Generation of a novel CD30+ B cell subset producing GM-CSF and its possible link to the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs ) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-ß further potentiated IL-4- and IL-13-induced GM-Beffs . Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-ß. GM-Beffs were enriched within CD20+ CD30+ CD38-/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+ CD1a+ CD14- CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs . Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

Increased Syk phosphorylation leads to overexpression of TRAF6 in peripheral B cells of patients with systemic lupus erythematosus.

OBJECTIVE: Activation of B cells is a hallmark of systemic lupus erythematosus (SLE). Syk and TRAF6 are key signaling molecules in B-cell activation through BCR and CD40/TLR, respectively. Nevertheless, whether expression of Syk and TRAF6 is altered in SLE B cells remains unknown. METHODS: Phosphorylation and/or expression of Syk and TRAF6 were analyzed by flow cytometry in peripheral blood mononuclear cells isolated from SLE patients. RESULTS: Pronounced phosphorylation and expression of Syk were noted in B cells from SLE patients compared with healthy donors. Levels of Syk phosphorylation correlated with the disease activity score. TRAF6 was significantly over-expressed in B cells of SLE patients as compared with healthy donors, and significant correlation of levels of TRAF6 expression and Syk phosphorylation was observed in SLE patients. Levels of TRAF6 expression were more pronounced in CD27+ memory B cells than in CD27-naïve B cells. In vitro treatment of SLE B cells with a Syk inhibitor (BAY61-3606) reduced Syk phosphorylation as well as TRAF6 expression. CONCLUSION: Our results suggest that the activated Syk-mediated TRAF6 pathway leads to aberrant activation of B cells in SLE, and also highlight Syk as a potential target for B-cell-mediated processes in SLE.

PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL.

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.

Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus.

OBJECTIVE: To analyze the Th1/Th2 balance of peripheral Th cells in patients with systemic lupus erythematosus (SLE). METHODS: The Th1:Th2 ratio was analyzed in 3 groups: SLE without proteinuria (group I; n = 23), SLE with proteinuria (group II; n = 31), and normal controls (group III; n = 24). Group II patients who had undergone renal biopsy were classified into 3 subgroups based on their renal histopathologic findings. The intracellular cytokine detection method with flow cytometry was used to quantitate Th1 and Th2 cells. RESULTS: There was no difference in the mean Th1:Th2 ratio between SLE patients (groups I and II) and healthy controls (group III). However, the mean value in group II was significantly higher than those in groups I and III. Moreover, within group II, the mean value in SLE patients who had diffuse proliferative lupus nephritis (World Health Organization class IV) was especially high. CONCLUSION: Although SLE has been considered to be a disease in which Th2 cells predominate, the Th1/Th2 balance of peripheral Th cells in SLE patients in the present study did not show a predominance of these cells. In contrast, among SLE patients with WHO class IV lupus nephritis, there was a strong predominance of Th1.

Antagonistic effects of an alternative splice variant of human IL-4, IL-4delta2, on IL-4 activities in human monocytes and B cells.

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.

Differential effects of interleukin-4 and interleukin-10 on nitric oxide production by murine macrophages.

OBJECTIVE: To study the effect of interleukin (IL)-4 and IL-10 on nitric oxide (NO) production by macrophages. MATERIALS AND METHODS: Elicited or resident peritoneal macrophages (PMO) and a macrophage cell line Raw264.7 were primed by IL-4 or IL-10 for 6 hours, and were further incubated in the presence of interferon (IFN)-gamma and/or lipopolysaccharide (LPS) for 48 hours. NO2- accumulation in the supernatant of cultured cells was used as an indicator of NO production and was determined by the standard Griess reaction adapted for microplates. The amount of tumor necrosis factor (TNF)-alpha in the culture supernatants was determined with a commercially available ELISA kit. The absorbance was measured at 450 nm with a microplate photometer. RESULTS: IL-4 inhibited NO production by murine macrophages of different sources and the macrophage cell line Raw264.7. In contrast, different macrophage populations showed differential responses to IL-10. After stimulation with LPS or IFN-gamma, IL-10 suppressed NO production by elicited PMO but enhanced NO production by resident PMO or by Raw264.7. Both IL-4 and IL-10 inhibited the production of TNF-alpha, which has been shown to play a crucial role in NO production. In the presence or the absence of blocking antibody to TNF-alpha, IL-10 always enhanced NO production by resident PMO. This result suggests that the inhibition of TNF-alpha production and the enhancement of NO production by resident PMO stimulated with IL-10 are independent, coexisting events. CONCLUSIONS: Factors other than TNF-alpha have been suspected to influence NO production by macrophages, and this study indicates that IL-10 may be a candidate cytokine for resident PMO.

