Estradiol differentially regulates calreticulin: a potential link with abnormal T cell function in systemic lupus erythematosus?

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease that affects women nine times more often than men. The present study investigates estradiol-dependent control of the calcium-buffering protein, calreticulin, to gain further insight into the molecular basis of abnormal T cell signaling in SLE T cells. METHODS: T cells were purified from blood samples obtained from healthy females and SLE patients. Calreticulin expression was quantified by real-time polymerase chain amplification. Calreticulin and estrogen receptor-α were co-precipitated and analyzed by Western blotting to determine if the proteins associate in T cells. RESULTS: Calreticulin expression increased (p = 0.034) in activated control T cells, while estradiol decreased (p = 0.044) calreticulin in resting T cells. Calreticulin expression decreased in activated SLE T cell samples and increased in approximately 50% of resting T cell samples. Plasma estradiol was similar (p > 0.05) among SLE patients and control volunteers. Estrogen receptor-α and calreticulin co-precipitated from nuclear and cytoplasmic T cell compartments. CONCLUSIONS: The results indicate that estradiol tightly regulates calreticulin expression in normal human T cells, and the dynamics are different between activated and resting T cells. The absence of this tight regulation in SLE T cells could contribute to abnormal T cell function.

Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity.

Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10(-7) M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0-60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression (P=0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0-2) compared with patients with moderate (4-6) or active (8-12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells.

Estrogen does not regulate CD154 mRNA stability in systemic lupus erythematosus T cells.

Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in CD154 expression in T cells from females with systemic lupus erythematosus (SLE). This present study investigates if the estrogen-dependent increase in CD154 expression is due to stabilization of the messenger RNA. T cells from female SLE patients and controls were cultured for 18 h in serum-free medium without and with estradiol 17-beta (10(-7) M). T cells were either unstimulated (resting) or were activated by further culture on anti-CD3 coated plates. Actinomycin D (25 microg/mL) was added to parallel cultures to inhibit new messenger RNA synthesis. CD154 messenger RNA stability was assessed by reverse-transcription polymerase chain amplification. Resting SLE (n = 10, P = 0.88) and normal (n = 7, P = 0.65) T cells showed no significant differences in message stability in response to estradiol. CD154 messenger RNA was also not significantly stabilized in activated SLE (n = 10, P = 0.15) or activated normal (n = 6, P = 0.077) T cells in response to estradiol. These findings indicate that the estrogen-dependent increase in CD154 in SLE T cells is not due to stability of the mRNA. These data are consistent with the postulate that estradiol stimulates CD154 transcription in SLE T cells.

Anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis: correlation of TNF-alpha serum level with clinical response and benefit from changing dose or frequency of infliximab infusions.

