Yosuke Tanaka

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.

Association between IL-4 genotype and IL-4 production in the Japanese population.

We have identified that there are only two IL-4 gene haplotypes (I and II) in the Japanese population. There are significant differences among three genotypes (I/I, I/II and II/II) in the IL-4 producing proportion of peripheral Th cells using intracellular cytokine detection assay. These results make it likely that IL-4 genotype could influence the type of immune response.

Inhibition by interleukin-10 of inducible cyclooxygenase expression in lipopolysaccharide-stimulated monocytes: its underlying mechanism in comparison with interleukin-4

Both interleukin-10 (IL-10) and IL-4 inhibited the prostanoid synthesis of lipopolysaccharide (LPS)-stimulated human monocytes, and their inhibition was shown to be based on a common mechanism to suppress the gene expression of inducible cyclooxygenase (COX). COX has been shown to exist in at least two distinct isoforms, designated COX-1 and COX-2, and their gene expressions exhibit different profiles. At both the protein and mRNA levels, the expression of COX-1 was constitutive and was not modulated by treatments with LPS, IL-10, or IL-4. In contrast, the expression of COX-2 was observed only after stimulation with LPS. IL-10 and IL-4 significantly inhibited LPS-induced COX-2 expression. Kinetic studies showed that they inhibited COX-2 mRNA expression within 1 hour after stimulation and that maximal inhibition was consistently observed at 5 hours. Moreover, the addition of cycloheximide (CHX) to LPS-stimulated monocytes resulted in a superinduction of COX-2 mRNA, whereas CHX almost abrogated the abilities of IL-10 and IL-4 to inhibit this gene expression. Experiments with actinomycin D showed that both cytokines accelerated the degradation of COX-2 mRNA. Furthermore, nuclear run-on experiments showed that both cytokines modestly inhibited LPS-induced COX-2 gene transcription. Thus, both cytokines seemed to regulate the COX-related pathway in a similar manner, although their receptor systems did not show any structural similarities. Considering recent findings showing that the drugs that exhibit a selective effect on COX-2 may be more preferable in inflammatory conditions, such biologic activities of IL-10 and IL-4 described above may offer useful tools in controlling inflammatory disorders in the future.

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 μg 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-α (TNF-α)) 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.

The combination of polymorphisms within interferon‐γ receptor 1 and receptor 2 associated with the risk of systemic lupus erythematosus

Genetic factors seem to play a significant role in susceptibility to systemic lupus erythematosus (SLE). We previously described the amino acid polymorphism (Val14Met) within the IFN‐γ receptor 1 (IFN‐γR1), and that the frequency of the Met14 allele in SLE patients was significantly higher than that of the healthy control population [Tanaka et al. (1999) Immunogenetics 49, 266–271]. We also found an amino acid polymorphism (Gln64Arg) within IFN‐γ receptor 2 (IFN‐γR2). Since the IFN‐γ receptor is a complex consisting of IFN‐γR1 and IFN‐γR2, we searched for the particular combination of two kinds of amino acid polymorphisms found within the IFN‐γ receptor which plays a prominent role in susceptibility to SLE. The greatest risk of the development of SLE was detected in the individuals who had the combination of IFNGR1 Met14/Val14 genotype and IFNGR2 Gln64/Gln64 genotype.

Association of the interferon-γ receptor variant (Val14Met) with systemic lupus erythematosus

Abstract Genetic factors seem to play a significant role in susceptibility to systemic lupus erythematosus (SLE). The purpose of this study was to investigate whether the amino acid polymorphism (Val14Met) found within the IFN-γ receptor gene (IFNGR1) plays a prominent role in susceptibility to SLE. We found Val14Met located at the COOH terminal of the signal peptide of the IFN-γ receptor. There was a significant difference in this polymorphism frequency between SLE patients and healthy populations. To clarify whether this amino acid substitution resulted in the alteration of the receptor function, we evaluated the induction of HLA-DR antigen expression on B cells by IFN-γ stimulation. There was also a significant difference in the induction of HLA-DR by IFN-γ stimulation between B cells. Furthermore, an intracellular cytokine assay indicated that the Th1/Th2 balance of Th cells bearing the variant receptor shifted to Th2. The genetic polymorphism found within the IFN-γ receptor gene (Val14Met) may result in a shift to Th2, and this shift may increase susceptibility to SLE.