Role of IL-10 in the crossregulation of prostaglandins and cytokines in monocytes.

In the present study we have focused mainly on the role of IL (interleukin)-10 in the crossregulation of prostaglandins and cytokines in human monocytes. We first determined the effects of tumor necrosis factor-alpha (TNF-alpha) and IL-10 on monocyte prostaglandin E2 (PGE2) production. Unstimulated monocytes constitutively produced a small but significant amount of PGE2 in the culture supernatants. Both TNF-alpha and lipopolysaccharide (LPS) caused a remarkable increase in monocyte PGE2 production. On the other hand, IL-10 alone was without effect on constitutive PGE2 production but drastically inhibited LPS-induced PGE2 production in monocytes. Moreover, this inhibitory effect of IL-10 was not simply attributable to its inhibition of TNF-alpha production in LPS-stimulated monocytes. Next, we determined the effect of PGE2 on TNF-alpha mRNA expression in monocytes. Treatment of monocytes with or without PGE2 showed no detectable TNF-alpha mRNA. Activation of monocytes by LPS resulted in a remarkable accumulation of TNF-alpha mRNA and PGE2 efficiently inhibited this expression. Finally, we determined the effect of PGE2 on IL-10 mRNA expression in monocytes. Similar to TNF-alpha mRNA, unstimulated monocytes showed no detectable IL-10 mRNA. Interestingly, PGE2 alone drastically induced IL-10 mRNA. Besides, activation of monocytes by LPS resulted in a remarkable accumulation of IL-10 mRNA, and PGE2 further enhanced this expression. These results indicate that TNF-alpha and PGE2 are key molecules for the induction of IL-10 in monocytes, and that IL-10, in turn, plays a crucial role in terminating the inflammatory cascade via downregulation of production of proinflammatory molecules including TNF-alpha and PGE2.

Activation of STAT5 by lipopolysaccharide through granulocyte-macrophage colony-stimulating factor production in human monocytes.

LPS is a potent stimulator of monocytes, inducing many of their functions. Although the details of how LPS exerts such functions remain largely unknown, transcription factors such as nuclear factor-kappaB, nuclear factor-IL-6, and activator protein-1 have been shown to be involved in this process. However, to date it has been thought that no known STAT molecule plays a role in the activation of monocytes by LPS. In this study we examined whether some known STAT molecule is stimulated by LPS, based on the finding that a GAS motif sequence is conserved in the promoter regions of human, mouse, and rat cyclo-oxygenase-2 (COX-2) genes. Consequently, LPS induced activation of STAT5 in human monocytes, and this STAT5 activation occurred in an indirect way via granulocyte-macrophage CSF (GM-CSF) secreted by LPS-stimulated monocytes. Expression of COX-2 protein was partially reduced by treatment of anti-human GM-CSF Ab. Activation of STAT5 was inhibited by either IL-10 or dexamethasone (Dex), but not by aspirin. IL-10 blocked activation of STAT5 indirectly by suppressing GM-CSF production, while Dex inhibited this activation both directly and indirectly. Taken together, these results suggest that in addition to other transcription factors, STAT5 plays an important role in activation of monocytes by LPS, and that STAT5 is another target for IL-10 and Dex to inhibit COX-2 expression in activated monocytes.