OBJECTIVE: To determine the relationship between serum TNF-alpha level and clinical response in rheumatoid arthritis patients treated by infliximab. This could be of value to predict clinical response to infliximab and to determine the optimal dose and interval between dosing of infliximab. RA patients who did not respond adequately to conventional doses (3 mg/kg) of infliximab were studied to see if increasing the dose or frequency of infliximab infusions would be more effective. METHODS: Fifty-five RA patients who fulfilled the American College of Rheumatology criteria and were receiving treatment by anti-TNF-alpha (infliximab 3 mg/kg body weight every 8 weeks) were evaluated by: clinical disease activity using the Richie score index before receiving their scheduled infliximab infusion. Serum level of TNF-alpha, as measured by competitive ELISA assay, was determined immediately before and 9-11 days after receiving infliximab. RA patients who did not respond adequately to treatment with infliximab were given either a larger dose of infliximab or given the infusion at six-week intervals. Their clinical response was then evaluated sixteen months later. RESULTS Patients were divided into 2 groups according to Richie score, active group with score > 10 (score 20.3 +/- 7.7 mean +/- standard deviation, n = 25) and inactive group with scores < or = 10 (score 4.1 +/- 3.2, n = 30). TNF-alpha serum levels pre-infliximab infusion were significantly higher in the active group 76.1 pg/ml than the inactive group 38.0 pg/ml (P < 0.02). Whereas TNF serum level significantly dropped post infliximab in the inactive group (P < 0.05), it did not drop in the active group. The mean level of the post-infusion TNF-alpha serum level was higher (76.6 +/- 93.4 ng/ml) in the-active than the mean level of the post-infusion serum TNF-alpha levels in the inactive group (26.4 ng/ml +/- 7.9) P < 0.01 using the t-test. Increasing the frequency was superior in RA patients' clinical outcome than increasing the dose of infliximab infusions. CONCLUSION: RA patients who responded well to infliximab and had inactive disease at the time of the study have lower levels of serum TNF-alpha which could be further suppressed by the recommended doses of infliximab. RA patients with active disease have higher serum levels of TNF-alpha which could not be suppressed after the recommended doses of infliximab infusion. Changing the frequency of infliximab infusions in the active group was more effective than increasing the dose of infliximab in inducing improved clinical outcome. We suggest that the lack of suppression of TNF-alpha in the active group could be due to inadequate dosing of infliximab or to the presence of a neutralizing antibody directed against infliximab. It remains to be seen if serum TNF-alpha levels could be used as a guide in determining the dose and intervals between dosing of anti-TNF therapy in RA in order to achieve the desired clinical response.

Gender differences in autoimmunity: molecular basis for estrogen effects in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women (9:1 compared to men). Estrogen is a female sex hormone that acts on target cells through specific receptor proteins and alters the rate of transcription of target genes. Experiments in our laboratory have shown that calcineurin steady-state mRNA levels and phosphatase activity increase when estrogen is cultured with SLE T cells. This estrogen-dependent increase is dose-dependent, hormone-specific and temporally regulated. Estrogen receptor antagonism by ICI 182,780 inhibits the increase in calcineurin mRNA and phosphatase activity, while cycloheximide has no effect suggesting that new protein synthesis is not required. Reverse transcription and polymerase chain amplification indicate that estrogen receptor-alpha and estrogen-beta are expressed in human T cells. However, calcineurin does not respond to estrogen stimulation in T cells from normal females, males and lupus males. Taken together, these results indicate a differential function of the estrogen receptor in women with lupus. A model is proposed that suggests estrogen, acting through the estrogen receptor, enhances T cell activation in women with lupus resulting in amplified T-B cells interactions, B cell activation and autoantibody production.

In vitro-activated human lupus T cells express normal estrogen receptor proteins which bind to the estrogen response element.

We have shown that estrogen receptor (ERalpha, ERbeta) transcripts are expressed in SLE and normal T cells. In this study, T cell nuclear extracts from female lupus patients and normal donors were tested for biologically active ER proteins capable of binding to the human estrogen response element (hERE) by electrophoretic mobility shift assays. When peripheral blood T cells were stimulated with 17beta-estradiol (E2), PMA and ionomycin, two major retarded bands in T cell nuclear extracts exhibited a migration pattern similar to slow migrating protein-ERE complexes in human breast cancer cell extracts. T cells cultured only with E2 did not have these complexes. The formation of the complexes was inhibited by competition with the hERE cold oligonucleotide and partially with anti-ERalpha antibodies. There was no notable difference in the migration pattern of ERE-binding proteins between the SLE and normal T cell extracts. Together, these results suggest that activated human T cells, whether lupus-derived or normal-derived, contain biologically active ERalpha proteins. Other factors may be responsible for differential sensitivity of lupus T cells to estrogen.

Estrogen increases CD40 ligand expression in T cells from women with systemic lupus erythematosus.

