Damage accrual related to pregnancies before and after diagnosis of systemic lupus erythematosus: a cross-sectional and nested case-control analysis from a lupus registry.

OBJECTIVE: The objective of this study was to evaluate the chronic damage associated with pregnancies before and after the diagnosis of systemic lupus erythematosus (SLE). METHODS: Using childbearing-aged female SLE patient data registered at the Okayama and Showa University Hospitals, a nested case-control analysis was performed to investigate the relationship between pregnancy and chronic damage using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). RESULTS: Pregnancy occurred in 22 patients before and 13 patients after the diagnosis of SLE in 104 eligible patients. Live births occurred in 82% (33/40) and 50% (9/18) of the pregnancies before and after the diagnosis of SLE, respectively. After matching age and disease duration, 33 case patients with chronic damage (SDI ≥ 1) and 33 control patients without chronic damage (SDI = 0) were selected. Hypertension was more frequent in cases than in controls (48% vs. 24%, p = 0.041). Pregnancies before and after the diagnosis of SLE were comparable between cases and controls (before the diagnosis: nine case patients and eight control patients; after the diagnosis: three case patients and five control patients; p = 1.00). Even after adjusting for hypertension using multivariate analysis, the pregnancies before and after the diagnosis were not significant predictors for chronic damage (odds ratio = 1.48 (95% confidence interval 0.33-6.65)), p = 0.60 of the pregnancy before the diagnosis; odds ratio = 0.78 (95% confidence interval 0.13-4.74), p = 0.78 of the pregnancy after the diagnosis). CONCLUSION: Pregnancies, either before or after the diagnosis of SLE, did not show any differences in chronic damage. Our results help alleviate fears regarding childbearing in female patients with SLE and their families.

The efficacy of add-on tacrolimus for minor flare in patients with systemic lupus erythematosus: a retrospective study.

OBJECTIVE: We have assessed the effectiveness of tacrolimus for minor flares in systemic lupus erythematosus (SLE) patients. METHODS: The medical records of 313 patients were retrospectively reviewed over a period of seven years, from 2006 to 2013. We enrolled patients with minor flare treated with add-on tacrolimus, without glucocorticoid (GC) intensification (tacrolimus group). Minor flare was defined as a ≥ 1-point increase in a total score between 3 and 11 in the SLE Disease Activity Index (SLEDAI). We enrolled as controls patients who were administered increased doses of GC for minor flare (GC group). All patients were followed for one year. The primary outcome measure was the proportion of responders. RESULTS: There were 14 eligible patients in the tacrolimus group and 20 eligible patients in the GC group. The mean SLEDAI at flare tended to be higher in the tacrolimus group than in the GC group (7.5 vs. 6.2, p = 0.085). A mean dose of 1.6 mg tacrolimus/day was administered for flare, while the mean GC dose was 13.7 mg/day in the GC group. The proportion of responders was 86% (12/14) in the tacrolimus group and 75% (15/20) in the GC group (p = 0.67). The mean dose of GC at 12 months was higher in the GC group than in the tacrolimus group (9.7 mg/day vs. 7.1 mg/day, p < 0.05). Only one patient discontinued tacrolimus because of fatigue after three months. CONCLUSION: Adding tacrolimus without increasing the GC dose may provide an effective treatment option for minor flares in patients with SLE.

Retention of tocilizumab and anti-tumour necrosis factor drugs in the treatment of rheumatoid arthritis.

OBJECTIVES: The retention of the anti-rheumatic agent tocilizumab (TCZ) has not been well documented in patients with rheumatoid arthritis (RA). We conducted an observational study to compare the retention of TCZ and anti-tumour necrosis factor (TNF) drugs in the treatment of patients with RA. METHOD: We reviewed continuation rates and causes of discontinuation of biological agents (biologics) by assessing medical records of patients with RA who were administered biologics at our institute from September 1999 to April 2012, using the Osaka University Biologics for Rheumatic Diseases (BiRD) registry. RESULTS: A total of 401 patients were included. TCZ, infliximab (IFX), etanercept (ETN), and adalimumab (ADA) were administered to 97, 103, 143, and 58 patients, respectively. There were some differences between the baseline characteristics of the groups. The median duration (range) of TCZ, IFX, ETN, and ADA administration was 2.5 (0.1-12.6), 1.9 (0.0-7.7), 2.9 (0.0-11.3), and 1.3 (0.0-3.4) years, respectively. Continuation rates for TCZ and ETN were significantly higher than those for IFX and ADA. Multivariate analyses showed that discontinuation due to lack or loss of efficacy was significantly less common in the TCZ group than in the other groups. Discontinuation due to overall adverse events was not significantly different between treatment groups. CONCLUSION: TCZ and ETN show better retention than IFX or ADA in the treatment of RA.

