The incidence and prevalence of systemic lupus erythematosus in Thrace, 2003-2014: A 12-year epidemiological study.

BACKGROUND: We estimated the prevalence and incidence, clinical features, treatment, and prognosis of systemic lupus erythematosus (SLE) patients in the Thrace region of Turkey. METHODS: We retrospectively evaluated 331 patients (307 female, 24 male, mean age 38.5 years) diagnosed with SLE between 2003 and 2014. Clinical features, treatments, and response to various treatment modalities were recorded. Our hospital has been the only tertiary referral center for rheumatological diseases for a mixed rural and urban population of 620,477 people (306,036 females, 314,411 males) for more than 16 years. RESULTS: The mean annual incidence of SLE was 4.44/100,000 (females, 8.4/100,000; males, 0.6/100,000). The overall prevalence of SLE was 51.7/100,000 (females, 97.7/100,000; males, 7/100,000). Major organ involvement was present in the following percentages: neurologic involvement: 20.1%; renal involvement: 28.2%; autoimmune hemolytic anemia: 9.6%; thrombocytopenia: 14.7%. Seventeen SLE patients (13 females, four males) died at a median follow-up of 48 months. The five-year survival was 94.5%, and the ten-year survival was 89.9%. According to Kaplan-Meier survival analysis, poor prognostic factors were: male gender (p = 0.015); smoking (p = 0.02); pleural involvement (p = 0.011); thrombocytopenia (p = 0.021); myocarditis (p = 0.028); renal involvement (p = 0.037); treatment with cyclophosphamide (p = 0.011); and an initial high SLEDAI score (>4) (p = 0.02). Lymphopenia at the time of diagnosis appeared as a favorable prognostic factor (p = 0.008). Cox regression analysis revealed myocarditis (OR: 20.4, p = 0.018) and age at diagnosis (OR: 1.11, p = 0.035) to be poor, and lymphopenia at the time of diagnosis to be good prognostic factors (OR:0.13, p = 0.031). CONCLUSIONS: The annual incidence and prevalence of SLE in the Thrace region of Turkey is lower than those reported in North America, however they are similar to those reported for European countries. Clinical manifestations appear to be milder, whereas survival was similar to those recorded in Western countries.

CAMP response element modulator a expression in patients with systemic lupus erythematosus.

T cells from patients with systemic lupus erythematosus (SLE) have high levels of cAMP response element modulator (CREM)-alpha which bind to the interleukin (IL-2) promoter and limit IL-2 production. In this case-controlled study, we show that CREM-alpha mRNA levels were higher in T cells from patients with SLE than controls while CREB mRNA levels did not differ between the two groups. CREM-alpha mRNA levels did not correlate with clinical characteristics, disease activity or treatment. Nevertheless, there was a trend for patients on high doses of corticosteroids to have low levels of CREM-alpha mRNA. The discovery of specific non-toxic medications that block the expression of CREM-alpha may prove useful in reversing the aberrant T cell function in SLE.

Defective production of functional 98-kDa form of Elf-1 is responsible for the decreased expression of TCR zeta-chain in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), the prototypic autoimmune disease, is characterized by defective expression of TCR zeta-chain. Elf-1 (E-74-like factor) is a member of the Ets (E-26-specific) family and is crucial for the basal transcription of TCR zeta-chain in Jurkat cells. We previously demonstrated that Elf-1 exists in the cytoplasm mainly as 80-kDa form and after phosphorylation and O-glycosylation it moves to the nucleus as a 98-kDa which binds DNA. We now demonstrate that Elf-1 is crucial for the transactivation of TCR zeta-chain promoter in normal and SLE T cells. Defective expression of TCR zeta-chain in SLE T cells is associated with two distinct molecular defects in the generation of the 98-kDa DNA binding Elf-1 form. In the first, the levels of the 98-kDa form were either decreased or absent. In the second, the apparent levels of the nuclear Elf-1 form were normal but included only two of the three bands into which the nuclear Elf-1 form separated in isoelectric focusing gels. Because both the transcription and the translation processes of Elf-1 gene are normal in SLE T cells, our data demonstrate that abnormal posttranslational mechanisms of the Elf-1 protein result in defective expression of functional Elf-1, and consequently, the transcriptional defect of TCR zeta-chain in patients of SLE.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3zeta expression.

