T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor zeta chain messenger RNA.

OBJECTIVE: T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells. METHODS: TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. RESULTS: DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. CONCLUSION: These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.

Protein kinase A RI beta subunit deficiency in lupus T lymphocytes: bypassing a block in RI beta translation reconstitutes protein kinase A activity and augments IL-2 production.

A profound deficiency of type I protein kinase A (PKA-I or RIalpha/beta2C2) phosphotransferase activity occurs in the T lymphocytes of 80% of subjects with systemic lupus erythematosus (SLE), an autoimmune disorder of unknown etiology. This isozyme deficiency is predominantly the product of reduced or absent beta isoform of the type I regulatory subunit (RIbeta). Transient transfection of RIbeta cDNAs from SLE subjects into autologous T cells that do not synthesize the RIbeta subunit bypassed the block, resulting in RIbeta subunit synthesis and restoration of the PKA-Ibeta (RIbeta2C2) holoenzyme. Transfected T cells activated via the T cell surface receptor complex revealed a significant increase of cAMP-activatable PKA activity that was associated with a significant increase in IL-2 production. These data demonstrate that a disorder of RIbeta translation exists, and that correction of the PKA-I deficiency may enhance T lymphocyte effector functions in SLE.

Fc epsilon receptor type I gamma chain replaces the deficient T cell receptor zeta chain in T cells of patients with systemic lupus erythematosus.

OBJECTIVE: T cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T cell receptor zeta chain, a critical signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative signaling mechanisms. We investigated whether Fcepsilon receptor type I gamma chain (FcepsilonRIgamma) could substitute for TCR zeta chain and contribute to T cell signaling in SLE. METHODS: T cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcepsilonRIgamma was investigated using immunoblotting, reverse transcriptase-polymerase chain reaction, and flow cytometry methods. Involvement of the FcepsilonRIgamma in T cell signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation. RESULTS: Western blotting and densitometric analysis showed that the expression of FcepsilonRIgamma in SLE T cells was 4.3-fold higher than in normal T cells (P < 0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcepsilonRIgamma+,CD3+, FcepsilonRIgamma+,CD4+, and FcepsilonRIgamma+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P < 0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcepsilonRIgamma antibody showed that FcepsilonRIgamma associates with the tyrosine kinase Syk and the CD3epsilon chain, suggesting that FcepsilonRIgamma is functionally involved in TCR signaling. CONCLUSION: These results demonstrate that the FcepsilonRIgamma chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcepsilonRIgamma chain in SLE T cells may account in part for the aberrant antigen receptor-initiated signaling and contribute to the diverse cellular abnormalities found in this disease.

Polymorphisms/mutations of TCR-zeta-chain promoter and 3' untranslated region and selective expression of TCR zeta-chain with an alternatively spliced 3' untranslated region in patients with systemic lupus erythematosus.

A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR zeta-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR zeta-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3' untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P = 0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P = 0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a -76T insertion compared to the published sequence, in all of the SLE samples and controls. RT-PCR analysis of the TCR zeta-chain 3' untranslated region showed a 344 bp product in addition to the expected 906 bp product. Cloning and sequence analysis of the 344 bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672-1233 of TCR zeta-chain mRNA. Unlike the nomal TCR zeta-chain, the expression of TCR zeta-chain with the alternatively spliced 344 bp 3' untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906 bp TCR zeta-chain 3' untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P = 0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3' untranslated region may affect the stability and/or transport of TCR zeta-chain mRNA, leading to its downregulation in SLE T cells.

