Administration of fasudil, a ROCK inhibitor, attenuates disease in lupus-prone NZB/W F1 female mice.

Accumulating evidence from murine studies suggests that the RhoA/ROCK pathway plays an important role in the development of autoimmune disorders. We previously demonstrated that ROCK inhibition ameliorates disease in MRL/lpr mice, a spontaneous model of lupus. This study aimed to explore the protective effects of the ROCK inhibitor fasudil in a distinct model of lupus, NZB/W F1 female mice, to assess the broad applicability of ROCK inhibition for the treatment of lupus. NZB/W F1 female mice were administered fasudil continuously in their drinking water starting at 18 or 24 weeks of age up until 44 weeks of age, or remained untreated. Fasudil treatment significantly improved survival and decreased proteinuria, particularly when treatment was started at 18 weeks. There was also a significant decrease in serum anti-dsDNA autoantibody production, glomerular IgG and C3 deposition, and glomerulonephritis. Analysis of the splenic lymphocyte compartment revealed reduced effector/memory CD4(+) T cell and plasma cell numbers in fasudil treated mice while the frequency of other B cell and T cell subsets was unchanged. These results thus indicate that fasudil can ameliorate disease in NZB/W F1 female mice, suggesting that ROCK inhibition might be broadly effective for the treatment of lupus.

Lineage-specific modulation of interleukin 4 signaling by interferon regulatory factor 4.

Interleukin (IL)-4 is an immunoregulatory cytokine that exerts distinct biological activities on different cell types. Our studies indicate that interferon regulatory factor (IRF)-4 is both a target and a modulator of the IL-4 signaling cascade. IRF-4 expression is strongly upregulated upon costimulation of B cells with CD40 and IL-4. Furthermore, we find that IRF-4 can interact with signal transducer and activator of transcription (Stat)6 and drive the expression of IL-4-inducible genes. The transactivating ability of IRF-4 is blocked by the repressor factor BCL-6. Since expression of IRF-4 is mostly confined to lymphoid cells, these data provide a potential mechanism by which IL-4-inducible genes can be regulated in a lineage-specific manner.

Interferon gamma signaling alters the function of T helper type 1 cells.

One mechanism regulating the ability of different subsets of T helper (Th) cells to respond to cytokines is the differential expression of cytokine receptors. For example, Th2 cells express both chains of the interferon gamma receptor (IFN-gammaR), whereas Th1 cells do not express the second chain of the IFN-gammaR (IFN-gammaR2) and are therefore unresponsive to IFN-gamma. To determine whether the regulation of IFN-gammaR2 expression, and therefore IFN-gamma responsiveness, is important for the differentiation of naive CD4(+) T cells into Th1 cells or for Th1 effector function, we generated mice in which transgenic (TG) expression of IFN-gammaR2 is controlled by the CD2 promoter and enhancer. CD4(+) T cells from IFN-gammaR2 TG mice exhibit impaired Th1 polarization potential in vitro. TG mice also display several defects in Th1-dependent immunity in vivo, including attenuated delayed-type hypersensitivity responses and decreased antigen-specific IFN-gamma production. In addition, TG mice mount impaired Th1 responses against Leishmania major, as manifested by increased parasitemia and more severe lesions than their wild-type littermates. Together, these data suggest that the sustained expression of IFN-gammaR2 inhibits Th1 differentiation and function. Therefore, the acquisition of an IFN-gamma-unresponsive phenotype in Th1 cells plays a crucial role in the development and function of these cells.

Th17 cells in rheumatoid arthritis and systemic lupus erythematosus.

Recent work has implicated a novel Th effector cell subset, the Th17 cell subset, in the development of both rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) because of the ability of Th17 cells to produce cytokines like IL-17 and IL-21 that can drive both inflammatory and humoral responses. In this review, we will discuss recent studies that have begun elucidating the factors that regulate the development of Th17 cells and provide a brief overview of the role of Th17 cells in RA and SLE.

Jak-STAT signaling induced by the v-abl oncogene.

The effect of the v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) on the Jak-STAT pathway of cytokine signal transduction was investigated. In murine pre-B lymphocytes transformed with A-MuLV, the Janus kinases (Jaks) Jak1 and Jak3 exhibited constitutive tyrosine kinase activity, and the STAT proteins (signal transducers and activators of transcription) normally activated by interleukin-4 and interleukin-7 were tyrosine-phosphorylated in the absence of these cytokines. Coimmunoprecipitation experiments revealed that in these cells v-Abl was physically associated with Jak1 and Jak3. Inactivation of v-Abl tyrosine kinase in a pre-B cell line transformed with a temperature-sensitive mutant of v-abl resulted in abrogation of constitutive Jak-STAT signaling. A direct link may exist between transformation by v-abl and cytokine signal transduction.

Lack of interferon gamma receptor beta chain and the prevention of interferon gamma signaling in TH1 cells.