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)

Multiple transcription factors regulate the inducible expression of the human complement receptor 2 promoter.

Complement receptor 2 (CR2) is regulated at the transcriptional level, but the promoter elements and the transcription factors that bind to them and contribute to its regulation are unknown. After documenting that PMA and cAMP induced the activity of the CR2 promoter by 10-fold, we conducted promoter truncation and mutagenesis experiments, in conjunction with shift assays, to determine the functionally important regions of the promoter and the proteins that bind to them. We identified two regions, separated by approximately 900 nucleotides, which together were responsible for inducible promoter activity. Mutagenesis of single promoter elements demonstrated a functional upstream stimulatory factor/E box in the TATA box-proximal region and three equally important, closely spaced, CREB/AP-1 half-sites in the upstream promoter region. The cAMP response element-binding protein (CREB)/AP-1 half-sites bound in vitro Jun and CREB that are induced by protein kinases A and/or C. The 900-nucleotide segment stretching between the above two regions had no functional impact on the induced transcription, and its deletion increased the promoter activity. Finally, a region upstream of the distal site had a repressor activity on CR2 transcription. Moreover, IL-4 induced binding of CREB and AP-1 to the upstream promoter elements and resulted in increased CR2 surface protein expression. These studies have characterized regions of the CR2 promoter and the transcription factors that bind to them and are crucial to induced CR2 expression. Our studies may provide insights to novel approaches to modulate B cell function by regulating CR2 gene transcription.

Protein kinase C-theta participates in the activation of cyclic AMP-responsive element-binding protein and its subsequent binding to the -180 site of the IL-2 promoter in normal human T lymphocytes.

IL-2 gene expression is regulated by the cooperative binding of discrete transcription factors to the IL-2 promoter/enhancer and is predominantly controlled at the transcriptional level. In this study, we show that in normal T cells, the -180 site (-164/-189) of the IL-2 promoter/enhancer is a p-cAMP-responsive element-binding protein (p-CREB) binding site. Following activation of the T cells through various membrane-initiated and membrane-independent pathways, protein kinase C (PKC)-theta phosphorylates CREB, which subsequently binds to the -180 site and associates with the transcriptional coactivator p300. Rottlerin, a specific PKC-theta inhibitor, diminished p-CREB protein levels when normal T cells were treated with it. Rottlerin also prevented the formation of p-CREB/p300 complexes and the DNA-CREB protein binding. Cotransfection of fresh normal T cells with luciferase reporter construct driven by two tandem -180 sites and a PKC-theta construct caused a significant increase in the transcription of the reporter gene, indicating that this site is functional and regulated by PKC-theta. Cotransfection of T cells with a luciferase construct driven by the -575/+57 region of the IL-2 promoter/enhancer and a PKC-theta construct caused a similar increase in the reporter gene transcription, which was significantly limited when two bases within the -180 site were mutated. These findings show that CREB plays a major role in the transcriptional regulation of IL-2 and that a major pathway for the activation of CREB and its subsequent binding to the IL-2 promoter/enhancer in normal T cells is mediated by PKC-theta.

ZAP-70 and SLP-76 regulate protein kinase C-theta and NF-kappa B activation in response to engagement of CD3 and CD28.

The transcription factor NF-kappaB is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-kappaB activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-kappaB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-kappaB activation was examined. NF-kappaB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-alpha) activated NF-kappaB normally. Protein kinase C (PKC) theta activation, which is required for NF-kappaB activation, also was defective in these cells. Reexpression of ZAP-70 restored PKCtheta and NF-kappaB activation in response to TCR and CD28 coengagement. p95(vav) (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKCtheta and NF-kappaB activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC theta and NF-kappaB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.