OBJECTIVE: To examine the in vitro effects of estrogen on CD40 ligand (CD40L) expression in peripheral blood T cells isolated from patients with systemic lupus erythematosus (SLE) and normal controls. METHODS: T cells from female patients with SLE and controls were cultured in serum-free medium without and with 2-fluoroestradiol. Some T cells were activated by further culture on anti-CD3 coated plates. Calcineurin was activated in some T cells by culture in ionomycin. Cell surface CD40L was quantitated by FACS analysis. mRNA expression was measured using semiquantitative PCR. RESULTS: Lupus T cells cultured in medium containing 2-fluoroestradiol showed a significant (p = 0.04) increase in the amount of CD40L on the cell surface, but not in the number of positive cells, compared to the same T cells cultured without estradiol. Estradiol did not significantly change CD40L expression on the surface of T cells from normal women. In addition, the difference in cell surface CD40L between T cells cultured without and with estradiol was significantly greater (p = 0.048) on SLE than on normal T cells. Culture of SLE T cells in medium containing 2-fluoroestradiol followed by T cell receptor (TCR) activation for 2 h using anti-CD3 resulted in a significant (p = 0.04) estrogen dependent increase in CD40L mRNA. The estrogen dependent increases in SLE T cell CD40L mRNA and cell surface protein were blocked by the estrogen receptor antagonist ICI 182,780. SLE and normal T cells pretreated with estradiol and cultured with ionomycin for 2 h to activate calcineurin showed no significant differences in CD40L mRNA. CONCLUSION: These results suggest that estradiol, working through the estrogen receptor, stimulates the expression of CD40L in unstimulated and activated SLE T cells. Estradiol effects may be exerted on multiple regulatory steps that control CD40L expression. The estrogen dependent increase in CD40L expression could hyperstimulate SLE T cells and thereby contribute to the pathogenesis of SLE.

Molecular mechanisms involved in the estrogen-dependent regulation of calcineurin in systemic lupus erythematosus T cells.

Previous experiments in our laboratory indicated that calcineurin expression and PP2B phosphatase activity increased when estrogen was cultured with SLE T cells but not with T cells from normal women. In this report we extended our findings to show that estrogen receptor (ER) antagonism by ICI 182,780 inhibited the estrogen-dependent increase in calcineurin mRNA and phosphatase PP2B activity indicating that estrogen action was mediated through the ER. Inhibition of de novo protein synthesis with cycloheximide suggested that the estrogen-dependent increase in T cell calcineurin mRNA was a direct effect of the ER and new protein synthesis was not required. Estrogen increased calcineurin mRNA in systemic lupus erythematosus (SLE) T cells at 6 h after the start of culture correlating with increased phosphatase activity at this same time. Phosphatase activity increased significantly (P < 0.02) in lupus T cells cultured for 8 h in estradiol-containing medium. Reverse transcription and polymerase chain amplification revealed that ER-beta and ER-alpha were expressed in female and male T cells from SLE patients and normal controls. However, calcineurin steady-state mRNA levels were unaffected by estradiol in cultured T cells from male SLE patients and normal male and female controls. These data indicate that estrogen, bound to the ER, evokes a direct increase in calcineurin expression in T cells from female lupus patients. This gender-specific response suggests that ER function is altered in women with the female predominant autoimmune disease, SLE.

Genome scan of human systemic lupus erythematosus: evidence for linkage on chromosome 1q in African-American pedigrees.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens including DNA, ribosomal P, Ro (SS-A), La (SS-B), and the spliceosome. Etiology is suspected to involve genetic and environmental factors. Evidence of genetic involvement includes: associations with HLA-DR3, HLA-DR2, Fcgamma receptors (FcgammaR) IIA and IIIA, and hereditary complement component deficiencies, as well as familial aggregation, monozygotic twin concordance >20%, lambdas > 10, purported linkage at 1q41-42, and inbred mouse strains that consistently develop lupus. We have completed a genome scan in 94 extended multiplex pedigrees by using model-based linkage analysis. Potential [log10 of the odds for linkage (lod) > 2.0] SLE loci have been identified at chromosomes 1q41, 1q23, and 11q14-23 in African-Americans; 14q11, 4p15, 11q25, 2q32, 19q13, 6q26-27, and 12p12-11 in European-Americans; and 1q23, 13q32, 20q13, and 1q31 in all pedigrees combined. An effect for the FcgammaRIIA candidate polymorphism) at 1q23 (lod = 3.37 in African-Americans) is syntenic with linkage in a murine model of lupus. Sib-pair and multipoint nonparametric analyses also support linkage (P < 0.05) at nine loci detected by using two-point lod score analysis (lod > 2.0). Our results are consistent with the presumed complexity of genetic susceptibility to SLE and illustrate racial origin is likely to influence the specific nature of these genetic effects.