Molecular basis of deficient IL-2 production in T cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by diverse cellular and biochemical aberrations, including decreased production of IL-2. Here we show that nuclear extracts from unstimulated SLE T cells, unlike extracts from normal T cells, express increased amounts of phosphorylated cAMP-responsive element modulator (p-CREM) that binds the -180 site of the IL-2 promoter. Nuclear extracts from stimulated normal T cells display increased binding of phosphorylated cAMP-responsive element binding protein (p-CREB) to the -180 site of the IL-2 promoter, whereas nuclear extracts from stimulated SLE T cells display primarily p-CREM and decreased p-CREB binding. In SLE T cells, p-CREM bound to the transcriptional coactivators, CREB binding protein and p300. Increased expression of p-CREM correlated with decreased production of IL-2. The transcription of a reporter gene driven by the -180 site was enhanced in normal T cells, but was suppressed in SLE T cells. These experiments demonstrate that transcriptional repression is responsible for the decreased production of IL-2 by SLE T cells.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-gamma gene and protein expression in lupus T cells.

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-gamma). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-gamma genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-gamma gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-gamma production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Molecular aberrations in human systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disorder that predominantly affects women during the childbearing years. Clinically, major organ systems are affected, including the skin, kidneys and nervous system. Genetic, hormonal, environmental and immunoregulatory factors contribute to the highly variable expression of the disease. Impaired cellular and humoral immune responses reflect disordered biochemical and molecular functions that might be determined genetically. Enhanced understanding of these molecular abnormalities should enable development of new, effective therapeutic agents in the near future.

mRNA mutations of type I protein kinase A regulatory subunit alpha in T lymphocytes of a subject with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by multiple T lymphocyte immune effector dysfunctions. Protein kinase A (PKA) isozymes contribute to the regulation of T cell immune effector functions. In SLE T cells, there is a profound deficiency of PKA-I isozyme activity characterized by both reduced RI alpha transcript and RI alpha protein levels. To identify a molecular mechanism(s) for this isozyme deficiency, we utilized single-strand conformation polymorphism (SSCP) analysis to detect structural changes in the cDNA. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band. Sequence analyses demonstrated that a shifted SSCP band from SLE T cells carried heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats-regions that are susceptible to transcript editing and/or molecular misreading. By contrast, no genomic mutations were identified. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in a subject with SLE. Mutant RI alpha transcripts are pathophysiolgically significant, for they can encode diverse, aberrant RI alpha isoforms, including truncated, dominant-negative subunits, resulting in deficient PKA-I activity. We propose that deficient PKA-I isozyme activity contributes to the pathogenesis of SLE by hindering effective signal transduction and impairing T cell effector functions.

Systemic lupus erythematosus in the elderly.

Aging modifies the clinical presentation and course of autoimmune disorders, although the mechanisms by which this occurs remain to be determined. Current evidence cited above supports the general concept that there is a natural senescence of the immune system. This evidence would suggest that somehow senescence directly affects gene expression, resulting in biochemical abnormalities that culminate in T-cell immunodysfunctions. This may be a principal factor that attenuates the autoimmune response to self-antigen and, therefore, the disease course. The authors speculate that there is a disorder primary to the T cell in SLE that is expressed as abnormal immunologic responses to self-antigens, resulting in autoimmunity. Although understanding of this primary T cell disorder is still limited, clinicians now know that the T cell harbors abnormal signaling pathways that reflect defective biochemical functions and seem to be genetically regulated. This aberrant signaling would be anticipated to affect both principal T cell subsets. It may hinder the capacity of cells, such as CD8 T cells, to effectively down-regulate the response of autoreactive CD4 helper T cells to autoantigens. Loss of self-regulation would manifest itself as loss of tolerance, a fundamental component of autoimmunity. The future challenge is to understand how aberrant signaling leads to loss of tolerance. Given this underlying genetic susceptibility in an aged individual whose T cells also are undergoing natural senescence, the authors suggest that it is conceivable that a stress factor may tip the balance in the favor of clinical disease. One such factor may be unspecified environmental stimuli. Yet another consideration is an intercurrent illness, such as an infection. It remains to be determined, however, what these environmental stimuli are and how they impact on the immune system to trigger disease.

