Systemic lupus erythematosus aggravates atherosclerosis by promoting IgG deposition and inflammatory cell imbalance.

BACKGROUND: Systemic lupus erythematosus (SLE) patients experience a premature and more severe presentation of coronary artery disease. The underlying mechanisms of accelerated coronary artery disease in SLE patients remain to be elucidated. METHODS: By using atherosclerosis combining a SLE murine model, we proved that the onset of SLE aggravates atherosclerosis. Although the onset of SLE reduced blood lipids slightly, immune deviation contributed to aggravated atherosclerosis in lupus mice. Lupus atheroma were characterized by inflammatory cell infiltration, such as gathered dendritic cells, macrophages, and IgG deposition. RESULTS: Decreased lymphocytes and magnified dendritic cells in the spleen were also observed in lupus mice. Hydroxychloroquine prevented atherosclerosis progression mainly by reversing immune status abnormality caused by SLE. Serum interferon alfa levels were not changed in lupus mice. CONCLUSION: These findings strongly suggested that anti-inflammatory therapies and hydroxychloroquine provide a new possible strategy for treating SLE patients with atherosclerosis.

Distinct patterns of heavy chain variable region subgroup use by human monoclonal autoantibodies of different specificity.

Using a panel of antibodies specific for H and L chain variable region subgroups, a panel of human monoclonal cold agglutinin (CA) and rheumatoid factor (RF) autoantibodies were analyzed. The vast majority of the two types of autoantibodies utilized VkIII L chains, many of which probably derive from the Humkv325 gene. However, while most RFs (77%) utilized VHI H chains, all the CAs used VHII subgroup H chains. These results are consistent with a model of autoantibody generation, wherein binding specificity is H chain defined in a set of antibodies that use a multipotential L chain.

A B cell superantigen-induced persistent "Hole" in the B-1 repertoire.

The bacterial toxin protein A from Staphylococcus aureus (SpA) interacts with B cell antigen receptors encoded by variable region heavy chain (V(H)) clan III genes via a V region framework surface that has been highly conserved during the evolution of the adaptive immune system. We have investigated the consequences of exposure to this prototypic B cell superantigen, and found that treatment of neonates or adults induces a T cell-independent deletion of a large supraclonal set of susceptible B cells that includes clan III/V(H) S107 family-expressing lymphocytes. In studies of different SpA forms, the magnitude of the induced deletion directly correlated with the V(H)-specific binding affinity/avidity. Upon cessation of SpA exposure, the representation of conventional splenic (B-2 subset) lymphocytes normalized; however, we found that the V(H) family-restricted deficit of peritoneal B-1 cells persisted. SpA treatment also induced a persistent loss of splenic S107-mu transcripts, with a loss of certain natural antibodies and specific tolerance to phosphorylcholine immunogens that normally recruit protective antimicrobial responses dominated by the S107-expressing B-1 clone, T15. These studies illustrate how a B cell superantigen can exploit a primordial Achilles heel in the immune system, for which B-1 cells, an important source of natural antibodies and host immune responses, have special susceptibility.

Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Genetic imprinting of autoantibody repertoires in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease distinguished by great heterogeneity in clinical manifestations and autoantibody expression. While only a handful of autoantibody specificities have proved useful for clinical diagnosis, to characterize complex lupus-associated autoantibody profiles more fully we have applied proteome microarray technology. Our multiplex microarrays included control ligands and 65-autoantigens, which represent diverse nuclear and cytoplasmic antigens recognized by disease-associated and natural autoantibodies. From longitudinal surveys of unrelated SLE patients, we found that autoantibody profile patterns can be patient-specific and highly stable overtime. From profiles of 38 SLE patients that included 14 sets of SLE twins, autoantibodies to the phospholipid neo-determinants, malondialdehyde (MDA) and phosphorylcholine (PC), which are exposed on apoptotic but not healthy cells, were among the most prevalent and highly expressed. We also found that immunoglobulin M (IgM) reactivity to MDA and PC ligands had significant direct correlations with DNA-containing antigens, while such a general relationship was not found with a panel of RNA-related antigens, or for IgG-autoantibodies. Significantly, hierarchical analysis revealed co-distribution/clustering of the IgM autoantibody repertoire patterns for six of 14 twin sets, and such patterns were even more common (10 of 14) for IgG autoantibody profiles. Our findings highlight the potentially distinct roles of IgM and IgG autoantibodies, as we postulate that the direct correlations for IgM autoantibodies to DNA antigens with apoptosis-related determinants may be due to co-expression arising from common pro-homeostatic protective roles. In contrast, the sharing of IgG autoantibody fingerprints by monozygotic twins suggests that lupus IgG autoantibodies can arise in predisposed individuals in genetically determined patterns.