Effect of IL-10 on collagen-induced arthritis in mice.

In the present study we investigated the effect of a potent anti-inflammatory cytokine, interleukin (IL)-10, on the development of collagen-induced arthritis (CIA) in mice. Each DBA1/J mouse was immunized with 200 micrograms of native collagen and followed by booster injections at 3 weeks. rmIL-10 was injected i.p. daily at a dose of 100 ng/mouse. Mice were divided into four groups according to the administration period of rmIL-10. As a result, a 48-day course of IL-10 treatment significantly suppressed the severity of arthritis. Among the 4 groups, the most pronounced suppression was observed in the group in which IL-10 was given from day 0 to 21. On the other hand, there were no significant differences in the serum IgG anti-type II collagen (CII) titers between the four groups. Moreover, the production of cytokines (IL-6 and tumor necrosis factor-alpha (TNF-alpha)) and other mediators (prostaglandin E2 (PGE2) and nitric oxide (NO)) by peritoneal macrophages seemed to show no clear correlation with the severity of arthritis in mice. These results raise the possibility that IL-10 might be a useful agent for suppressing the progression and the development of CIA in mice.

Suppression of superoxide anion production by interleukin-10 is accompanied by a downregulation of the genes for subunit proteins of NADPH oxidase.

Interleukin-10 (IL-10) inhibited the production of superoxide anion (02-) by both unactivated and interferon-gamma (IFN-gamma)-activated human monocytes. Simultaneous addition of IL-10 with IFN-gamma at the start of incubation was necessary for an optimal inhibitory effect. The degree of inhibition was substantially comparable to that of IL-4, and the combination of suboptimal concentrations of IL-10 and IL-4 produced an additive effect. A similar effect was also obtained when viral IL-10 (vIL-10) was used instead of IL-10. The inhibitory effect of IL-10 was accompanied by the reduced accumulation of transcripts for heavy chain subunit of cytochrome b558 (gp9l-phox) and 47-kD cytosolic factor (p47-phox), components of the O2--generating NADPH oxidase system. Reduction of the mRNAs was distinct within 24 hours. On the other hand, the induced O2- production by human monocytic leukemia cell lines (THP-1 and HL60) was not inhibited by IL-10. The amount of gp9l-phox and p47-phox mRNAs remained unchanged even in the presence of excess amount of IL-1O. Taken together, these results suggest that IL-10 inhibits 02- production by downregulation of the gp9l-phox and p47-phox genes in human monocytes.

[Inhibitory effects of interleukin (IL) -10 and viral IL-10 (vIL-10) on the functions of monocytes/macrophages].

In this study, we examined the effects of IL-10 and vIL-10 on the production of superoxide anion (O2-) and nitric oxide (NO) by human monocytes and mouse macrophages. At an optimal concentration, human IL-10 (hIL-10) and vIL-10 significantly inhibited the production of interferon (IFN)-gamma by stimulated human peripheral blood mononuclear cells (PBMNCs). They also efficiently inhibited the production of O2- by both unstimulated and IFN-gamma-activated human monocytes. Mouse IL-10 (mIL-10) also significantly inhibited the production of NO by lipopolysaccharide (LPS) and IFN-gamma-stimulated mouse peritoneal macrophages. Moreover, the production of O2- and NO was effectively suppressed whether the IL-10 was added before or together with the stimulus, indicating that this cytokine acts primarily at an early stage of monocyte/macrophage activation by IFN-gamma and LPS. We also examined the effects of IL-4 and transforming growth factor (TGF)-beta on the production of O2- and NO by human monocytes and mouse macrophages, and found that they significantly inhibited both the production of O2- by human monocytes and the production of NO by mouse macrophages. Moreover, a combination of any two of IL-10, IL-4 and TGF-beta caused an additive effect on the inhibition of O2- production by human monocytes. These results indicated that IL-10 suppresses monocyte/macrophage activation either indirectly via an inhibition of the synthesis of IFN-gamma, a potent monocyte/macrophage activator, by PBMNCs, or directly via the deactivation of monocytes/macrophages. Moreover IL-10 may act in concert with IL-4 and TGF-beta to suppress monocyte/macrophage activation in vivo.