Gender differences in autoimmune diseases: estrogen increases calcineurin expression in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) predominantly affects women (9:1 compared to men) of childbearing age and often decreases its intensity in postmenopausal women, suggesting that sex hormones play a role in its pathogenesis. Comparison of steady-state levels of calcineurin mRNA using RNase protection assays revealed increased calcineurin expression in response to estradiol in cultured T cells from nine female lupus patients. Calcineurin mRNA levels did not increase significantly in T cells from eight age-matched normal control female volunteers. Estrogen-dependent calcineurin mRNA increased in a dose-dependent fashion, while progesterone and dexamethasone did not increase calcineurin mRNA in patient cells. Lupus T cell calcineurin mRNA increased in response to estradiol at 6 h but not at 3 h. Calcineurin phosphatase activity increased in lupus T cell extracts after incubation of cells with estradiol, while phosphatase activity in normal T cells was unaffected by estrogen. Calcineurin expression in T cells from patients with vasculitis and rheumatoid arthritis taking medications similar to those taken by the lupus patients was unaffected by estradiol. This study provides the first evidence for a molecular marker of estrogen action in lupus patients and suggests that estrogen-dependent changes in lupus T cell calcineurin could alter proinflammatory cytokine gene regulation and T-B cell interactions.

Isolation of anti-nucleosome antibodies from the plasma of lupus nephritis patients.

Anti-nucleosome antibodies, which recognise conformational epitopes consisting of histone and DNA in chromatin, have been described in autoimmune diseases. In this study, an attempt was made to isolate anti-nucleosome antibodies from the anti-DNA-depleted plasma IgG of two lupus patients either with or without nephritis by nucleohistone affinity chromatography. The purified nucleohistone-binding antibodies bound to nucleohistone in a specific manner and contained enriched anti-histone antibodies. However, adsorption of the purified antibodies with histone revealed that the nephritis patient-derived antibodies contained nucleohistone-specific antibodies. Although such purified antibodies may not recognise native structures of nucleosomes, this chromatography may provide a method to isolate and determine the fine specificity of anti-nucleosome antibodies in various autoimmune diseases.

Peripheral blood T cells and monocytes and B cell lines derived from patients with lupus express estrogen receptor transcripts similar to those of normal cells.

OBJECTIVE: To identify and characterize estrogen receptor (ER) transcripts expressed in immune cells of patients with systemic lupus erythematosus (SLE) and healthy donors. METHODS: Peripheral blood monocytes and T cells were prepared from patients with SLE (n = 6) and healthy donors (n = 8). T cells were separated into CD4 and CD8. Some monocytes and T cells were stimulated with estradiol, PMA, and ionomycin. Epstein-Barr virus-transformed B cell lines (n = 7) and B cell hybridomas (n = 2) established from patients with SLE and a healthy individual were used as a B cell source. These cells were examined for ER mRNA by reverse transcription nested polymerase chain reaction. Amplified cDNA were sequenced by standard methods. RESULTS: In all cells tested, ER mRNA was expressed without prior in vitro stimulation. Partial sequences from exons 1-8 were nearly identical to the published sequence of the human ER mRNA. There were no notable differences in the ER transcripts between patients and healthy controls. Variant receptor transcripts lacking exon 5 or exon 7, which encodes the hormone binding domain, were identified in the majority of the cells. Precise deletion of the exons suggests that they are alternatively spliced transcripts. Whether the detected transcripts are translated into functional receptor proteins remains to be determined. In vitro stimulation did not affect ER mRNA expression. The presence of variants did not correlate with disease activity or medication. CONCLUSION: Monocytes, T cells, and B cells in patients express transcripts of the normal wild type ER and the hormone binding domain variants in vivo.