Variable region diversity in human circulating antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b. Preferential usage of two types of VH3 heavy chains.

Antibodies against capsular polysaccharides are important in the defense against many pathogenic bacteria. To determine the mechanism for the variability in responses to polysaccharides, a panel of well characterized serologic reagents that identify diagnostic primary amino acid sequences in the framework and hypervariable regions of heavy (H) and light (L) chains were created to characterize the variable region diversity in circulating human antibodies. 10 normal adult volunteers were immunized with the type b capsular polysaccharide of Haemophilus influenzae (Hib PS). By immunoblot analyses each individual was found to use at least three different variable L (VL) families, but all had preferential usage of VH3-derived H chains. Four individuals had lesser populations of VH1-derived H chains and three had populations of VH4-derived H chains, but anti-Hib PS antibodies derived from the VH2, VH5, and VH6 families were not detected. The anti-Hib PS antibodies from all subjects were also identified by serologic markers for two specific types of VH3 H chains. These H chains are structurally related to the 20P1 and 30P1 VH genes that are preferentially rearranged in the early human repertoire. These findings document the VH restriction of physiologic responses to Hib PS immunization, and demonstrate a technique to directly assess the structural and genetic diversity of specific serum antibodies.

Superantigen properties of a human sialoprotein involved in gut-associated immunity.

Protein Fv (pFv) is a recently described 175-kD gut-associated sialoprotein with a potent capacity for augmentation of antibody-dependent immune functions. To investigate the molecular basis for Fab-mediated binding of pFv, we evaluated a panel of 52 monoclonal IgM and found that approximately 40% bound pFv. Whereas the majority (> or = 75%) of V H3 and V H6 IgM strongly bound pFv, only a small minority (< 20%) of IgM from other V H families bound pFv, and these antibodies had weaker binding interactions. Inhibition studies suggested that all binding occurred at the same (or overlapping) site(s) on pFv. Surface plasmon resonance studies demonstrated binding affinity constants up to 6.7 x 10(8) M-1 for pFv. Biopanning of IgM and IgG Fab phage-display libraries with pFv preferentially selected for V H3 and V H6 antibodies, but also obtained certain V H4 IgM. V H sequence analyses of 36 pFv-binding antibodies revealed that binding did not correlate with CDR sequence, JH, or L chain usage. However, there was preferential selection of pFv binders with V H CDR3 of small size. These studies demonstrate that a protein which enhances immune defense in the gut has structural and functional properties similar to known superantigens.

Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity.

The immune response to oxidized LDL (OxLDL) may play an important role in atherogenesis. Working with apoE-deficient mice, we isolated a panel of OxLDL-specific B-cell lines that secrete IgM Abs that specifically bind to oxidized phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC). These Abs block uptake of OxLDL by macrophages, recognize similar oxidation-specific epitopes on apoptotic cells, and are deposited in atherosclerotic lesions. The Abs were found to be structurally and functionally identical to classic "natural" T15 anti-PC Abs that are of B-1 cell origin and are reported to provide optimal protection from virulent pneumococcal infection. These findings suggest that there has been natural selection for B-1 cells secreting oxidation-specific/T15 antibodies, both for their role in natural immune defense and for housekeeping roles against oxidation-dependent neodeterminants in health and disease.

Repertoire cloning of lupus anti-DNA autoantibodies.

To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.

Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance.

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

Idiotypic and subgroup analysis of human monoclonal rheumatoid factors. Implications for structural and genetic basis of autoantibodies in humans.

Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated "sequence-dependent" reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity.

Neo-self antigens and the expansion of B-1 cells: lessons from atherosclerosis-prone mice.