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-α (TNF-α) 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-α 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-α production in LPS-stimulated monocytes. Next, we determined the effect of PGE2 on TNF-α mRNA expression in monocytes. Treatment of monocytes with or without PGE2 showed no detectable TNF-α mRNA. Activation of monocytes by LPS resulted in a remarkable accumulation of TNF-α mRNA and PGE2 efficiently inhibited this expression. Finally, we determined the effect of PGE2 on IL-10 mRNA expression in monocytes. Similar to TNF-α 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-α 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-α and PGE2.

Selective DNA-binding activity of interleukin-10-stimulated STAT molecules in human monocytes

It has been demonstrated that interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) have various reverse effects on macrophages; however, the molecular mechanism of this difference has not been fully understood. In this study, we analyzed the binding activity of IL-10- and IFN-gamma-activated STAT molecules to two kinds of GAS-motif sequences. IL-10-activated STAT1 could bind to the GAS-motif sequence in the promoter region of the Fcgamma receptor, but not to that in the promoter region of the COX-2 gene, whereas IFN-gamma-activated STAT1 and STAT5 could bind to both sequences. IL-10 inhibited IFN-gamma-induced STAT activation without newly synthesized protein. We further demonstrated that aspirin, but not dexamethasone, suppressed IFN-gamma-induced STAT activation. Taken together, these results suggest that IL-10-activated STAT1 has a specificity in binding to the GAS-motif sequences, whereas IFN-gamma-activated STAT1 and STAT5 have a broader spectrum in binding to the GAS-motif sequences. This may explain the difference between IL-10 and IFN-gamma in biological activity, and the inhibitory effect of IL-10 on IFN-gamma activities.

Transplantation of mesenchymal stem cells ameliorates secondary osteoporosis through interleukin-17-impaired functions of recipient bone marrow mesenchymal stem cells in MRL/lpr mice.

IntroductionSecondary osteoporosis is common in systemic lupus erythematosus and leads to a reduction in quality of life due to fragility fractures, even in patients with improvement of the primary disorder. Systemic transplantation of mesenchymal stem cells could ameliorate bone loss and autoimmune disorders in a MRL/lpr mouse systemic lupus erythematosus model, but the detailed therapeutic mechanism of bone regeneration is not fully understood. In this study, we transplanted human bone marrow mesenchymal stem cells (BMMSCs) and stem cells from exfoliated deciduous teeth (SHED) into MRL/lpr mice and explored their therapeutic mechanisms in secondary osteoporotic disorders of the systemic lupus erythematosus model mice.MethodsThe effects of systemic human mesenchymal stem cell transplantation on bone loss of MRL/lpr mice were analyzed in vivo and ex vivo. After systemic human mesenchymal stem cell transplantation, recipient BMMSC functions of MRL/lpr mice were assessed for aspects of stemness, osteogenesis and osteoclastogenesis, and a series of co-culture experiments under osteogenic or osteoclastogenic inductions were performed to examine the efficacy of interleukin (IL)-17-impaired recipient BMMSCs in the bone marrow of MRL/lpr mice.ResultsSystemic transplantation of human BMMSCs and SHED recovered the reduction in bone density and structure in MRL/lpr mice. To explore the mechanism, we found that impaired recipient BMMSCs mediated the negative bone metabolic turnover by enhanced osteoclastogenesis and suppressed osteoblastogenesis in secondary osteoporosis of MRL/lpr mice. Moreover, IL-17-dependent hyperimmune conditions in the recipient bone marrow of MRL/lpr mice damaged recipient BMMSCs to suppress osteoblast capacity and accelerate osteoclast induction. To overcome the abnormal bone metabolism, systemic transplantation of human BMMSCs and SHED into MRL/lpr mice improved the functionally impaired recipient BMMSCs through IL-17 suppression in the recipient bone marrow and then maintained a regular positive bone metabolism via the balance of osteoblasts and osteoclasts.ConclusionsThese findings indicate that IL-17 and recipient BMMSCs might be a therapeutic target for secondary osteoporosis in systemic lupus erythematosus.

Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.