Takuya Kawakita

Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis

To clarify the behavioral and pathological features of spontaneous scratching of NC mice with mite-induced chronic dermatitis, we investigated the spontaneous and pruritogen-evoked scratching of NC mice. Although the frequency of scratching of NC mouse did not increase under specific pathogen-free environment, it gradually and markedly increased from 3 to 6 weeks after transfer to conventional environment. The onset of increase in spontaneous scratching was similar to that of dermatitis development and the elevation of plasma concentration of immunoglobulin E. At chronic stage (16 weeks after environment change), the frequency of spontaneous scratching was roughly parallel to the degree of dermatitis, but not to the plasma concentration of immunoglobulin E. The spontaneous scratching of NC mice with dermatitis was inhibited by distraction and the opioid antagonist naltrexone, suggesting that the scratching is itch-associated response. An intradermal injection of serotonin, but not histamine and substance P, elicited scratching of the injected site. Methysergide and cyproheptadine inhibited the serotonin-induced scratching but not spontaneous scratching. The results suggest that marked elevation of plasma immunoglobulin E is not always the cause of spontaneous itch-associated response of NC mice with dermatitis. Serotonin, histamine and substance P may not play an important role in spontaneous itch-scratch response at a chronic stage.

Accelerated recovery from cyclophosphamide-induced leukopenia in mice administered a Japanese ethical herbal drug, Hochu-ekki-to.

The effect of a Japanese ethical herbal drug, Hochu-ekki-to (HOT), on recovery from leukopenia induced by cyclophosphamide (CY) was investigated. Daily oral administration of 1000 mg/kg HOT into CY-treated mice significantly prevented decrease of leukocyte numbers in the peripheral blood and accelerated recovery from leukopenia. Ginsenoside Rgl extracted from Ginseng radix, a major herb of HOT, was one of the active ingredients. HOT increased numbers of neutrophils and monocytes in the peripheral blood compared with CY-treated control. Moreover, HOT augmented the resistance against Pseudomonas aeruginosa infection. The number of colony-forming units in the spleen (CFU-S) also increased in HOT-treated mice. The frequencies of IL-3-, GM-CSF- and IFN-gamma-producing cells increased in the spleen, bone marrow, liver and IEL on HOT treatment, and HOT clearly augmented the expressions of IL-3, GM-CSF and IFN-gamma mRNA in the spleen, bone marrow, liver and IEL except IL-3 and IFN-gamma mRNA in the IEL. These results suggest that HOT enhances the production of hematopoietic lymphokines, stimulates the proliferation of hematopoietic progenitor cells and consequently accelerates recovery from leukopenia in CY-treated mice. Additionally, IFN-gamma which HOT-augmented the production may contribute the protective effect against the bacterial infection by activating of phagocyte cells.

Suppression of IgE production in mice treated with a traditional Chinese medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to)

The ability of a traditional herbal medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to, HOT), to suppress IgE production was investigated. BALB/c mice were intraperitoneally immunized with aluminium hydroxide adsorbed with DNP-KLH (DNP-KLH + alum). When oral administration of HOT was begun just after immunization, the serum level of antigen-specific IgE was significantly decreased, although those of antigen-specific IgG1 and IgG2a were not influenced. In the culture of spleen cells obtained 14 days after immunization with DNP-KLH, antigen-specific IgE and IgG1 production by the cells of the HOT-treated mice was significantly suppressed compared to that in immunized mice. Furthermore, in the combination culture with CD4+ T cells and B cells separated from spleen cells, IgE production by the cells from immunized mice was inhibited by replacement of their corresponding cell population with either CD4+ T cells or B cells of HOT-treated mice. Additionally, production of interleukin 2 (IL-2) and IL-4 was significantly suppressed in HOT-treated mice but not that of IFN-gamma in comparison to the immunized mice. These results suggested that HOT decreased the IgE level in serum by inhibiting the development of IL-4-producing CD4+ T cells.