V gene sequences of lupus-derived human IgM anti-ssDNA antibody: implication for the importance of the location of DNA-binding amino acids.

Binding and structural characteristics of human IgMk anti-ssDNA antibody 7B3 were determined. 7B3 was derived from Epstein-Barr virus-transformed peripheral blood B cells of a lupus nephritis patient. Purified 7B3 bound ssDNA from various species, but not dsDNA or structurally unrelated antigens. The relative avidity of 7B3 was high in comparison with IgM anti-DNA antibodies previously described by other investigators. Sequence analysis showed that 7B3 used VH26/D35/JH3 and Humkv328h5/JK1 germline genes, and had a few mutations in the complementarity determining regions (CDRs). No arginine was expressed in the heavy-chain CDR3. However, the putative DNA contact sites, based on the previous crystallographic and computer modeling studies, were occupied by mutated or germline-derived basic and polar amino acids. These results suggest that a minimally mutated IgM anti-ssDNA antibody with a paucity of arginines could display monospecificity and high avidity if DNA-binding amino acids are enriched at the critical DNA contact sites.

Estrogen decreases in vitro apoptosis of peripheral blood mononuclear cells from women with normal menstrual cycles and decreases TNF-alpha production in SLE but not in normal cultures.

Estrogen has been suspected of causing changes in the lupus disease process by an as yet undetermined mechanism. In vitro apoptosis of peripheral blood mononuclear cells (PBMCs) in short-term unstimulated cultures of systemic lupus erythematosus (SLE) cells is accelerated compared to that in cells from normal individuals. To determine whether estrogen might be involved in regulating the rate of apoptosis in lupus, PBMCs or T cells from women with or without normal menstrual cycles were cultured for 16-20 hr with or without 30 ng/ml estradiol. The rate of apoptosis of the cells was measured, and supernatants of these cultures were tested for various cytokines known to affect apoptosis directly or indirectly. Compared to untreated cultures, estrogen significantly reduced in vitro apoptosis of both patient (P < 0.05, n = 12) and normal (P < 0.001, n = 14) PBMCs if the donors had normal menstrual cycles. Estrogen did not decrease apoptosis of noncycling patient (n = 8) nor of normal (n = 11) cells. Apoptosis of T cells cultured alone was not affected by estrogen. Supernatants from patients' estrogen-treated PBMCs had significantly less TNF-alpha than untreated cultures (P < 0.05, n = 12). TNF-alpha levels from normals' cell cultures were unchanged. Changes in hormone status (hysterectomy or menopause) alter estrogen-sensitive apoptosis, which may be mediated through monocytes. Estrogen-induced decreases in apoptosis combined with decreased TNF-alpha production in the presence of estrogen may allow survival of auto-immune cells in SLE patients.

Presence of a variant form of the estrogen receptor in peripheral blood mononuclear cells from normal individuals and lupus patients.

Estrogen may participate in the pathogenesis of systemic lupus erythematosus (SLE) via its intracellular receptor. To investigate the presence of various isoforms of the estrogen receptor (ER) in SLE we isolated RNA from mononuclear cells of lupus patients and normal controls. Using RT-PCR we were able to identify both the full length wild-type form and an isoform of the ER which precisely lacks exon V in both patient and normal individuals. Our results, although limited, suggest that normal individuals can express both the wild-type and truncated version at the same time, whereas lupus patients only express either the wild-type or the truncated ER. This finding may lead to a better understanding of the reasons for the prevalence of lupus in females and of the estrogenic effects on SLE disease activity.