Association of deficient type II protein kinase A activity with aberrant nuclear translocation of the RII beta subunit in systemic lupus erythematosus T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by abnormal T cell signal transduction and altered T cell effector functions. We have previously observed a profound deficiency of total protein kinase A (PKA) phosphotransferase activity in SLE T cells. Here we examined whether reduced total PKA activity in SLE T cells is in part the result of deficient type II PKA (PKA-II) isozyme activity. The mean PKA-II activity in SLE T cells was 61% of normal control T cells. The prevalence of deficient PKA-II activity in 35 SLE subjects was 37%. Deficient isozyme activity was persistent over time and was unrelated to SLE disease activity. Reduced PKA-II activity was associated with spontaneous dissociation of the cytosolic RIIbeta2C2 holoenzyme and translocation of the regulatory (RIIbeta) subunit from the cytosol to the nucleus. Confocal immunofluorescence microscopy revealed that the RIIbeta subunit was present in approximately 60% of SLE T cell nuclei compared with only 2-3% of normal and disease controls. Quantification of nuclear RIIbeta subunit protein content by immunoprecipitation and immunoblotting demonstrated a 54% increase over normal T cell nuclei. Moreover, the RIIbeta subunit was retained in SLE T cell nuclei, failed to relocate to the cytosol, and was associated with a persistent deficiency of PKA-II activity. In conclusion, we describe a novel mechanism of deficient PKA-II isozyme activity due to aberrant nuclear translocation of the RIIbeta subunit and its retention in the nucleus in SLE T cells. Deficient PKA-II activity may contribute to impaired signaling in SLE T cells.

Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression.

Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

High prevalence of T cell type I protein kinase A deficiency in systemic lupus erythematosus.

OBJECTIVE: To estimate the prevalence of protein kinase A type I isozyme (PKA-I) deficiency in a cohort of systemic lupus erythematosus (SLE) patients, and to establish whether the isozyme deficiency is associated with SLE disease activity. METHODS: Thirty-five SLE patients and 35 age-, sex-, and race-matched normal controls were studied. Fifteen subjects were restudied on at least 3 occasions over a 4-year interval. Clinical disease activity was estimated by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and the T cell activation markers CD25+ and HLA-DR+ were quantified by flow cytometry. PKA-I isozyme activities were quantified in enriched T cells. Statistical analyses were performed by Student's t-test, Mann Whitney U test, and Pearson product moment test. RESULTS: The mean PKA-I activity in SLE T cells (540 pmoles/minute/mg of protein) was significantly lower than that in control T cells (1,578 pmoles/ minute/mg of protein) (P<0.001). The prevalence of isozyme deficiency in this cohort was 80%. During a 4-year interval, PKA-I activities remained significantly reduced, whereas SLEDAI scores significantly improved. There was no relationship between deficient PKA-I activity and either SLEDAI scores or the proportion of T cells bearing CD25+ or HLA-DR+ activation markers. CONCLUSION: There is a high prevalence of deficient T cell PKA-I isozyme activity in SLE that persists over time and is independent of SLE disease activity.

Association analysis of exonic variants of the gene encoding the GABAB receptor and idiopathic generalized epilepsy.

The gene encoding the GABAB receptor (GABABR1) maps close to the HLA-F locus on chromosome 6p21.3 in the same region to which a major susceptibility locus for common subtypes of idiopathic generalized epilepsy (IGE), designated as EJM1, has been localized. Moreover, animal models suggest that the GABAB receptor plays a critical role in the epileptogenesis of absence seizures. Accordingly, the present association study tested the candidate gene hypothesis that genetic variants of the human GABABR1 gene confer susceptibility to common subtypes of IGE. Three DNA sequence variants in exons 1a1, 7, and 11 of the GABABR1 gene were assessed by PCR-based restriction fragment length polymorphisms in 248 unrelated probands of German descent, comprising 72 patients with juvenile myoclonic epilepsy (JME), 46 patients with idiopathic absence epilepsy (IAE), and 130 control subjects without a history of epileptic seizures and lack of generalized spike-wave discharges in their electroencephalogram. The results revealed no evidence for an allelic association of any of the GABABR1 sequence variants with either JME or IAE (P > 0.18). Thus, we failed to demonstrate that any of the three exonic GABABR1 variants themselves, or other so-far unidentified mutations, which are in strong linkage disequilibrium with the investigated variants, are involved in the pathogenesis of common IGE subtypes.