Activation of murine peritoneal macrophages by intraperitoneal administration of a traditional chinese herbal medicine, xiao-chai-hu-tang (Japanese name: Shosaiko-To)

Macrophage activation by a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), was investigated. Intraperitoneal (i.p.) administration of shosaiko-to into (BALB/c x DBA/2)F1 mice resulted in marked activation of macrophages with respect to phagocytic and lysosomal enzyme activities (acid phosphatase and N-acetyl-beta-D-glucosaminidase) compared with the control. The maximal responses were induced by an i.p. injection of 3 mg shosaiko-to 4 days previously. Enhanced activities induced by shosaiko-to were also seen in C3H/HeJ mice, which is a non-responder strain to bacterial lipopolysaccharide (LPS). Significant macrophage accumulation in the peritoneal cavity and increased lysosomal enzyme activities were observed in mice injected with shosaiko-to. Shosaiko-to exhibited significant cytostasis-inducing activity. In addition, the administration of shosaiko-to led to a moderate expression of Ia antigen on the surface of peritoneal macrophages. These results suggest that shosaiko-to is a potent macrophage activator.

Augmentation of NK Activity after Oral Administration of a Traditional Chinese Medicine, Xiao-Chai-Hu-Tang (Shosaiko-To)

We have shown that a traditional Chinese medicine, Xiao-chai-hu-tang (Japanese name: Shosaiko-to) augments natural killer (NK)1 activity in mice. The maximum augmentation of NK activity in the peripheral blood and liver was observed at 12 hr after administration of Shosaiko-to. NK activity was augmented by Shosaiko-to dose-dependently. The augmentation became significantly positive at a dose of 500 mg/kg, and the maximum effect was observed at a dose of 1000 mg/kg. The augmentation of NK activity appeared at first in the liver from 6 hr after administration of Shosaiko-to and became detectable later in the peripheral blood from 12 hr after the administration. Activation of NK cells by Shosaiko-to may occur in the liver and subsequently the activated NK cells may be supplied to the peripheral blood. Changes in percentages of cell surface markers (asialo GM1, CD3, CD4, CD8) after Shosaiko-to treatment were hardly detected, but augmentation of NK activity induced by Shosaiko-to was abrogated by anti-asialo GM1 antibody treatment before the cytotoxicity assay. In addition, cytotoxic activity to P-815 target cells was not detected in Shosaiko-to treated mice. Augmentation of NK activity by Shosaiko-to is probably mediated by functional activation of classical NK cells of asialo GM1+ phenotype. These results suggest that augmentation of NK activity in the liver is one of mechanisms involved in clinical efficacy of Shosaiko-to in patients with virus chronic hepatitis.

Role of b-lymphocytes in the immunopharmacological effects of a traditional chinese medicine, xiao-chai-hu-tang (shosaiko-to)

We previously reported that a traditional Chinese medicine, Xiao-chai-hu-tang (Japanese name: Shosaiko-to), induced interferon (IFN) activity in the serum of mice after intraperitoneal (i.p.) administration. In the present study in which murine spleen cells were cultured in vitro with Shosaiko-to, B-cells isolated by anti-immunoglobulin-coated plates were confirmed to generate IFN in response to Shosaiko-to stimulation. IFN activity was induced in the serum after i.p. administration of Glycyrrhizae radix, Scutellariae radix, Bupleuri radix and Pinelliae tuber which are included in Shosaiko-to as its constituent. Such an IFN-inducing activity was confirmed to exist in methanol-insoluble fractions of these extracts derived from Shosaiko-to and these constituents but not in methanol-soluble fractions. These four extracts as well as Shosaiko-to, induced interleukin 6 (IL-6) in the serum after the administration. In in vitro stimulation of spleen cells, Shosaiko-to and extracts of Glycyrrhizae radix, Bupleuri radix and Pinelliae tuber showed mitogenic activity, but an extract of Scutellariae radix with in vivo IFN-inducing activity did not. B-cells appear to participate in the immunopharmacological effects of Shosaiko-to through mitogenic activity, IFN induction and the effect of IL-6.

Activation of murine peritoneal macrophages by saikosaponin A, saikosaponin D and saikogenin D

Macrophage activation by saikosaponins and saikogenins was investigated and compared with that by other saponins and macrophage stimulants. Saikosaponins a and d induced a marked cell accumulation in the peritoneal cavity when administered intraperitoneally. Among saikosaponins and saikogenins tested, saikosaponin d significantly activated peritoneal macrophages in terms of enhancement of phagocytic activity, increased level of cellular lysosomal enzyme (acid phosphatase), induction of cytostatic activity and expression of Ia antigen on the cell surface. The activities of saikosaponin d were much stronger than those of typical saponins ginsenoside Rg1 and glycyrrhizin and almost comparable with or somewhat weaker than those of lipopolysaccharide, a streptococcal preparation OK-432 and formalin-killed Propionibacterium acnes, indicating that saikosaponin d is a potent macrophage activator.

