Mitsuru Naiki

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c+B220+ subset expresses Ly6Chigh and MHC class IIlow in contrast with previously described thymic DC (CD11c+B220– cells). Freshly isolated thymic CD11c+B220+ cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo‐proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN‐α following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL‐12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN‐producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.

Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non‐treated ones by 2‐D PAGE in the pI range of 4.0–7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI‐TOF/TOF MS and subsequent protein database searching. They included unc‐18 protein homolog 67K, collapsin response mediator protein (CRMP)‐2 and CRMP‐4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post‐translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.

Increased Frequency of Pre- Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for a Developmental Block in B Cell Differentiation

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor component Igα (mb-1). Furthermore, levels of expression of the Rug2, λ5 and Igβ (B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

Hypertension Associated with Reduced Plasma Thrombomodulin Levels and a Hypercoagulable State in Rats

The plasma thrombomodulin (TM) level, an indicator of systemic endothelial cell damage, was measured in spontaneously hypertensive rats (SHR), deoxycorticosteron acetate (DOCA)‐induced hypertensive rats and normotensive Wistar–Kyoto (WKY) rats to clarify its changes in hypertension. Plasma TM levels, measured by enzyme‐linked immuno‐sorbent assay, decreased with aging (5–20‐weeks‐old) in both SHR and WKY, and they were lower in SHR than age‐matched WKY in all ages examined. Deoxycorticosteron acetate‐induced hypertensive WKY also showed decreased TM levels compared with normotensive WKY. Accelerated coagulation and fibrinolysis shown by the increases in thrombin–antithrombin complex (TAT) and D‐dimer levels were observed in both groups of hypertensive rats. These results suggest that hypertension may decrease plasma TM levels and induce a hypercoagulable state in rats.

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17s structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations.

NEUROTROPIN INHIBITS EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE) IN LEWIS RATS

The effects of Neurotropin, a substance extracted from the inflammatory dermis of rabbits inoculated with Vaccinia virus, for experimental allergic encephalomyelitis (EAE) in Lewis rats, a model for human multiple sclerosis (MS), was studied. The peptide defined by residues 68-84 (MB 68-84) which corresponds to the encephalitogenic portion of the guinea pig myelin basic protein (MBP) in complete adjuvant H37Ra (CFA) was injected into the hind foot pad of each rat. Neurotropin significantly suppressed the clinical and histological expression of actively induced EAE when administered i.p. daily from day 0 to day 6 after immunization. In addition, passive EAE induced by precultured spleen cells from rats immunized with MB 68-84 in CFA was also suppressed by daily administration of Neurotropin after cell transfer. Neurotropin treatment significantly suppressed the delayed-type hypersensitivity (DTH) response to MB 68-84. Furthermore, the ability of spleen cells from Neurotropin-treated rats to transfer EAE was significantly lower than that of saline-treated rats. It seemed that the suppression may be due to the inhibition of the activation by MB 68-84 of sensitized spleen cells, as demonstrated by proliferative response to MB 68-84. However, no difference was observed in Con A-induced proliferative response of the spleen cells between Neurotropin- and saline-treated rats. These findings indicate that Neurotropin inhibits EAE by suppressing the immune responses to encephalitogenic MBP with little non-specific suppression.

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.

2-Buten-4-olide (2-B4O) inhibits type II collagen-induced arthritis in Lewis rats

2-Buten-4-olide (2-B4O) is an endogenous substance which suppresses appetite and/or food intake. We studied its effect on type II collagen-induced arthritis (CIA) in Lewis rats, an animal model for human rheumatoid arthritis. Bovine type II collagen with incomplete Freunds adjuvant was injected intradermally into Lewis rats to induce CIA. 2-B4O (50 or 100 mg/kg) significantly inhibited the expression of the clinical symptoms when administered i.p. daily from day 1 to 21 after immunization. Furthermore, administration of 2-B4O daily from day 15 to 21 significantly reduced the severity of symptoms in established CIA. In addition, the progression of soft tissue swelling and articular bone erosions were suppressed by daily administration of 2-B4O. 2-B4O also significantly suppressed the delayed-type hypersensitivity (DTH) response to type II collagen at doses of 50 and 100 mg/kg. Finally 2-B4O significantly inhibited the formation of anti-type II collagen antibody at a dose of 100 mg/kg, but not at 50 mg/kg. These results suggest that 2-B4O has the strong inhibitory effects and therapeutic usefulness effects on CIA through the suppression of immune responses to type II collagen.

Suppression of acute experimental allergic encephalomyelitis by neurotropin: clinical, histopathologic, immunologic and immunohistochemical studies.

The effect of neurotropin, an extract isolated from the inflamed skin of rabbits inoculated with Vaccinia virus was examined on acute experimental allergic encephalomyelitis (EAE) in Lewis rats. A dose of 40 mg per kg body weight of neurotropin was administered intraperitoneally for 7 days post-inoculation. The severity of clinical signs of acute EAE was decreased by the administration of neurotropin. Histopathologic evaluation showed that lesion severity of EAE in neurotropin-treated rats was less than that seen in untreated rats. Blood lymphocyte subset analysis revealed that in comparison to untreated EAE rats, in neurotropin-treated rats, the percentage of OX6+ (Ia antigen) cells was lower and the W3/25+ (helper T cell): OX8+ (suppressor/cytotoxic T cell) cell ratio was greater during the period of peak inflammation. Immunohistochemical examination of neurotropin-treated rats demonstrated that OX6+ and W3/25+ cells within EAE lesions were fewer and that OX8+ cells in lesions occurred in greater numbers than those in untreated rats. These findings suggest that the OX8+ cells in the inflammatory lesions may have been induced by neurotropin treatment and that the suppressive effects on the disease may have been causally related to their presence.

Neurotropin inhibits accumulation of eosinophils induced by allergen through the suppression of sensitized T-cells

A non-protein extract isolated from the inflamed dermis of rabbits inoculated with vaccinia virus (Neurotropin) has been clinically used in Japan as an analgesic and anti-allergic drug. To clarify its anti-allergic mechanism, the effect of Neurotropin on eosinophil accumulation induced by ragweed pollen extract or platelet-activating factor (PAF) was examined in BALB/c mice. Neurotropin inhibited the T-cell-dependent accumulation of eosinophils induced by allergen in a dose-dependent manner when administered during sensitization. However, Neurotropin was unable to inhibit the T-cell-independent accumulation of eosinophils induced by PAF. A T-cell transfer experiment was performed to address the inhibitory mechanism. A marked accumulation of eosinophils was observed when recipients were injected i.p. with allergen and Nylon wool column-passed splenic T-cells from the sensitized donor mice. However, significant accumulation of eosinophils was not observed when sensitized donor mice were administered with Neurotropin but not saline. Taken together, these results suggest that Neurotropin inhibits the accumulation of eosinophils induced by allergen via the suppression of sensitized T-cell induction, or alternatively by interfering with T-cell function.