Diminished levels of protein kinase A RI alpha and RI beta transcripts and proteins in systemic lupus erythematosus T lymphocytes.

Deficient type I protein kinase A phosphotransferase activity occurs in the T cells of 80% of subjects with systemic lupus erythematosus (SLE). To investigate the mechanism of this deficient isozyme activity, we hypothesized that reduced amounts of type I regulatory (RI) isoform transcripts, RIalpha and RIbeta, may be associated with a diminution of RIalpha and/or RIbeta protein. Sixteen SLE subjects with a mean (+/-1 SD) SLE disease activity index of 12.4 +/- 7.2 were studied. Controls included 16 normal subjects, six subjects with primary Sjögren's syndrome (SS), and three subjects with SS/SLE overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells expressed mRNAs for all seven R and catalytic (C) subunit isoforms. Quantification of mRNAs by competitive PCR revealed that the ratio of RIalpha mRNA to RIbeta mRNA in normal T cells was 3.4:1. In SLE T cells there were 20 and 49% decreases in RIalpha and RIbeta mRNAs (RIbeta; p = 0.008), respectively, resulting in an RIalpha:RIbeta mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in RIbeta mRNA compared with normal controls (p = 0.01). Immunoblotting of normal T cell RIalpha and RIbeta proteins revealed a ratio of RIalpha:RIbeta of 3.2:1. In SLE T cells, there was a 30% decrease in RIalpha protein (p = 0.002) and a 65% decrease in RIbeta protein (p < 0.001), shifting the ratio of RIalpha:RIbeta protein to 6.5:1. T cells from 25% of SLE subjects lacked any detectable RIbeta protein. Analysis of several lupus T cell lines demonstrated a persistent deficiency of both proteins, excluding a potential effect of disease activity. In conclusion, reduced expression of RIalpha and RIbeta transcripts is associated with a decrement in RIalpha and RIbeta proteins and may contribute to deficient type I protein kinase A isozyme activity in SLE T cells.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane.

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4(+) CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Clinical expression of autoimmune diseases in older adults.

Aging modifies the clinical presentation and course of autoimmune disorders. In the older person who carries a complement of predisposing genes, environmental factors may alter a senescent immune system and trigger the onset of autoimmunity. In this article about autoimmune diseases of the older adult, the authors discuss the epidemiology, clinical presentations, laboratory and radiographic findings, and management of systemic lupus erythematosus, primary Sjogren's syndrome, and idiopathic inflammatory myopathies.

Mapping, genomic structure, and polymorphisms of the human GABABR1 receptor gene: evaluation of its involvement in idiopathic generalized epilepsy.

Neurophysiological and pharmacological studies suggest a major role of the GABAB receptor in the epileptogenesis of absence seizures. The gene encoding the human GABABR1 receptor (GABABR1) has recently been mapped to human chromosome 6p21.3 by in situ hybridization, a region that harbors a susceptibility locus (EJM1) for idiopathic generalized epilepsy (IGE). We investigated the hypothesis that the GABABR1 gene (GABBR1) represents a candidate gene for EJM1 by: (1) defining the precise localization approximately 130 kilobases telomeric to the HLA-F locus, (2) by characterizing its genomic organization, and (3) by mutation screening of the entire coding region of GABBR1 in 18 German patients with juvenile myoclonic epilepsy (JME) who were derived from families with evidence for linkage to chromosome 6p21.3 (cumulative lod score Z=3.17 at HLA-DQ). The GABAB receptor gene consists of 22 translated exons. The two alternative transcripts, GABABR1a and GABABR1b, are derived from the same locus but they differ in their alternative 5'-exons. Mutation analyses in JME revealed several DNA sequence polymorphisms, two of which result in amino acid changes occurring in all IGE-affected members of two families. However, clinically unaffected relatives did carry the same variations, excluding these amino acid substitutions as the cause for IGE in these families.