Expression of the c-kit ligand and interleukin 6 genes in mouse bone marrow stromal cell lines.

The expression of c-kit ligand and interleukin 6 (IL-6) genes in mouse bone marrow-derived stromal cell lines was examined using quantitative polymerase chain reaction (PCR) analysis based on the design of an internal DNA control. The stromal cells studied included the 14F1.1 endothelial-adipocytes that support long-term hemopoiesis and two additional cell lines (MBA-1, MBA-13) which do not have this function. All the cell lines expressed c-kit ligand gene constitutively, and this expression was not increased by lectins. On the other hand, the expression of the IL-6 gene was markedly induced in all the lines by lipopolysaccharide (LPS) and by phorbol 12-myristate 13 acetate (PMA). The constitutive expression of c-kit ligand in 14F1.1 cells was the lowest among the three cell lines studied and could be increased by stimulation with IL-4. Thus, we observed some quantitative differences among the cell lines in their expression of cytokine genes. However, the unique capacity of 14F1.1 cells to support in vitro hemopoiesis cannot thus far be explained solely on the basis of the ability of these cells to secrete cytokines which are not produced by other stromal cell lines. c-kit ligand may be necessary, but its presence alone is not sufficient for 14F1.1 cells to support prolonged hemopoiesis.

Transfection of interferon-gamma gene in a mouse bone marrow stromal preadipocyte cell line causes apoptotic cell death.

It has been reported that bone marrow and serum of patients with aplastic anemia or chronic myeloproliferative disorders contain an abnormal concentration of cytokines. In the present study, we tried to isolate mouse bone marrow stromal cell lines that were stably transformed with a variety of cytokine genes and that expressed them constitutively. From mouse bone marrow stromal cell lines MBA-1, MBA-13, and 14F1.1, we isolated clones secreting interleukin-3 (IL-3), IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte (G)-CSF. Interferon-gamma (IFN-gamma)-producing stable transformants could not be established from 14F1.1 cells in spite of repeated transfection trials. At early stages of transfection, 14F1.1 cells did secrete IFN-gamma; however, exogenously added mouse IFN-gamma could not inhibit 14F1.1 cell growth. We discovered that chromosomal DNA isolated from 14F1.1 after transfection with the mouse IFN-gamma gene was fragmented. This is characteristic of cells undergoing apoptotic cell death. DNA fragmentation was also observed in 14F1.1 cells transfected with the human IFN-gamma gene. These results indicate that intracellular IFN-gamma induces apoptotic cell death of 14F1.1 stromal cells.

Epstein-Barr virus BCRF1 gene product (viral interleukin 10) inhibits superoxide anion production by human monocytes.

Due to its similar biological activities to interleukin 10 (IL-10), Epstein-Barr virus (EBV) BCRF1 gene product (viral IL-10: vIL-10) has recently been recognized as an analogue of authentic IL-10. Preincubation of human monocytes with vIL-10, like human IL-10, induced smaller amounts of interferon-gamma (IFN-gamma) mRNA in activated human peripheral blood mononuclear cells (PBMNCs) than nonpreincubation, indicating that vIL-10 acts principally on monocytes. Since the activation of monocytes and their generation of oxidative products are regulated by various cytokines, we examined the effects of vIL-10 on superoxide anion (O2-) production by human PBMNCs and monocytes. Not only PBMNCs but also monocytes preincubated with vIL-10 showed a smaller production of O2-. Inhibition was achieved in a dose-dependent fashion and increased gradually after incubation with vIL-10. Additions of IFN-gamma, macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), which prime monocyte activation and induce O2- production, were also affected by the reciprocal effect of vIL-10. Thus, vIL-10 production by EBV-infected cells may be involved in the development of EBV-related disorders.