The pathogenesis of atherosclerosis involves an inflammatory process that is modulated by the immune system, and within these complex responses we have discerned a possible role for an archetypic B-1 clone. We speculate that due to their immunogenicity and in vivo distribution the "neo"-self determinants created in oxidatively modified LDL are highly stimulatory for certain B-1 cell clones. These neo-self determinants, which can be created chemically, by somatic processes, may in fact represent the molecular analogues of somatic maturation, or even aging. These changes, including those on non-protein antigens induced by oxidative metabolism, amongst others, create neo-determinants against which the host no doubt can not develop rigorous B-cell tolerance. The onset of expression of these oxidative neo-determinants relatively late in development may well serve a useful function for the highly evolved mammalian immune system, as targeting by evolutionarily selected B-1 clones may facilitate the amplification of other useful antibody-mediated physiologic functions. As in the case of the T15 clone, these antibodies may aid in protection against common microbial pathogens. Hence we postulate that during the evolution of the adaptive immune system the neo-self antigenic milieu may have been exploited for the natural selection of primordial clonal specificities. The T15 B-1 clone may then illustrate a common paradigm in which there has been natural selection based on utility for the defense of the individual from environmental threats, as well as for possible "housekeeping" role(s) and the maintenance of cellular homeostasis.

Human-derived anti-oxidized LDL autoantibody blocks uptake of oxidized LDL by macrophages and localizes to atherosclerotic lesions in vivo.

Autoantibodies to oxidation-specific epitopes of low density lipoprotein (LDL), such as malondialdehyde-modified LDL (MDA-LDL), occur in plasma and atherosclerotic lesions of humans and animals. Plasma titers of such antibodies are correlated with atherosclerosis in murine models, and several such autoantibodies have been cloned. However, human-derived monoclonal antibodies to epitopes of oxidized LDL (OxLDL) have not yet been reported. We constructed a phage display antibody library from a patient with high plasma anti-MDA-LDL titers and isolated 3 monoclonal IgG Fab antibodies, which specifically bound to MDA-LDL. One of these, IK17, also bound to intact OxLDL as well as to its lipid and protein moieties but not to those of native LDL. IK17 inhibited the uptake of OxLDL by macrophages and also bound to apoptotic cells and inhibited their phagocytosis by macrophages. IK17 strongly immunostained necrotic cores of human and rabbit atherosclerotic lesions. When (125)I-IK17 was injected intravenously into LDL receptor-deficient mice, its specific uptake was greatly enriched in atherosclerotic plaques versus normal aortic tissue. Human autoantibodies to OxLDL have important biological properties that could influence the natural course of atherogenesis.

The murine clan V(H) III related 7183, J606 and S107 and DNA4 families commonly encode for binding to a bacterial B cell superantigen.

Superantigens, by virtue of their unconventional binding interactions with Ag receptors, can simulate a large subset of mature lymphocytes in the repertoire. Recent studies have documented that in vivo exposure to the model bacterial B cell superantigen, Staphylococcal protein A (SpA), induces large scale effects on murine B-cell clonal selection by mechanism(s) that include deletion of supra-clonal sets. While the structural bases for the immunomodulatory properties of several T-cell superantigens have been well characterized, the requirements for murine Fab-binding of SpA remain incompletely defined. To investigate these structural requirements, a series of direct binding and inhibition studies were performed with a large panel of Moabs of diverse variable region gene usage. These studies confirm previous reports that superantigen binding is completely restricted to the products of clan V(H) III-related families, that include the small S107 and J606 families, and we also demonstrated that usage of the related small DNA4 family commonly correlates with weaker binding activity. Furthermore, our results document that genes from the largest clan V(H) III family, 7183, commonly encode for Fab-mediated binding of SpA, while antibodies from five other VH families, J558, Q52, Sm7, VH11 and VH12, did not display Fab-mediated SpA binding activity. By contributing to the essential foundation for understanding of the structural basis for binding interactions, these findings will aid interpretation of evolving observations regarding the clonal fates induced by in vivo B-cell superantigen exposure.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis.

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

The incidence of a new human cross-reactive idiotype linked to subgroup VHIII heavy chains.

Cross-reactive idiotypes (CRI) on human rheumatoid factors (RF), which are identified by murine monoclonal antibodies (mAb), have proved useful in defining both the incidence and the structural characteristics of these autoantibodies. In this study, a new murine anti-idiotypic reagent, mAb B6, has been used to identify and define the expression of a distinct heavy chain CRI. The B6 CRI was found on 20% of monoclonal IgM (16 of 81), but on only 5% of monoclonal IgA (1 of 20) and on no monoclonal IgG. In addition, this CRI was expressed exclusively on a subset of Ig derived from the VHIII protein variable region subgroup. In immunoblotting experiments, the mAb B6 bound directly to the heavy (H) chains of CRI positive proteins. The B6 CRI was found frequently on monoclonal IgM-RF molecules, and the mAb B6 could inhibit the binding of the RF to its IgG antigen. It was also demonstrated that Staphylococcus aureus protein A (SpA), which has recently been shown to bind to the F(ab) region of VHIII molecules, could block the interaction of some B6 CRI positive IgM to the anti-CRI. These experiments suggest that the B6 CRI is a marker for one or a few VHIII genes and that it is expressed commonly on IgM paraproteins, many of which have RF activity.

Human kappa light chain subgroup analysis with synthetic peptide-induced antisera.

All human kappa light chains belong to one of four subgroups, classified according to their amino acid sequences or by reactivity with adsorbed heteroantisera. The structural basis for the subgroup distinction by antisera is unknown. Therefore, to create anti-kappa subgroup antibodies with predefined specificity, we immunized rabbits with synthetic peptides which correspond to sequences within the first framework region of prototype kappa I, II, III, and IV light chains. The peptide-induced antisera recognized primary sequence-dependent kappa subgroup determinants. They correctly predicted the amino acid sequence in the first framework region of two kappa light chains. By Western immunoblotting and enzyme-linked immunoassay the antisera also identified previously typed, monoclonal light chains of different subgroups with complete specificity. These reagents, define a site of kappa subgroup distinction and represent a potent tool for the characterization of light chain heterogeneity.

Herpes zoster in patients with treated Wegener's granulomatosis. A possible role for cyclophosphamide.

In review of the ongoing protocol for the treatment of Wegener's granulomatosis with cyclophosphamide at te National Institutes of Health, an increased incidence of herpes zoster infection was noted. There were a total of nine episodes in seven of a total of 65 patients with a 255 patient year follow-up. The infections occurred while the patients were in complete clinical remission during immunosuppressive therapy. Cutaneous dissemination was noted in two episodes, but no visceral or central nervous system involvement was noted despite continuation of immunosuppressive therapy. The major causal factor of the increased incidence of herpes zoster appeared to be the cyclophosphamide therapy.

Human antibody responses to bacterial antigens: studies of a model conventional antigen and a proposed model B cell superantigen.

We have investigated the human antibody repertoires that bind to two different classes of bacterial antigens. Immunization with the conventional antigen, type b capsular polysaccharide of Haemophilus influenzae Hib PS, uniformly induces IgA and IgG responses dominated by clones that use heavy chains structurally related to two subsets of VH3 genes, while in a minority of subjects antibodies from the VH1 or VH4 families are co-induced. In contrast, the "alternative binding site" of Staphylococcal Protein A (SPA) represents an unconventional determinant, because; (i) SPA is bound by a large proportion of non-immune IgM, IgA and IgG F(ab')2, (ii) SPA is bound only to Fab from the VH3 family, which can be encoded by at least four different germline genes, (iii) SPA binding is independent of VL usage, (iv) by flow cytometry SPA is bound by > 15% of tonsilar B cells, but not to T cells. (v) In vitro stimulation with an SPA containing mitogen induces the preferentially production of Ig bearing a VH3 marker. Taken together, these studies characterize a VH family restricted binding interaction that is distinct from the properties associated with conventional antigens such as Hib PS. Based on these data we propose that SPA represents a prototype for a B cell superantigen.

B-cell superantigens: molecular and cellular implications.

B cell superantigens are proteins that are capable of immunoglobulin variable region mediated binding interactions with the naive B cell repertoire at frequencies that are orders of magnitude greater than occur for conventional antigens. Within this review we discuss recent observations regarding the molecular basis of these interactions and the distribution of superantigen binding capacities in different human B cell populations. These findings and current predictions regarding the relevance of these proteins to the physiologic development of immune repertoires are also discussed.