Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW x BXSB)F1 mice and human systemic lupus erythematosus

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW x BXSB)F1 (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.     

Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW × BXSB)F1 mice and human systemic lupus erythematosus

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW × BXSB)F₁ (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

The presence of autoantibodies specific for NZB serum factors in adult NZB mice and the establishment of monoclonal autoantibodies against these humoral factors

Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.     

Heparin inhibits the binding of beta 2-glycoprotein I to phospholipids and promotes the plasmin-mediated inactivation of this blood protein. Elucidation of the consequences of the two biological events in patients with the anti-phospholipid syndrome.

The phospholipid-binding plasma protein beta2-glycoprotein I (beta2-GPI) is the primary antigen recognized by the circulating autoantibodies in patients with the "anti-phospholipid syndrome" (APS). Although heparin is routinely used in the treatment and prophylaxis of APS patients, the primary heparin-binding site within beta2-GPI has not been identified. More importantly, how heparin exerts its beneficial effects in vivo in APS patients has not been deduced at the molecular level. Using an expression/site-directed mutagenesis approach, we now show that the positively charged site that resides in the first domain of beta2-GPI is not the primary heparin-binding site. Rather it is the second positively charged site located within the fifth domain of the protein that also binds to phospholipids. Lys(284), Lys(286), and Lys(287) in this domain are essential for the interaction of beta2-GPI with heparin. These data indicate that beta2-GPI binds to heparin in a relatively specific manner even though the affinity for the interaction is rather low. Lys(317) resides in the center of the high affinity phospholipid-binding site. Surprisingly, heparin at concentrations that can be achieved in vivo during anticoagulation therapy greatly enhances the plasmin-mediated cleavage of the Lys(317)-Thr(318) site in beta2-GPI. Because the cleaved form cannot bind to phospholipids effectively, the combined actions of heparin and plasmin result in a diminished ability of beta2-GPI to recognize phospholipids. This, in turn, decreases the prothrombotic activity of the endogenous circulating anti-beta2-GPI antibodies in the patients. Thus, heparin exerts its beneficial effects in APS patients by at least two distinct mechanisms.     

Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor.

Anticardiolipin antibodies (aCL) found in sera from patients with SLE react with cardiolipin (CL) in the presence of a 50-kDa serum cofactor. The cofactor, which was identified to be beta 2-glycoprotein I by sequencing the N-terminal amino acids, not only enhances CL binding by antibodies in SLE but also depresses it by antibodies associated with syphilis. Cofactor-dependent binding of aCL in SLE to solid phase CL was competitively inhibited by the simultaneous addition of fluid phase CL but was unaffected by either prior or simultaneous addition of a high excess of the cofactor. Binding of aCL in syphilis to solid phase CL was competitively inhibited by either addition of the cofactor or fluid phase CL. aCL in SLE reacted with CL, PS, and PA in the presence of cofactor. In contrast, biotinyl-cofactor bound directly to these anionic phospholipids (PL) and also to PG. These results show that the cofactor-CL complex bears an epitope that confers recognition specificity for aCL in SLE, in contrast with direct CL recognition by syphilitic aCL. The direct binding of the cofactor to PL suggests that the cofactor dependence of aCL binding to PL is due to recognition by aCL of a unique epitope generated upon the formation of the cofactor-CL complex.     

Cellular basis of in vitro anti-DNA antibody production: evidence for T cell dependence of IgG-class anti-DNA antibody synthesis in the (NZB X NZW)F1 hybrid

An in vitro system was designed to measure anti-DNA antibody synthesis, and the cellular basis of this autoantibody production in NZB X NZW (B/W)F1 (B/W F1) mice was analyzed. The spleen cells from old B/W F1 mice contained a number of B cells that spontaneously produced anti-DNA antibodies of both IgM and IgG classes in the absence of stimulants, thereby demonstrating that these B cells had been activated in vivo. These activated B cells could be removed by Sephadex G-10 column (G-10) filtration. Such G-10-passed, homogeneously small B cells were activated by the stimulant lipopolysaccharide (LPS) and produced both IgM and IgG class anti-DNA antibodies. The G-10-passed cells contained both B and T cells, and the cytotoxic treatment of the cells with monoclonal antibodies to T cells, anti-Thy-1 and anti-L3T4, abolished the LPS-induced IgG class, but not IgM class, anti-DNA antibody syntheses. Thus, the LPS-induced production of IgG class anti-DNA antibodies in B/W F1 mice is regulated by T cells. Reconstitution experiments revealed the requirement of T-B cell contact but not of the proliferative response of T cells. Moreover, there was no apparent adherent cell requirement. Such IgG class anti-DNA antibodies were produced only by spleen cells from old B/W F1 mice, but not from young B/W F1, NZB, NZW, and C57BL/6 mice. Like IgM class anti-DNA antibodies, LPS-induced synthesis of polyclonal IgM was T cell-independent. Only a slight reduction in the polyclonal IgG synthesis was observed after the G-10-passed cells had been treated with anti-Thy-1 antibody plus complement. This study should facilitate investigation of cell to cell interactions in the formation of autoantibodies and their correlations to immunologic abnormalities in autoimmune disease.     