Viral interleukin 6 stimulates human peripheral blood B cells that are unresponsive to human interleukin 6.

Cellular responsiveness to human interleukin 6 (hIL6) requires the expression of two receptor molecules: IL6-specific receptor (CD126'IL6R') and a nonspecific signal-transducing molecule (CD130'gp130'). Regulation of responsiveness to hIL6 is generally controlled by CD126'IL6R' expression. A viral homologue of hIL6 (vIL6) is encoded by human herpesvirus-8 and has biologic activity similar to hIL6 on a number of cell lines. vIL6 differs from hIL6 in its receptor utilization, requiring only CD130'gp130'. Total human B cells isolated from peripheral blood, which are predominantly CD126'IL6R'-negative, as well as sorted CD126'IL6R'-negative B cells, could be stimulated by recombinant vIL6, but not by hIL6, as indicated by induction of IL6-like signaling (STAT3 phosphorylation). This suggests that the ability of vIL6 to stimulate B cells expressing little or no CD126'IL6R' allows it to act on a larger pool of target B cells, compared to human IL6.

Receptor engagement by viral interleukin-6 encoded by Kaposi sarcoma-associated herpesvirus.

Receptor usage by viral interleukin-6 (vIL-6), a virokine encoded by Kaposi sarcoma- associated herpesvirus, is an issue of controversy. Recently, the crystal structure of vIL-6 identified vIL-6 sites II and III as directly binding to glycoprotein (gp)130, the common signal transducer for the IL-6 family of cytokines. Site I of vIL-6, however, comprising the outward helical face of vIL-6, where human IL-6 (hIL-6) would interact with the specific alpha-chain IL-6 receptor (IL-6R), is accessible and not occupied by gp130. This study examined whether this unused vIL-6 surface is available for IL-6R binding. By enzyme-linked immunosorbent assay, vIL-6 bound to soluble gp130 (sgp130) but not to soluble IL-6R (sIL-6R). Using plasmon surface resonance, vIL-6 bound to sgp130 with a dissociation constant of 2.5 microM, corresponding to 1000-fold lower affinity than that of hIL-6/sIL-6R complex for gp130. sIL-6R neither bound to vIL-6 nor affected vIL-6 binding to gp130. In bioassays, vIL-6 activity was neutralized by 4 monoclonal antibodies (mAbs) recognizing a domain within vIL-6 site I, mapped to the C-terminal part of the AB-loop and the beginning of helix B. The homologous region in hIL-6 participates in site I binding to IL-6R. In addition, binding of vIL-6 to sgp130 was interfered with specifically by the 4 neutralizing anti-vIL-6 mAbs. Based on the vIL-6 crystal structure, the vIL-6 neutralizing mAbs map outside the binding interface to gp130, suggesting that they either produce allosteric changes or block necessary conformational changes in vIL-6 preceding its binding to gp130. These results document that vIL-6 does not bind IL-6R and suggest that conformational change may be critical to vIL-6 function.

Preferential and persistent activation of the STAT1 pathway in rheumatoid synovial fluid cells.

OBJECTIVE: Inflammatory cytokines such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor-alpha are produced in great quantities in inflamed rheumatoid joints. However, little is known about the pathogenic significance of each cytokine in the proliferative synovitis and destruction of bone and joint. We investigated the role of cytokine receptor signals transduced into cells at the foci of rheumatoid inflammation. METHODS: Synovial fluid (SF) cells from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were examined for the activation of a group of cytokine receptor signaling molecules, signal transducers and activators of transcription (STAT). RESULTS: DNA binding of STAT1 in SF cells was observed in 8 out of 14 patients with RA, but in none of the 10 patients with OA studied, and this was prevented by preincubation of these cells with neutralizing anti-IL-6 antibody. IL-6 activated both STAT1 and STAT3 in normal peripheral blood (PB) leukocytes, and preferentially STAT1 in rheumatoid SF cells. Moreover, STAT1 activation in rheumatoid SF cells appeared to be continuous, in contrast to the transient activation in normal PB leukocytes. CONCLUSION: STAT1 and STAT3 are differentially regulated in response to IL-6 in different cell types. The continuous STAT1 activation may be of pathogenic significance in the progression and persistence of RA.

Paramagnetic water-soluble metallofullerenes having the highest relaxivity for MRI contrast agents.