The ability of interferon gamma (IFN-gamma) to inhibit the proliferation of type 2 T helper cells (TH2), but not that of type 1 (TH1) cells, suggests that helper cell subsets might differ in their activation of the IFN-gamma signaling pathway. The IFN-gamma-inducible signal transducing factor (STF-IFN gamma) was activated in murine TH2 but not in TH1 cell clones, because in the latter the second chain of the IFN-gamma receptor (accessory factor 1 or IFN-gamma R beta) was absent. Thus, TH1 cells use receptor modification to prevent the activation of STF-IFN gamma and achieve an IFN-gamma-resistant state.

Intravenous pulse cyclophosphamide treatment of severe lupus nephritis: a prospective five-year study.

Despite its widespread use, there are only a few published studies of the use of intravenous high dose pulse cyclophosphamide in systemic lupus nephritis. There are few data about the long-term efficacy and safety of this form of therapy. This study evaluates the clinical efficacy, toxicity, and effects on renal morphology of this regimen in patients with severe lupus nephritis followed prospectively over a five-year period. Twenty consecutive patients with severe active lupus nephritis were enrolled in a treatment regimen of six monthly intravenous pulses of cyclophosphamide (0.5 to 1 g/m2) together with high dose corticosteroid therapy which was rapidly tapered. Efficacy was assessed by improvement or stabilization of clinical, serologic and renal functional parameters. Repeat renal biopsies were performed in 15 patients. Potential toxicity related to therapy was documented. Over the first six months of treatment, this regimen resulted in improvement of clinical activity, lupus serology, stabilization of renal function and decreased proteinuria in 19/20 patients. Nephrotic syndrome remitted in 8/10 patients by one year. Over five years of follow-up, there were five treatment failures defined as a doubling of serum creatinine over baseline. At five years, 3 patients required renal replacement therapy. Elevated plasma creatinine at time of first biopsy, degree of proteinuria, histologic activity and chronicity were not statistically correlated with treatment failure. Patients who failed to respond to this treatment were, however, more likely to have diffuse proliferative lupus nephritis (WHO Class IV) lesions on initial biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage-specific modulation of IFN-regulatory factor 4 function by Krüppel-type zinc finger proteins.

Optimal humoral responses depend on the activation of Ag-specific B cells, followed by their progression toward a fully differentiated phenotype. Acquisition of stage-appropriate patterns of gene expression is crucial to this differentiation program. However, the molecular mechanisms used by B cells to modulate gene expression as they complete their maturation program are poorly understood. IFN-regulatory factor 4 (IRF-4) plays a critical role in mature B cell function. Using the transcriptional regulation of the human B cell activation marker CD23 as a model system, we have previously demonstrated that IRF-4 is induced in response to B cell-activating stimuli and that it acts as a transactivator of CD23 gene expression. We have furthermore found that IRF-4 function can be blocked by B cell lymphomas 6 (BCL-6) protein, a Krüppel-type zinc finger repressor normally expressed in germinal center B cells. However, CD23 expression is known to be down-regulated in plasma cells despite high level expression of IRF-4 and the lack of BCL-6, suggesting that in plasma cells the IRF-4-mediated induction of CD23 is prevented by its interaction with a distinct repressor. In this set of studies, we demonstrate that IRF-4 interacts with B lymphocyte-induced maturation protein/positive regulatory domain I-binding factor 1 (Blimp1/PRD1-BF1), a Krüppel-type zinc finger protein whose expression correlates with terminal B cell differentiation. Functional studies indicate that Blimp1, like BCL-6, can block IRF-4-transactivating ability. These findings thus support a model whereby IRF-4 function is modulated in a stage-specific manner by its interaction with developmentally restricted sets of Krüppel-type zinc finger proteins.

IFN-alpha activates Stat6 and leads to the formation of Stat2:Stat6 complexes in B cells.

IFN-alpha consists of a family of highly homologous proteins, which exert pleiotropic effects on a wide variety of cell types. The biologic activities of IFN-alpha are mediated by its binding to a multicomponent receptor complex resulting in the activation of the Janus kinase-STAT signaling pathway. In most cell types, activation of Stat1 and Stat2 by IFN-alpha leads to the formation of either STAT homo-/heterodimers or of the IFN-stimulated gene factor 3 complex composed of Stat1, Stat2, and p48, a non-STAT protein. These distinct transcriptional complexes then target two different sets of cis-elements, gamma-activated sites and IFN-stimulated response elements. Here, we report that IFN-alpha can activate complexes containing Stat6, which, until now, has been primarily associated with signaling by two cytokines with biologic overlap, IL-4 and IL-13. Induction of Stat6 complexes by IFN-alpha appears to be cell type specific, given that tyrosine phosphorylation of Stat6 in response to IFN-alpha is predominantly detected in B cells. Activation of Stat6 by IFN-alpha in B cells is accompanied by the formation of novel Stat2:Stat6 complexes, including an IFN-stimulated gene factor 3-like complex containing Stat2, Stat6, and p48. B cell lines resistant to the antiproliferative effects of IFN-alpha display a decrease in the IFN-alpha-mediated activation of Stat6. Activation of Stat6 as well as of Stat2:Stat6 complexes by IFN-alpha in B cells may allow modulation of target genes in a cell type-specific manner.

