosaccharides with terminal N-acetyl-galactosamine, mannose and glucuronic acid. The authors report that SAP can bind to influenza A virus and inhibit agglutination of erythrocytes mediated by the virus subtypes H1N1, H2N2 and H3N2. SAP also inhibits the production of haemagglutinin (HA) an the cytopathogenic effect of influenza A virus in MDCK cells. The binding of SAP to the virus requires physiological calcium concentrations and is blocked by specific SAP antibodies. Denaturated and renaturated SAP retained inhibition of HA. Electron microscopy shows Ca(2+)-dependent binding of SAP to spikes on the viral envelope and immunoblotting indicates that SAP binds to a 50-55 kDa peptide corresponding to the mass of the HA1 peptide. Of several monosaccharides tested only D-mannose interfered with SAP's inhibition of both HA and infectivity. The glycosaminoglycans heparan sulfate and heparin, which bind SAP, reduced SAPs binding to the virus. The results indicate that the inhibition by SAP is due to steric effects when SAP binds to terminal mannose on oligosaccharides localized close to the sialic acid-binding site of the HA trimer.

An in vitro cellular system for generation of AA-amyloid.

Different conditions for establishing a cell culture system for generation of AA-amyloid were investigated. The most effective system was based on peritoneal macrophages from CBA/J mice that had received repeated injections of Hammersten casein, with subsequent cultivation of the cells at high density, high levels of acute phase serum, and neutral pH. Staining with Congo red, thioflavin T, and anti-AA revealed amyloid-like structures associated with macrophage clusters. The structures increased in number and size from day 2 to 6 of cell cultivation. The concentration of apoSAA in the culture medium fell markedly in the amyloid-producing cell cultures, while the SAP concentration was not reduced. The described cell culture system can be useful in studies of the influence of chaperone molecules and other factors or the formation and degradation of amyloid fibrils.

Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins: enhanced binding at slightly acid pH.

Serum amyloid P component (SAP), a member of the pentraxin family of proteins, binds calcium-dependently to several ligands including glycosaminoglycans (GAG's). We have investigated the influence of pH on the Ca2(+)-dependent binding of SAP to solid phase GAG's and amyloid fibril proteins (AA and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under conditions of slightly lowered pH.

Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase.

Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy. Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate that firm binding of C1q and C4bp to SAP requires that SAP is presented on a solid phase, that C1q and C4bp react with sites distinct from the heparin binding site, and that C1q and collagen I share binding sites on SAP. Immobilized native SAP, aggregated SAP and SAP-heparansulphate complexes induced no detectable complement activation.

Isolation and characterization of porcine mannan-binding proteins of different size and ultrastructure.

The authors report on the purification and characterization of mannan- binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan-Sepharose, protein A- and anti-porcine IgM-Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed gamma 1-gamma 2-electrophoretic mobility. The MBP designated pMBP-28 had a molecular mass of 28 kDa when analysed on SDS-PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP-28 revealed an oligomeric protein similar to rodent MBP-A and human MBP but with a predominance of penta- and hexameric molecules. Another protein designated pMBP-27 was composed of peptides of 27 kDa and had an Mr of 300-350 kDa on gel filtration chromatography. Electron microscopy of pMBP-27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N-terminal 26(pMBP-27) and 24(MBP-28) amino acid residues showed 54% and 58% identity with human MBP.pMBP-28 showed a higher degree of sequence similarity to rat and mouse MBP-A (60% identity) than to mouse and rat MBP-C (41-45% identity). Both pMBPs exhibited Ca2+-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was not sequenced.

Complement activation by malignant B cells from patients with chronic lymphocytic leukaemia (CLL).

It has previously been reported that the expression of the complement receptors CR1 (CD35) and CR2 (CD21) on malignant B cells in CLL is reduced compared with the expression on normal B cells, while deposition of complement C3 fragments, as a consequence of alternative pathway (AP) activation of complement, is observed on mononuclear cells from patients with B CLL. Following our demonstration that normal B cells are capable of activating the AP of complement in a CR2-dependent fashion, we have chosen to re-examine the complement-activating ability of B CLL cells in relation to their altered phenotype with respect to CR2 and the complement regulatory membrane proteins, CR1, decay accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46). Flow cytometry was used to measure expression of complement receptors and regulatory proteins on CD5+ B cells from CLL patients, as well as the deposition of C3 fragments occurring both in vivo and after in vitro AP activation. We have confirmed the reduced expression of CR1 and CR2 on CLL cells and have shown that AP activation in the presence of homologous, normal serum was reduced on B CLL cells compared with normal B cells. The degree of AP activation correlated directly with CR2 expression. In addition, we observed that CLL cells bear in vivo-deposited C3d,g, although at a significantly lower level than normal B cells.

Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction.

We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-alpha 2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with alpha 2M.

Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE).

It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that normal B cells are capable of activating the alternative pathway (AP) of complement in a CR2-dependent fashion. In this study we have investigated whether disturbances in this activity may be related to the altered phenotype of SLE B cells. Flow cytometry was used to measure expression of complement receptors and regulatory proteins on B cells from SLE patients, as well as the deposition of C3 fragments occurring in vivo or after in vitro AP activation. We have confirmed, for a proportion of the patients studied, reduced expression of CR1 and CR2 on B cells, and shown a consistency between low CR2 expression and reduced in vitro AP activation in the presence of homologous, normal serum. In addition, the B cells, like erythrocytes, bear raised levels of in vivo-deposited C3dg, but not C3b fragments, compared with normal B cells. The erythrocytes from SLE patients were unable to inhibit in vitro AP activation by B cells in homologous serum. Finally, we demonstrated an inverse relationship between SLE disease activity index (SLEDAI) and the expression of complement receptor 2 (CR2) on SLE B cells. Thus, determination of CR2 on B cells may emerge as an additional laboratory tool in the assessment of SLE activity.

Native human serum amyloid P component is a single pentamer.

Serum amyloid P component (SAP) and C-reactive protein (CRP) are members of the pentraxin protein family. SAP is the precursor protein to amyloid P component present in all forms of amyloidosis. The prevailing notion is that SAP in circulation has the form of a double pentameric molecule (decamer) whereas CRP is a single pentameric molecule. We have investigated by gel permeation chromatography the M(r) of SAP in freshly collected human serum and of SAP purified by carbohydrate affinity chromatography and anion exchange chromatography. SAP was monitored by quantitative immunoelectrophoresis and ELISA, and SAP peak fractions were analysed by use of SDS-PAGE, Western blotting, and electron microscopy. The results indicate that native SAP circulates as a single pentamer, a part of which forms complexes with C4b-binding protein. The properties of SAP changed during purification as indicated by rocket immunoelectrophoresis and electron microscopy. Thus, electron micrographs of purified SAP showed a predominance of decamers. However, the decamer form of SAP reversed to single pentamers when purified SAP was incorporated into SAP-depleted serum.