Familial clustering of end-stage renal disease in blacks with lupus nephritis.

The factors that determine a patient's susceptibility to specific target organ involvement in systemic lupus erythematosus (SLE) remain unknown. Lupus nephritis can be a particularly devastating complication, with an increased mortality and the risk of progressive renal damage resulting in end-stage renal disease (ESRD). This analysis was performed to determine whether renal disease aggregated in select families or was a sporadic complication in patients with SLE. We compared the family history of ESRD in 50 patients with SLE complicated by lupus nephritis with 37 controls who had SLE but lacked nephritis after a mean follow-up duration of more than 11 years. The frequency of relatives with ESRD in the lupus nephritis cases was compared with that in controls using Fisher's exact test (significance at P < or = 0.05). Fifty percent (25) of the 50 lupus nephritis patients were black and 50% (25) white, in contrast to 35% (13) and 65% (24) of the 37 lupus non-nephropathy controls, respectively. A first-, second-, or third-degree relative with ESRD was present in 16% (eight) of the 50 lupus nephritis cases and in 0% of the 37 SLE non-nephropathy controls (P = 0.019, Fisher's exact test, two-tail). Twenty-eight percent (seven) of the 25 black patients with lupus nephritis had relatives with ESRD compared with 0% of the 13 black lupus non-nephritis controls (P = 0.07). Only one of the eight relatives with ESRD had SLE or a collagen vascular disease. Lupus nephritis patients and the non-nephritis controls had similar ages (mean +/- SD: 38.5 +/- 10.0 years v 46.6 +/- 11.8 years; P = 0.28), family sizes (6.27 +/- 2.61 first-degree relatives v 6.35 +/- 3.25 first-degree relatives; P = 0.16), and duration of SLE (9.26 +/- 5.94 years v 11.35 +/- 6.43 years; P = 0.60). Familial clustering of ESRD was observed in black patients with SLE who had nephritis. This was unlikely to be related to differences in patient age, family size, or duration of SLE. This data, coupled with the known familial aggregation of ESRD in blacks with hypertensive and diabetic ESRD, supports the contention that genetic factors contribute to the familial clustering. The presence of relatives with etiologies of ESRD other than SLE suggests that there is an inherited susceptibility to progressive renal failure, independent of the etiology of ESRD.

B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events.

To understand the molecular mechanisms that are responsible for the B cell overactivity that is observed in patients with SLE, we have conducted experiments in which the surface immunoglobulin (sIg)-mediated early cell signaling events were studied. The anti-sIgM-mediated free intracytoplasmic calcium ([Ca2+]i) responses were significantly higher in SLE B cells compared with responses of normal individuals and to those of patients with other systemic autoimmune rheumatic diseases. The anti-IgD mAb induced [Ca2+]i responses were also higher in lupus B cells than in controls. The magnitude of anti-sIgM-mediated Ca2+ release from intracellular stores was also increased in B cells from SLE patients compared with normal controls. The amount of inositol phosphate metabolites produced upon crosslinking of sIgM was slightly higher in patients with lupus than in normal controls, although the difference was not statistically significant. In contrast, the degree of anti-sIgM-induced protein tyrosine phosphorylation was obviously increased in lupus patients. Our study demonstrates clearly for the first time that SLE B cells exhibit aberrant early signal transduction events, including augmented calcium responses after crosslinking of the B cell receptor and increased antigen-receptor-mediated phosphorylation of protein tyrosine residues. Because the above abnormalities did not correlate with disease activity or treatment status, we propose that they may have pathogenic significance.