Gamma chain-associated cytokine receptors signal through distinct transducing factors.

The IL-2, IL-4, and IL-7 signaling pathways have been shown to utilize shared components. The receptors for these cytokines are composed of ligand-specific binding chains that associate with a shared signaling subunit, the common gamma (gamma c) chain. In addition, IL-2, IL-4, and IL-7 induce activation of a common set of nonreceptor tyrosine kinases, Jak-1 and Jak-3. We have further investigated the signaling events induced by these cytokines and find that the gamma c-associated receptors activate distinct signal transducing factors (STFs). In addition, we show that a 94-kDa STAT-related protein (p94) is activated in response to IL-2 and IL-7, but not IL-4. These data indicate that IL-2, IL-4, and IL-7 activate distinct signaling molecules which might be differentially recruited to the receptor complex by the ligand-specific units of the IL-2, IL-4, and IL-7 receptors.

STF-IL-4: a novel IL-4-induced signal transducing factor.

The mechanism by which interleukin-4 (IL-4) regulates the expression of particular genes is unknown. We have determined that IL-4 induces a DNA binding factor (termed STF-IL-4) which has a strong affinity for an IFN-gamma activation site (GAS). Interestingly, STF-IL-4 also binds to the IL-4 responsive promoter for the Ig heavy chain germline epsilon transcript. The IL-4 dependent activation of STF-IL-4 is rapid, does not require protein synthesis and results in the sequential appearance of binding activity first in the cytoplasm and then later in the nucleus. Activation of STF-IL-4 is sensitive to tyrosine kinase inhibitors and the active factor is tyrosine phosphorylated. This pattern of activation is similar to the activation of interferon-induced transcription factors. STF-IL-4 appears to be a new member of a growing family of cytokine-induced transcriptional regulators.

Signaling pathways mediated by the TNF- and cytokine-receptor families target a common cis-element of the IFN regulatory factor 1 promoter.

CD40 activation of B cells is strongly influenced by the presence of cytokines. However, the molecular basis for the interplay between these distinct stimuli is not clearly delineated. IFN regulatory factor 1 (IRF-1) is a transcription factor activated by either CD40 or cytokines. We have found that these different sets of signals target a common cis-acting element in the promoter of this gene, the IRF-1 gamma-activated site (GAS). Targeting of the IRF-1 GAS is not confined to activation via CD40 but extends to other stimuli that mimic the CD40 signaling cascade, like TNF-alpha and EBV. In contrast to induction of STATs by cytokines, the IRF-1 GAS-binding complex activated by CD40, TNF-alpha, or EBV contains Rel proteins, specifically p50 and p65. In this system, simultaneous exposure to CD40L together with either IL-4 or IFN-gamma does not lead to the activation of novel Rel/STAT complexes. Given the importance of IRF-1 in a variety of biologic functions from proliferation to apoptosis, our findings support the notion that modulation of IRF-1 levels may be a critical control point in B cell activation.

T-BAM/CD40-L on helper T lymphocytes augments lymphokine-induced B cell Ig isotype switch recombination and rescues B cells from programmed cell death.

An important component of T cell help for B lymphocyte differentiation is the contact-dependent signaling mediated by the T cell-B cell activating molecule (T-BAM/CD40-L), an activation-induced surface membrane protein on CD4+ T helper cells in lymphoid follicles that interacts with the B cell surface molecule, CD40. The present study dissects the roles of T-BAM/CD40-L in helper function by means of a neutralizing anti-T-BAM/CD40-L mAb (5c8), a T-BAM/CD40-L-expressing T cell tumor subclone (Jurkat D1.1), and a T-BAM/CD40-L-responsive IgM+ B cell tumor of germinal center origin (RAMOS 266). Like activated T cells, D1.1 cells induce B cells to synthesize IgG, IgA, and IgE in a process that is specifically inhibited by the mAb 5c8. Although rIL-4 alone, but not Jurkat D1.1, induces IgH C gamma mRNA transcripts in RAMOS 266, the T-BAM/CD40-L molecule on D1.1 acts on rIL-4-primed RAMOS B cells to augment expression of C gamma transcripts. In addition, IgG+ RAMOS 266 clones were expanded from D1.1- and rIL-4-stimulated cultures that had undergone deletional IgH isotype switch recombination events. Furthermore, T-BAM/CD40-L signals delivered by the D1.1 clone dramatically rescue RAMOS 266 from mAb anti-IgM-induced apoptosis. Taken together, these data support the idea that T-BAM/CD40-L plays important roles in inducing Ig isotype switch recombination and the clonal selection of isotype-switched B cells.

Interleukin 4 signals through two related pathways.

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.