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.

Immune cell signaling in lupus.

The fate of the lymphocyte is determined by integration of signals delivered after the binding of antigen to the surface antigen receptor, signals delivered by cytokines that bind to their surface receptors, and signals initiated after the engagement of other surface receptors, known as costimulatory molecules. The summation of this input determines whether the immune cell will become stimulated, ignore the signal (anergy), or die (apoptosis). Antigen-receptor signaling events are abnormal in lupus lymphocytes, manifested by increased calcium responses and hyperphosphorylation of several cytosolic protein substrates. Further down, at the gene transcription level, the activity of the nuclear factor kappaB is decreased. These events are underwritten by defective T cell receptor zeta chain expression, overexpression of the gamma chain of the Fc(epsilon)RI that functions as an alternate of zeta chain, and decreased p65 -Rel A protein that is responsible for the inducible NFkappaB activity. Accumulated research data have enabled us to begin deciphering the molecular basis of the abnormal lupus lymphocyte and may lead to the development of new medicinal treatments for lupus.

Serum regulates the expression of complement receptor 2 on human B cell lines.

Complement receptor 2 (CR2) participates in the regulation of B cell responses to antigen. In this study we report that treatment of IM-9 B lymphoblastoid cells or Raji Burkitt's lymphoma cells with 10% heat-inactivated fetal bovine serum for 24 hr increased both the CR2 mRNA level and CR2 surface protein expression more than two-fold. No change in the CR2 expression level was observed if cells were cultured in serum-free medium. The CD19 mRNA level decreased after 24 hr independently of the presence of serum. The serum-stimulated increase in CR2 expression was not due to changes in the proliferative capacity of the cells and could not be mimicked by various cytokines. However, IFN-gamma as well as OKB7, a CR2-specific monoclonal antibody, blocked the serum-induced increase in CR2 expression at the mRNA level. Our data show for the first time that factors in serum induce the expression of the CR2 gene and that signals initiated by IFN-gamma and OKB7 interfere with the serum-induced changes. Because stimuli that alter CR2 expression can influence the extent of the B cell response to antigen-C3d complexes, serum factors may play a role in regulating the responsiveness of B cells.

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.

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains.

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.

Enhanced and sustained activation of human B cells by anti-immunoglobulin conjugated to the EBV glycoprotein gp350.

We coupled a monoclonal anti-human IgD to the gp350 gylcoprotein of Epstein-Barr virus, which has been shown to bind to the complement receptor 2 (CR2), and compared its B cell stimulatory ability to that of anti-Ig and to a multivalent anti-Ig-dextran conjugate. The anti-Ig-gp350 conjugate stimulated higher levels of human B cell proliferation in vitro than did anti-Ig or anti-Ig conjugated to control viral protein, comparable to the proliferation stimulated by the multivalent anti-Ig-dextran. This enhanced proliferation was dependent on binding of the conjugate to CR2, inasmuch as an anti-CD2 antibody blocked the enhanced proliferative response. This enhanced proliferative response was associated with prolonged elevations of intracellular ionized calcium, which was comparable to the response stimulated by anti-Ig-dextran. These findings suggest the use of gp350 as a carrier molecule for weakly immunogenic peptides or antigens which, when bound to gp350, would enhance B cell clonal expansion and activation of antigen-specific B cells.

Regulation of heat shock protein 72 kDa and 90 kDa in human breast cancer MDA-MB-231 cells.