Water-soluble gadolinium (Gd) endohedral metallofullerenes have been synthesized as polyhydroxyl forms (Gd@C(82)(OH)(n)(), Gd-fullerenols) and their paramagnetic properties were evaluated by in vivo as well as in vitro for the novel magnetic resonance imaging (MRI) contrast agents for next generation. The in vitro water proton relaxivity, R(1) (the effect on 1/T(1)), of Gd-fullerenols is significantly higher (20-folds) than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) at 1.0 T close to the common field of clinical MRI. This unusually high proton relaxivity of Gd-fullerenols leads to the highest signal enhancement at extremely lower Gd concentration in MRI studies. The strong signal was confirmed in vivo MRI at lung, liver, spleen, and kidney of CDF1 mice after i.v. administration of Gd-fullerenols at a dose of 5 micromol Gd/kg, which was 1/20 of the typical clinical dose (100 micromol Gd/kg) of Gd-DTPA.

SOCS-1/SSI-1-deficient NKT cells participate in severe hepatitis through dysregulated cross-talk inhibition of IFN-gamma and IL-4 signaling in vivo.

Suppressor of cytokine signaling-1 (SOCS-1), also known as STAT-induced STAT inhibitor-1 (SSI-1), is a negative feedback molecule for cytokine signaling, and its in vivo deletion induces fulminant hepatitis. However, elimination of the STAT1 or STAT6 gene or deletion of NKT cells substantially prevented severe hepatitis in SOCS-1-deficient mice, while administration of IFN-gamma and IL-4 accelerated its development. SOCS-1 deficiency not only sustained IFN-gamma/IL-4 signaling but also eliminated the cross-inhibitory action of IFN-gamma on IL-4 signaling. These results suggest that SOCS-1 deficiency-induced persistent activation of STAT1 and STAT6, which would be inhibited by SOCS-1 under normal conditions, may induce abnormal activation of NKT cells, thus leading to lethal pathological changes in SOCS-1-deficient mice.

Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1)/suppressor of cytokine signaling 1 (SOCS1) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation.

Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1) is known to function as a negative feedback regulator of cytokine signaling, but it is unclear whether it is involved in other biological events. Here, we show that SSI-1 participates and plays an important role in the insulin signal transduction pathway. SSI-1-deficient mice showed a significantly low level of blood sugar. While the forced expression of SSI-1 reduced the phosphorylation level of insulin receptor substrate 1 (IRS-1), SSI-1 deficiency resulted in sustained phosphorylation of IRS-1 in response to insulin.Furthermore, SSI-1 achieves this inhibition both by binding directly to IRS-1 and by suppressing Janus kinases. These findings suggest that SSI-1 acts as a negative feedback factor also in the insulin signal transduction pathway through the suppression of IRS-1 phosphorylation.

Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice.

Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-gamma, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of gammadelta T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common gamma-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common gamma-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.

Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor alpha-induced cell death in fibroblasts.

Signal transducers and activators of transcription (STAT)-induced STAT inhibitor-1 [SSI-1; also known as suppressor of cytokine signaling-1 (SOCS-1)] was identified as a negative feedback regulator of Janus kinase-STAT signaling. We previously generated mice lacking the SSI-1 gene (SSI-1 -/-) and showed that thymocytes and splenocytes in SSI-1 -/- mice underwent accelerated apoptosis. In this paper, we show that murine embryonic fibroblasts lacking the SSI-1 gene are more sensitive than their littermate controls to tumor necrosis factor-alpha (TNF-alpha)-induced cell death. In addition, L929 cells forced to express SSI-1 (L929/SSI-1), but not SSI-3 or SOCS-5, are resistant to TNF-alpha-induced cell death. Furthermore L929/SSI-1 cells treated with TNF-alpha sustain the activation of p38 mitogen-activated protein (MAP) kinase. In contrast, SSI-1 -/- murine embryonic fibroblasts treated with TNF-alpha show hardly any activation of p38 MAP kinase. These findings suggest that SSI-1 suppresses TNF-alpha-induced cell death, which is mediated by p38 MAP kinase signaling.

IFN regulatory factor-1-mediated transcriptional activation of mouse STAT-induced STAT inhibitor-1 gene promoter by IFN-gamma.

STAT-induced STAT inhibitor-1 (SSI-1), also referred to as suppressor of cytokine signaling-1 and JAK-binding protein, is a member of a new family, the members of which are negative regulators of cytokine signals. SSI-1 is induced by various cytokines; however, the transcriptional mechanism of the SSI-1 gene is not fully understood. Here, we showed that transcription of the mouse SSI-1 gene was initiated from six adjoining sites accompanying three GC boxes and a single GC box-like element near them, but not from the TATA box or an initiator sequence. We also showed that IFN-gamma induced SSI-1 mRNA more strongly than IL-6 in NIH-3T3 fibroblasts and that this IFN-gamma effect was mediated by Stat1. To determine the signal pathway downstream of Stat1, transcriptional activities of several mutant promoters were examined. The region mediating stimulatory effect of IFN-gamma to the gene transcription was localized to the -88/-60 region containing three tandem GAAA units, named variant IFN-gamma-responsive element (VIRE), while four IFN-gamma activation site (GAS)-like elements located far upstream were not related to the IFN-gamma response. Gel-shift assays revealed that IFN-gamma induced IFN regulatory factor-1 (IRF-1) binding to VIRE, but not that of IRF-2 or three components of ISGF3. Furthermore, forced expression of IRF-1 mimicked and that of IRF-2 inhibited the stimulatory effect of IFN-gamma on SSI-1 gene transcription. Finally, mouse embryonal fibroblasts lacking IRF-1 showed impaired SSI-1 mRNA induction by IFN-gamma. These results demonstrated that IRF-1, which is induced by activation of Stat1, mediated transcriptional activation of the SSI-1 gene by IFN-gamma via VIRE.

Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice.

Growth, differentiation, and programmed cell death (apoptosis) are mainly controlled by cytokines. The Janus kinase-signal transducers and activators of transcription (JAK-STAT) signal pathway is an important component of cytokine signaling. We have previously shown that STAT3 induces a molecule designated as SSI-1, which inhibits STAT3 functions. To clarify the physiological roles of SSI-1 in vivo, we generated, here, mice lacking SSI-1. These SSI-1-/- mice displayed growth retardation and died within 3 weeks after birth. Lymphocytes in the thymus and spleen of the SSI-1-/- mice exhibited accelerated apoptosis with aging, and their number was 20-25% of that in SSI-1+/+ mice at 10 days of age. However, the differentiation of lymphocytes lacking SSI-1 appeared to be normal. Among various pro- and anti-apoptotic molecules examined, an up-regulation of Bax was found in lymphocytes of the spleen and thymus of SSI-1-/- mice. These findings suggest that SSI-1 prevents apoptosis by inhibiting the expression of Bax.

Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling.

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.

Cloning and functional analysis of new members of STAT induced STAT inhibitor (SSI) family: SSI-2 and SSI-3.

Upon the corresponding ligand's stimulation, the cytokine receptors activate several signal pathways: JAK-STAT pathway, Ras-MAP kinase pathway and so on. Recently, we demonstrated that one of the STAT3 (signal transducer and activator of transcription-3) target genes could suppress the function of STAT3 and designated as SSI-1(STAT induced STAT inhibitor-1). SSI-1 is thought to play a critical role in negative feedback control of JAK-STAT signaling pathway. In the present study, we identified two novel human genes which products have homologous region in their SH2 domain and its COOH-terminal region to mouse SSI-1. Northern blotting analysis and functional studies demonstrated that SSI-2 and SSI-3 mRNA were also induced by cytokine stimulation and their forced expression in mouse myeloid leukemia cell, M1, suppressed the apoptotic effect of LIF, like SSI-1. We also demonstrated the structure of human SSI-1.

Structure and function of a new STAT-induced STAT inhibitor.

The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3. Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK-STAT pathway induced by cytokine stimulation.

Leptin receptor (OB-R) oligomerizes with itself but not with its closely related cytokine signal transducer gp130.

Leptin (OB) exerts weight-reducing effects in mice. The structure of the receptor for this factor, OB-R, is considerably similar to those of gp130, the common signal transducing receptor component for the interleukin-6 (IL-6) family of cytokines, and leukemia inhibitory factor receptor (LIFR). Since the IL-6 family of cytokines signal through gp130 homodimer or gp130/LIFR heterodimer, we have examined in this study the possible involvement of gp130 and LIFR in leptin signaling through OB-R. Leptin stimulation induces tyrosine phosphorylation of neither gp130 nor LIFR, while LIF stimulation does both. As examined by using two differently epitope-tagged OB-R molecules, the spontaneous homo-oligomerization of OB-R has been elucidated. Ba/F3 cells, which do not express gp130, are non-responsive to leptin and exhibit increased DNA synthesis in response to leptin after transfection of OB-R cDNA alone. OB-R appears to transduce the signal via its homo-oligomerization without interaction with gp130 or LIFR.