The Lyb-3+5+ subset of B cells is not required for lupus-like autoantibody formation caused by graft-vs-host reaction

We asked the question whether or not the Lyb-3+5+ B cell subset, which is lacking in CBA/N immune defective mice, is required for the lupus-like autoantibody formation caused by graft-vs-host reaction (GVHR). (CBA/N X DBA/2)F1 male defective mice injected with DBA/2 T cells produced IgG autoantibodies to the same extent as did nondefective F1 mice suffering from GVHR. Although a very small number of DBA/2 B cells might have contaminated the T cell inocula, it was shown that these were B cells of the defective F1 mice that produced autoantibodies during the GVHR. This was demonstrated by detecting autoantibodies carrying an immunoglobulin allotype of the F1 recipient. Furthermore, the defective F1 male mice injected with CBA/N lymphoid cells, which were lacking Lyb-3+5+ B cells, also produced autoantibodies. Isotype analysis of antinuclear antibodies revealed that some of them belonged to IgG3 isotype. It was concluded that the ontogenically late-appearing B cell subset is not required for GVH autoimmunity.     

Beneficial effects of treatment with transglutaminase inhibitor cystamine on macrophage response in NZB/W F1 mice

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F(1) mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F(1) mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

Ia specificities on parental and hybrid cells of an I-A mutant mouse strain

Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.     

The BM12 mutation and autoantibodies to dsDNA in NZB.H-2bm12 mice

Molecular and genetic tools have been used to shed light on the genes that contribute to susceptibility to murine lupus and the mechanisms that lead to immunopathology. The MHC genes and their products have been consistently shown to contribute toward the development of disease. To understand the contribution of MHC-class II genes, our laboratory had derived two inbred strains of mice, NZB.H-2bm12 and NZB.H-2b. These new colonies of mice were studied and compared in the 10th generation backcross; inbreeding was serially followed by H-2 typing, responses to beef/porcine insulin, and the presence of the B6 Ig allotype, IgG2ab. Of great interest is the finding that NZB.H-2bm12, in contrast to NZB.H-2b or NZB (H-2d), mice develop high titer autoantibodies to dsDNA. This result is unique because NZB (H-2d) mice, unliked NZB x NZW (NZB/W F1) or NZB x SWR (SNF1) hybrids do not develop autoantibodies to dsDNA, even after immunization. NZB mice, in contrast, are characterized only by autoantibodies to ssDNA. Our observation is also striking because the gene conversion that resulted in the I-A beta bm12 mutation occurred at amino acid residues 68, 71, and 72 of I-E beta b. Recently the contribution of NZW to accelerated autoimmunity in the NZB x NZW F1 hybrid has also been linked to H-2 and a single amino acid change at amino acid 72 of I-E beta. Thus, amino acid residue 72 may be a hot spot for disorders of immune regulation when superimposed on the appropriate genetic background. NZB mice expressing the I-Abm12 mutation will allow specific dissection of the requirements for autoantibody production to dsDNA uncomplicated by heterozygosity.     

A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that beta(2)-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for beta(2)-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by beta(2)-GPI and subsequently by anti-beta(2)-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both beta(2)-GPI and an anti-beta(2)-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-beta(2)-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with beta(2)-GPI. We suggest that autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL and Abs may be present in APS.     

Monoclonal anti-deoxyribonucleic antibodies. I. Isotype and specificity studies

Ten monoclonal anti-DNA antibodies generated in three separate fusion experiments performed using nonimmunized B/W spleen cells were studied. Their antigenic specificities were demonstrated to be identical and directed against a conformational determinant of the B helical form of double-stranded DNA (dsDNA). These data suggest that autoantibodies to dsDNA in B/W mice could constitute a homogeneous population.     

Identification of ribosomal protein autoantigens

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Monoclonal antibodies against bovine growth hormone.

A number of mouse x mouse hybridomas producing monoclonal antibodies (MAbs) against bovine growth hormone (bGH) were prepared by fusion of spleen cells from bGH-primed mice (Balb/c) with non-secretory mouse myeloma cells (PAIOP3) and characterized. MAbs obtained from three fusion experiments belonged to IgM, IgG1 and IgG2b class/subclass of antibodies. Cross-reaction studies indicated that generated antibodies were against three different epitopes of bGH. VIA6E8 (IgG1) and VIIB2E11C9 (IgM) did not cross-react with ovine prolactin (oPRL), ovine leutinizing hormone (oLH) and porcine follicle stimulating hormone. Antibody VIB3C9E8 (IgM) exhibited cross-reaction with oPRL and oLH. Antibody VIC1F9 (IgG2b) cross reacted with oPRL. All MAbs were against conformational epitopes of bGH.     

Cross-reactivity between major histocompatibility complex class II antigens in mice NOD and an islet antigen with 58 kDA molecular weight

The NOD mouse has been recently developed as a model for autoimmune insulin-dependent diabetes mellitus. The NOD mouse is further characterized by a genetic linkage with genes mapping within the major histocompatibility complex at the I-A locus. The present work demonstrates the presence in NOD mice of circulating autoantibodies specific for a 58 kDa antigen which is present in membrane extracts prepared from the murine insulin-secreting tumoral cell line Rin5F. This 58 kDa antigen shows a cross reactivity with NOD mouse class II antigens as indicated by its recognition by anti-I-A(NOD) monoclonal antibodies.     

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.     

Omega-carboxyl variants of 7-ketocholesteryl esters are ligands for beta(2)-glycoprotein I and mediate antibody-dependent uptake of oxidized LDL by macrophages

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that beta(2)-GPI specifically binds to oxidized LDL (oxLDL) and that the beta(2)-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, and T. Koike. 2001. A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697-709). In the present study, we demonstrate that omega-carboxylated 7-ketocholesteryl esters are critical for beta(2)-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by beta(2)-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of beta(2)-GPI and an anti-beta(2)-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC), also showed a significant interaction with beta(2)-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with beta(2)-GPI. Thus, omega-carboxyl variants of 7-ketocholesteryl esters can mediate anti-beta(2)-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL.