Autoantibodies to the constitutive 73-kD member of the hsp70 family of heat shock proteins in systemic lupus erythematosus.

Serum from patients with systemic lupus erythematosus (SLE) frequently contain IgM and IgG autoantibodies to the constitutively expressed 73-kD/pI 5.5 member of the hsp70 family of heat shock proteins, as determined by one-dimensional (SDS-PAGE) and two-dimensional (IEF/SDS-PAGE) immunoblotting, and by solid-phase SLE Ig immunoprecipitation experiments using hsp70 protein-specific mAbs as probes. Autoantibodies to hsp70 also were detected in a minority of sera from patients with other rheumatic or viral diseases, but not in normal sera. These data may provide additional insight into etiologic and pathophysiologic mechanisms in this and related autoimmune disorders.

A depression of cell-mediated immunity to measles antigen in patients with systemic lupus erythematosus.

Using the direct migration inhibition test, response to measles antigen in patients with systemic lupus erythematosus (SLE) was found to be decreased when compared with that of normal subjects. No alteration was observed in similar experiments using parainfluenza type 1 and rubella antigens. The specific decrease in measles antigen effect showed no obvious correlation with activity of SLE or with the presence of lymphocytotoxic antibodies. Whether the specificity of the decrease in reactivity is due to some particular relationship between the measles virus or antigen and SLE, or to the possibility that measles reactivity is a more sensitive indicator of a generalized defect of cell-mediated immunity, remains unclear.

Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus.

Nearly one-third of IgM antilymphocyte autoantibody-positive sera from patients with systemic lupus erythematosus (SLE) contain IgM antibodies to one or more 180-220-kD molecules (p180, p190, p205, and p220) in blots of glycoproteins purified from T cells by wheat germ agglutinin affinity chromatography. Identity of these IgM targets with multiple isoforms of CD45 was established by their specific depletion from T cell glycoproteins by immunoprecipitation with T191, a monoclonal antibody (mAb) that reacts with an epitope common to all CD45 isoforms. Although the anti-CD45 autoantibodies recognize higher molecular weight isoforms primarily, antigenic specificity in this system is quite heterogeneous and includes multiple distinct CD45 isoforms on different types of T cells that are, at least in part, different from those reactive with mAbs 2H4 and UCHL-1. Because CD45 is a major membrane protein tyrosine phosphatase that plays a critical role in antigen-induced T cell activation, the present data may be relevant to some of the antilymphocyte antibody-mediated immunologic abnormalities that characterize SLE and related autoimmune diseases.

Constitutive expression of a groEL-related protein on the surface of human gamma/delta cells.

Rabbit antibodies to hsp58 (P1), the human homologue of the Escherichia coli stress protein groEL, react specifically in indirect immunofluorescence and complement-dependent microcytoxicity experiments with a cell surface antigen expressed constitutively by T cell lines bearing gamma/delta receptors. This anti-hsp58-reactive antigen is not demonstrable on T cells that express alpha/beta receptors or on various cells that lack T cell receptors. Certain evidence was obtained to suggest that the target antigen on the surface of gamma/delta T cells is a approximately 77-kD protein distinct from intracellular hsp58 and known members of the hsp70 stress protein family. While the exact nature and significance of this anti-hsp58-reactive protein remain to be determined, these data may help to clarify the roles of groEL-related stress proteins and gamma/delta cells that recognize groEL homologous in immunologic defense against infection and in autoimmune disease.

Inhibition of soluble antigen-induced T cell proliferation by warm-reactive antibodies to activated T cells in systemic lupus erythematosus.

One of the fundamental immunologic characteristics of systemic lupus erythematosus (SLE) is a depressed T cell proliferative response to various specific and nonspecific stimuli. Both intrinsic cellular defect(s) and inhibitory influences of humoral factors, e.g., antilymphocyte autoantibodies or immune complexes, have been postulated to underly this functional abnormality. Because patient serum can induce SLE-like T cell dysfunction in normal cells, an extrinsic mechanism is probably responsible, but the nature and site of action of this humoral activity has not been defined. This laboratory recently described a novel antibody specific for activated T cells in SLE, which raised the possibility that suppression of T cell proliferation by SLE serum involved antibodies directed to surface determinants expressed during the process of activation. In experiments to examine this concept further, relatively warm-reactive antibodies to T cell blasts were found to inhibit strongly the well-characterized T cell response to tetanus toxoid. These antibodies were distinct from conventional cold-reactive IgM antibodies to resting T cells, which exhibited little inhibitory activity. Inhibition involved noncytotoxic effects on early activation events at the level of the responding T cell, which markedly reduced the expression of receptors for interleukin 2. Inhibitory effects on antigen-pulsed macrophages or on T cells already committed to proliferate were not demonstrable. Anti-T blast antibodies were characteristic of active SLE and were detected only occasionally in patients with inactive disease or non-SLE rheumatic disorders. Although the exact antigenic specificity was not identified, considerable evidence was obtained against the presence of antibodies to Ia and certain other surface determinants of functional relevance. Our observations concerning the suppressive effects of anti-T blast antibodies in SLE serum on the T cell response to tetanus toxoid should provide new insight into mechanisms of in vivo T cell dysfunction in this and other immunologic disorders.

Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. Association of high avidity antinative DNA antibody with glomerulonephritis.

Significant differences in both specificity and avidity of anti-DNA antibodies were observed in the sera of groups of patients with active systemic lupus erythematosus glomerulonephritis, active systemic lupus erythematosus without nephritis, and in IgG eluates obtained by DNAase digestion of isolated glomeruli from glomerulonephritic kidneys. With methylated albumin-kieselguhr fractionated 3H-HeLa DNA as a source of native or single-strand DNA antigen in a modified Farr assay, an increased level of antibody to native DNA was associated with active systemic lupus erythematosus, particularly active nephritis. The avidity of antinative DNA estimated from plots of the reciprocals of bound and free antigen according to the Sips distribution formula was significanly lower in active glomerulonephritis sera than in sera from patients with active systemic lupus erythematosus without nephritis. However, antinative DNA of uniformly high avidity was found in the glomerular eluates. Avidity of single-strand DNA antibodies did not differ in the various patient groups. The data stronly supprot a major role for high avidity antinative-DNA in DNA/antiDNA immune complex-induced glomerular injury in systemic lupus erythematosus.

Specific concentration of polynucleotide immune complexes in the cryoprecipitates of patients with systemic lupus erythematosus.

Although the association of cryoglobulinemia with hypocomplementemia and tissue injury in systemic lupus erythematosus is well recognized, composition of cryoprecipitates in terms of circulating antigens and antibodies in this disease is less clear. To clarify this question, cryoprecipitates from patients with SLE were examined with sensitive assay techniques for certain antipolynucleotide antibodies and DNA antigen. DNA antibodies were highly enriched relative to serum levels in the majority of cryoprecipitates. DNA antigen was also demonstrable. Antibody to ribonucleoprotein, although less frequently present, was similarly enriched in certain cryoprecipitates. In contrast, anti-double strand RNA, which was commonly detectable in relatively high titer in serum, was only minimally concentrated in a minority of cryoprecipitates. Absorption experiments using red blood cells heavily coated with polynucleotide antigen indicated that a major proportion of the IgG in certain cryoprecipitates was specific antibody. The data strongly suggest that the cryoprecipitates in systemic lupus erythematosus represent circulating immune complexes that are soluble at 37 degrees C and come out of solution in the cold. The marked concentration of immune complexes in the cryoglobulin offers a simple and direct method for determination of the nature of the complexes. The accumulated evidence obtained in the present study indicates that these complexes closely reflect, in their composition, the circulating immune complexes which are most significant pathogenetically in renal tissue injury.

Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I.

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.

Analyses of lymphocytes from patients with rheumatoid arthritis and systemic lupus erythematosus. Occurrence of interfering cold-reactive antilymphocyte antibodies.

Large percentages of the lymphocytes from some patients with rheumatoid arthritis and systemic lupus erythematosus were densely covered with Ig demonstrable by immunofluorescence, which was occasionally present in the form of caps. The amount and character of the Ig staining depended largely on the procedures used in the isolation and washing of the lymphocytes. Cold-reactive antilymphocyte antibodies present in many sera wre primarily responsible for these variations. Overnight culture of the lymphocytes proved to be an efficient procedure for the removal of adsorbed antibody. Some evidence was also obtained for the presence of circulating immune complexes and exogenous rheumatoid factor molecules on the lymphocyte surface. Thus on freshly isolated cells the demonstration of surface Ig proved to an unreliable marker of bone marrow-derived (B) cells in these disease: the actual percent of B cells with intrinsic surface Ig was often markedly decreased. In patients with systemic lupus erythematosus, this reduction was in agreement with the low numbers of cells that had a receptor for aggregated IgG. The mean percentage of thymus-derived (T) cells in both diseases was slightly greater than the normal level.The concentrations of lymphocytes in joint fluids from patients with rheumatoid arthritis were often greater than levels found in blood. T cells primarily accounted for this increase. The T cells typically formed unusually dense rosettes with sheep erythrocytes. B lymphocytes were proportionally much diminished. Evidence was obtained for the existence of a major joint fluid lymphocyte population that lacked all assayed surface markers.

Stress proteins, autoimmunity, and autoimmune disease.

At birth, the immune system is biased toward recognition of microbial antigens in order to protect the host from infection. Recent data suggest that an important initial line of defense in this regard involves autologous stress proteins, especially conserved peptides of hsp60, which are presented to T cells bearing gamma delta receptors by relatively nonpolymorphic class lb molecules. Natural antibodies may represent a parallel B cell mechanism. Through an evolving process of "physiological" autoreactivity and selection by immunodominant stress proteins common to all prokaryotes, B and T cell repertoires expand during life to meet the continuing challenge of infection. Because stress proteins of bacteria are homologous with stress proteins of the host, there exists in genetically susceptible individuals a constant risk of autoimmune disease due to failure of mechanisms for self-nonself discrimination. That stress proteins actually play a role in autoimmune processes is supported by a growing body of evidence which, collectively, suggests that autoreactivity in chronic inflammatory arthritis involves, at least initially, gamma delta cells which recognize epitopes of the stress protein hsp60. Alternate mechanisms for T cell stimulation by stress proteins undoubtedly also exist, e.g., molecular mimicry of the DR beta third hypervariable region susceptibility locus for rheumatoid arthritis by a DnaJ stress protein epitope in gram-negative bacteria. While there still is confusion with respect to the most relevant stress protein epitopes, a central role for stress proteins in the etiology of arthritis appears likely. Furthermore, insight derived from the work thus far in adjuvant-induced arthritis already is stimulating analyses of related phenomena in autoimmune diseases other than those involving joints. Only limited data are available in the area of humoral autoimmunity to stress proteins. Autoantibodies to a number of stress proteins have been identified in SLE and rheumatoid arthritis, but their pathogenetic significance remains to be established. Nevertheless, the capacity of certain stress proteins to bind to multiple proteins in the nucleus and cytoplasm both physiologically and during stress or injury to cells, suggests that stress proteins may be important elements in the "immunogenic particle" concept of the origin of antinuclear and other autoantibodies. In short, this fascinating group of proteins, so mysterious only a few years ago, has impelled truly extraordinary new lines of investigation into the nature of autoimmunity and autoimmune disease.

Intrathecal IgG synthesis and blood-brain barrier impairment in patients with systemic lupus erythematosus and central nervous system dysfunction.

Paired serum and cerebrospinal fluid specimens from 19 patients with SLE and central nervous system dysfunction were studied with respect to cerebrospinal fluid IgG index (a measure of intrathecal IgG synthesis), isoelectric focusing using immunoperoxidase staining techniques to detect oligoclonal IgG, and determination of the cerebrospinal fluid/serum albumin quotient (Q albumin) as a measure of blood-brain barrier integrity. Twenty-five patients without neurologic disease and 70 patients with a variety of non-SLE neurologic disorders were also studied for comparison. Of most interest was the observation that 42 percent of the patients with SLE had cerebrospinal fluid oligoclonal IgG, usually in association with elevation of the cerebrospinal fluid IgG index. In addition, two of the cerebrospinal fluid specimens that exhibited oligoclonal IgG also had increased titers of alpha-interferon. Q albumin was normal (under 9.0) in 12 of 13 patients with SLE, who had seizure, psychosis, or cranial neuropathy as principal central nervous system manifestations (mean +/- SD = 5.3 +/- 2.4), but was significantly elevated (mean +/- SD = 27.4 +/- 18.8, p less than 0.001) in five of six patients with diffuse, major central nervous system injury, for example, encephalopathy with coma, transverse myelopathy, paraparesis. Blood-brain barrier impairment was not correlated either with presence of circulating immune complexes or with other clinical or serologic evidence for extra-central nervous system disease activity. Taken together, the data suggest that, within the limitations of the techniques used, impairment of the blood-brain barrier in SLE may be secondary to the central nervous system lesion, rather than a result of systemic immune complex injury. In addition, substantial evidence is provided for an ongoing humoral immune response within the central nervous system in this disorder, which, in certain patients, may be associated with the production of intrathecal alpha-interferon.

Studies of twins with systemic lupus erythematosus. A review of the literature and presentation of 12 additional sets.

To assess the role of genetic factors in systemic lupus erythematosus (SLE), 12 twon pairs (seven definitely monozygotic, three definitely dizygotic) of which one or both twins had SLE, were studied and compared to 17 twin pairs (12 definitely monozygotic) previously described. In the present series, four of seven (57 per cent) definitely monozygotic pairs were clinically concordant for SLE, satisfying the preliminary criteria of the American Rheumatism Association (ARA). Concordance for the presence of antinuclear factor (ANF) and hypergammaglobulinemia was 71 and tinuclear factor (ANF) and hypergammaglobulinemia was 71 and 87 per cent, respiectively. These data closely agree with those on the 12 definitely monozygotic sets previously described. All three of the dizygotic sets in the present series were discordant for clinical SLE, although one clinically well twin had marked serologic abnormalities. Comparison of these data with thos from other first degree relatives of out twins clearly suggests a strong genetic component in the pathogenesis of SLE. The relative contribution of nongenetic and environmental factors to the expression of the disease is discussed.

Utility of protease-digested human peripheral blood lymphocytes for the detection of lymphocyte-reactive alloantibodies by indirect immunofluorescence.

Human peripheral blood lymphocytes were digested briefly with protease prior to application of indirect immunofluorescence techniques for detecting alloantibodies in sera of patients with chronic renal failure on maintenance hemodialysis. Background staining of intrinsic surface IgM and cytophilic IgG bound to Fc receptors was eliminated or greatly reduced, enabling detection of B cell specific antibodies, including cold-reactive types not demonstrable by conventional immunofluorescence or complement-dependent lymphocytotoxicity. The antigenicity of HLA and other surface membrane determinants was not decreased by protease, although reactivity with certain sera was enhanced. In experiments comparing indirect immunofluorescence using protease-treated cells with complement-dependent lymphocytotoxicity and antibody-dependent, lymphocyte-mediated cytotoxicity assays, indirect immunofluorescence was more sensitive and comprehensive, but not less specific, in defining alloantibodies of a variety of types.

The 8.5-kb PstI allele of the stress protein gene, Hsp70-2: an independent risk factor for systemic lupus erythematosus in African Americans?

SLE is dramatically more prevalent in persons of African descent than in other populations. Several genes in the class III region of the MHC have been considered as potential susceptibility loci for this disorder, but the primary association(s) remains unknown. The stress protein gene, hsp70-2, is of special interest in this regard because it encodes a protein functionally relevant to antigen processing and presentation and has itself been identified as a putative susceptibility locus in organ-specific autoimmune diseases in Caucasians. To clarify the relationship of the hsp70-2 gene to SLE in African Americans, genomic DNA from 46 patients and 42 appropriately matched control subjects was analyzed for an RFLP of the hsp70-2 gene using the probe pH2.3 and the restriction endonuclease PstI, which identifies alleles of 8.5 and 9.0 kb. The 8.5-kb hsp70-2 allele was associated with SLE in this population (X2 = 8.2473, p = 0.0044). This association was not due to linkage disequilibrium with the C4A deletion or with HLA-DR3, as has been reported in Caucasians with IDDM. These data suggest that the 8.5-kb hsp70-2 allele may be an independent susceptibility marker for SLE in African Americans.

Specificity of autoantibodies to histone H1 in SLE: relationship to DNA-binding domains.

Autoantibodies in the sera of patients with systemic lupus erythematosus were examined with respect to their specificity for proteolytic fragments of histone H1 that retain, or do not retain, DNA-binding domains. 16 of 31 sera contained IgG and IgM antibodies to histone H1. IgM antibodies to H1 in 8 sera (50%) were directed at 18 kD and 20 kD alpha-chymotrypic H1 fragments that bore binding sites for DNA, as identified by staining immunoblots containing the fragments with ssDNA plus 6/0, a mouse monoclonal antibody against ssDNA, IgM with this type of histone H1 specificity did not react with comparably-sized V8 protease fragments of H1. IgM antibodies to H1 in the other patients were directed against entirely different epitopes which were preserved in V8 protease digests of H1. In serial studies of three patients during different phase of their SLE, the level of antibodies against the 18 kD and 20 kD histone H1 fragments varied in parallel with the level of anti-ssDNA antibodies in one and varied inversely in the other two. The data suggest that a significant proportion of autoantibodies to histone H1 are directed at a limited number of epitopes localized to H1 fragments containing DNA-binding sites.

Cryoglobulinemia.

A large number of disease states are characterized by cryoglobulinemia. Quantification and immunochemical classification of cryoglobulins in serum provide information of diagnostic and pathophysiologic utility. Thus, type I cryoglobulins consist of a monoclonal immunoglobulin of a single class and are associated with lymphoproliferative disorders, such as multiple myeloma. Type II (mixed) cryoglobulins contain monoclonal IgM or rheumatoid factor and polyclonal IgG, and occur in patients with Waldenstrom's macroglobulinemia or chronic active hepatitis, for example. In type III cryoglobulins, both the IgM and rheumatoid factor and the IgG components are polyclonal. A large number of autoimmune or infectious diseases exhibit type III cryoglobulinemia. In certain well-studied situations, type II and type III cryoglobulins have been shown to contain antigen-antibody complexes directly involved in tissue injury in vivo, e.g., DNA and anti-DNA in systemic lupus erythematosus.

Pain in fibromyalgia.

Just as our caveman forebears were frail in the face of predatory animals, we are frail in today's society of childhood neglect or abuse, bumper-to-bumper traffic, frustration at work, and multiple daily hassles. The same neuroendocrine systems and pain regulatory mechanisms that protected early man during acute stress are still encoded in our genome, but may be maladaptive in psychologically and physiologically vulnerable people faced with chronic stress. Many patients with fibromyalgia become vulnerable because of the long-lasting psychological and neurophysiological effects of negative experiences in childhood. Ill-equipped with positive cognitive, emotional, and behavioral skills as adults, they display maladaptive coping strategies, low self-efficacy, and negative mood when confronted with the inevitable stressors of life. Psychological distress ensues, which reduces thresholds for pain perception and tolerance (already relatively low in women) even further. Converging lines of psychological and neurobiological evidence strongly suggest that chronic stress-related blunting of the HPA, sympathetic, and other axes of the stress response together with associated alterations in pain regulatory mechanisms may finally explain the pain and fatigue of fibromyalgia. Vulnerable people who can be classified by the ACR criteria as having fibromyalgia do not have a discrete disease. They are simply the most ill in a continuum of distress, chronic pain, and painful tender points in the general population.

Autoregulation of Tcr V region epitopes in autoimmune disease.

Normal individuals possess low levels of autoantibodies specific for certain peptide defined regions of T-cell receptor (Tcr) variable regions, particularly CDR1 and Fr3. These regions are predicted to be exposed on the surface of the native molecule and, by analogy and comparison with immunoglobulins, correspond to public idiotype determinants. The anti-Tcr idiotype antibodies appear to be ubiquitous and we propose that they play a role in the regulation of T-cell function. To delineate the parameters of expression of these antibodies, we characterized anti-Tcr antibody activity in normal individuals, in those suffering from the autoimmune diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and in patients with non-autoimmune arthritis (osteoarthritis) as a disease control. There were significant increases in autoantibody levels in the autoimmune patients. There was also variation in isotype and the particular variable regions recognized. IgM autoantibodies directed against a few peptide defined determinants were elevated in RA, whereas SLE patient sera showed high levels of IgG binding to a broad spectrum of Tcr peptides.

The University of North Carolina Arthritis Center.

The University of North Carolina Arthritis Center combines the broadly-based research agenda of the Thurston Arthritis Research Center with comprehensive interdisciplinary clinical programs in rheumatology, orthopaedics, and pediatric rheumatology. In keeping with the University's long tradition of service to the people of North Carolina, a primary aim of the Center is to provide the citizens of this state with the best available arthritis care and prevention strategies. The approach here is twofold. New knowledge is created by laboratory investigation of basic disease mechanisms, by clinical studies of new therapies, by social and behavioral research to better understand how patients and their families cope and adjust to chronic arthritis, and by health services research that examines arthritis from a societal perspective. This information, together with advances in rheumatology and related fields from Duke and other institutions, is then applied to optimum clinical and educational services for North Carolina patients and their physicians.

Studies on antilymphocyte antibodies in patients with rheumatoid arthritis and systemic lupus erythematosus.

Anti-lymphocyte antibodies have been demonstrated in patients with SLE and RA by immunofluorescence. They differ from those arising through iso-immunization by being cold reactive and chiefly of the IgM class. Separate anti specificities were shown for determinants on either T or B cells.