It has been shown that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. In this study, we conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MDA-MB-231 cells. Using [35S]methionine incorporation and Western immunoblots, we established that heat shock increased production of HSP-72 and -90. Cells exposed to 44 degrees C for 20 min displayed increased expression of HSP-72 and -90, that reached a maximum 3-7 h later and returned to baseline levels within 24 h. The synthesis of both HSP-72 and -90 was attenuated when cells were exposed to heat shock in medium devoid of Ca2+ or pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. Similarly, synthesis of HSP-72 and -90 was inhibited when cells were treated with the protein kinase A inhibitor, H89. These data indicate that Ca2+ and PKA are involved in the regulation of HSP-72 and -90 protein synthesis. Levels of HSP-72 mRNA in cells exposed to heat shock increased, suggesting that the heat-induced increase in HSP-72 occurs at the transcriptional level. Also, heat shock caused phosphorylation and translocation from the cytosol to the nucleus of heat shock factor 1 (HSF 1), a transcription factor for heat shock protein synthesis. Removal of external Ca2+ or treatment with a PKA inhibitor prevented the phosphorylation and the translocation of HSF 1. Cells overexpressing HSP-72 and -90 induced by exposure to a sublethal temperature displayed cytoprotection from thermal injury. Removal of external Ca2+ and treatment with BAPTA or H89 prior to exposure to sublethal heat shock that reduced the amount of HSP-72 and -90 production still protected cells from subsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MDA-MB-231 cells was 175+/- nM. Heat shock increased [Ca2+]i in a time-and temperature-dependent manner. Exposure of cells to 44 degrees C for 20 min increased [Ca2+]i by 234+/-13%, which subsequently returned to baseline levels within 30 min. Removal of external Ca2+ eliminated the increase, indicating that the increase in [Ca2+]i was due to Ca2+ influx. Pretreatment of the cells with H89 but not GF-109203X for 30 min led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 and -90 are regulated by [Ca2+]i and PKA activity in MDA-MB-231 cells. Kiang JG, Gist ID, Tsokos GC: Regulation of Heat Shock Protein 72 kDa and 90 kDa in Human Breast Cancer MDA-MB-231 Cells.

Biochemical requirements for the expression of heat shock protein 72 kda in human breast cancer MCF-7 cells.

Heat shock alters the susceptibility of tumor cells to chemotherapeutic agents. Cultured breast cancer MCF-7 and MDA-MB-231 cells that express high levels of heat shock protein 70 and 27 kDa are resistant to treatment with certain anticancer drugs. These findings indicate that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MCF-7 cells exposed to heat shock by intracellular free Ca2+ and protein kinase C. Cells exposed to 44 degrees C for 20 min displayed increased expression of HSP-72 and GRP-94, that reached a maximum 4-5 h later and returned to baseline levels within 24 h. Levels of HSP-72 mRNA in cells exposed to heat shock increased, suggesting that the heat-induced increase in HSP-72 occurs at the transcriptional level. The synthesis of HSP-72 but not GRP-94 was inhibited when cells were exposed to heat shock in medium devoid of Ca2+ and attenuated by more than 50% when cells were pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. HSP-72 synthesis was enhanced when cells were treated with the protein kinase C inhibitor, GF-109203X. These data indicate that Ca2+ and PKC are involved in regulation of HSP-72 synthesis. However, removal of external Ca2+ and treatment with BAPTA, GF-109203X, or exposure to sublethal heat shock protected cells from subsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MCF-7 cells was 156 +/- 16 nM (n = 29). Heat shock increased [Ca2+]i in a time- and temperature-dependent manner. Exposure of cells to 44 degrees C for 20 min increased [Ca2+]i by 234 +/- 13%, which subsequently returned to baseline levels within 120 min. Removal of external Ca2+ eliminated the increase, indicating that the increase in [Ca2+]i was due to Ca2+ influx. Pretreatment of the cells with BAPTA or GF-109203X for 30 min or a sublethal heat shock to allow HSP-72 overexpression led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 but not GRP-94 is regulated by [Ca2+]i and PKC activity. The cytoprotection produced by chelation of Ca2+, GF-109203X, or HSP-72 overexpression is probably due to their ability to attenuate the [Ca2+]i response to heating.

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.