Binding affinity and quantity of estrogen receptor in peripheral blood monocytes of patients with systemic lupus erythematosus.

Binding affinity and quantity of the estrogen receptor in monocytes of patients with systemic lupus erythematosus (SLE) were studied. The tritiated-estradiol binding assay was performed using peripheral blood adherent cells (> 95% monocytes) derived from six lupus patients (SLEDAI score: 2-30) and five age-comparable normal women during the mid-follicular phase of the menstrual cycle. Dissociation constant (Kd) and number of binding sites (Ro) were estimated by Scatchard analysis. The specificity and sensitivity of the assay were verified by using estrogen receptor-positive ZR75-1 human breast cancer cells. Kd and Ro of the type I receptor for the SLE patients were 12.2 +/- 6.5 (nM) and 69.0 +/- 42.4 (x 1000/cell), respectively, while those of the normals were 14.5 +/- 3.7 and 86.8 +/- 23.4, respectively. Three patients displayed relatively low Kd or Ro values. While those low values fell within the mean -3s.d. of the normal controls, precise statistical comparison was not possible. No clear correlation between the receptor parameters and the SLEDAI scores was noted. Although further studies of a larger number of samples are needed to conclude, these results suggest that peripheral blood monocytes of SLE patients express the estrogen receptor whose Kd and Ro are similar to those of normals.

V gene sequences of human anti-ssDNA antibodies secreted by lupus-derived CD5-negative B cell hybridomas.

V gene sequences encoding two lupus-derived human monoclonal IgMk anti-ssDNA antibodies (2F7 and 1A6) and CD5 mRNA expression by the corresponding hybridomas were investigated. Both antibodies displayed V gene sequences nearly in germline configuration compared with their putative germline counterparts. It appeared that 2F7 used hv3019b9/HUD-3/JH6 and 12La/Jk2, while 1A6 utilized HHG19/D31-HUD-3/JH2 and Humkv328h5/Jk1. Assessment of R/S mutation ratios suggested that 2F7 and 1A6 have not undergone the antigen-driven somatic mutation. The HCDR3 featuring arginine appeared to be important in determining the anti-ssDNA specificity. CD5 mRNA was negative in both hybridomas. Since 2F7 was previously shown to be monospecific and of high affinity, these results provide the molecular basis of such unique immunochemical characteristics of the IgM anti-ssDNA antibody. Germline V genes and N sequences may be selected to confer such anti-ssDNA specificity during V gene rearrangement, which might involve CD5-negative B cells.

Anti-(DNA-histone) antibodies in active lupus nephritis.

OBJECTIVE: To detect and characterize anti-(DNA-histone) antibodies in patients with active lupus nephritis. METHODS: Calf thymus double stranded DNA was reassociated with histone in vitro. Polynucleosomes were prepared from chicken erythrocyte nuclei, calf thymus nucleohistone, and human peripheral blood mononuclear cells. Anti-DNA activity was depleted from purified IgG using DNA-cellulose. Binding of the adsorbed IgG to various (DNA-histone) related antigens was measured by ELISA. Antigen specificity was assessed by inhibition assays and adsorption studies using histone and nucleosome conjugated sepharose beads. RESULTS: Anti-(DNA-histone) antibodies were detected in 3 of 5 patients with active lupus nephritis. They specifically recognized determinants consisting of both DNA and histone, but not DNA or histone alone. Anti-(DNA-histone) antibodies largely overlapped with antinucleosome antibodies and appeared to react with nucleosomes released by apoptotic human mononuclear cells. CONCLUSION: Anti-(DNA-histone) antibodies are present in active lupus nephritis and largely represent antinucleosome antibodies. They may contribute to the pathogenesis of nephritis by forming immune complexes with apoptosis related nucleosomes.