Effect of a traditional chinese herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to) on hematopoietic stem cells in mice

Cell dynamics after intraperitoneal (i.p.) and oral administration of a traditional herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to, NYT), were investigated. When NYT was injected i.p. into C3H/He mice, numbers of spleen and peritoneal cells significantly increased in a dose-dependent manner and showed high levels from 4 to 21 days. Two peaks in the total cell number were observed on days 7 and 14 in the peritoneal cavities and spleen of C3H/He mice administered NYT. A marked accumulation of PMN cells in the peritoneal cavity and spleen was detected at 7 days after injection. The numbers of macrophages and lymphocytes also increased by i.p. administration of NYT. The thymus cell number decreased transiently between 4 and 7 days and thereafter returned to the control level. No significant change in the cell number of lymph nodes was observed. Such cellular accumulation was also detected in C3H/HeJ mice, a nonresponder strain to bacterial endotoxin, and athymic nude mice. The activity of colony-forming units in the spleen (CFU-S) of C3H/He as well as C3H/HeJ mice was markedly augmented by i.p. administration of NYT. NYT induced significant CSF production as detectable by its activity in the sera. In addition, oral administration of NYT for 10 days induced a significant increment of peripheral leukocytes and spleen cells and enhanced CFU-S activity of bone marrow cells as induced by i.p. administration, indicating that NYT acts on hematopoietic stem cells capable of differentiating to lymphocytes, macrophages and PMN cells into the periphery.

Induction of interferon after administration of a traditional chinese medicine, xiao-chai-hu-tang (shosaiko-to)

We examined the ability of a traditional chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to) to induce IFN in mice. A maximum activity (105 units/ml) of interferon (IFN) appeared in the serum of mice 16 h after intraperitoneal (i.p.) treatment with 250 mg/kg of shosaiko-to. Addition of polymyxin B did not abrogate the ability of shosaiko-to to induced serum IFN. The IFN was identified as IFN-alpha/beta by neutralizing test using anti-IFN alpha/beta antibodies. Pretreatment of mice with carrageenan suppressed the IFN induction by shosaiko-to, whereas the IFN induction by shosaiko-to was impaired neither in mice treated with anti-asialo-GM1 antibody nor in T-cell-deficient athymic nude mice. IFN was produced in vitro by spleen cells obtained from shosaiko-to treated mice. Moreover, spleen cells from untreated mice could also produce IFN when they were cultured with shosaiko-to. Additionally, serum IFN was also induced by the adoptive transfer of spleen cells from shosaiko-to treated mice to normal mice. On the other hand, peroral administration of shosaiko-to also induced IFN-alpha/beta in the serum. While IFN activity induced by i.p. administration of shosaiko-to declined after repeated treatments, the activity induced by its peroral administration did not decline during a long term treatment. These results showed that shosaiko-to is an IFN-alpha/beta inducer capable of repeated peroral administration.

Protective effect of a traditional Japanese medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to), on the restraint stress-induced susceptibility against Listeria monocytogenes.

In this study, the effect of traditional Japanese (Chinese) medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to, HOT), on the restraint stress treatment (RST)-induced susceptibility against Listeria monocytogenes (L. monocytogenes) was examined. When RST was performed every day for 10 h from the day of infection, the bacterial numbers were increased at 3 and 5 days after the infection. Oral pretreatment with HOT for 7 days prevented such increases. Pretreatment with HOT prevented the suppression of antigen-specific IFN-gamma production by RST. HOT also prevented suppression of macrophage accumulation, including MHC-class II positives, in the peritoneal cavity and their bactericidal activity by RST. HOT suppressed the serum corticosterone level elevated by RST in infected mice. Taken together, the suppression of corticosterone using HOT participates in the prevention of suppressions of the bactericidal activity of macrophages, migration of macrophages and antigen-specific IFN-gamma production of Th1 cells by RST. Our findings suggest that HOT is a useful drug for patients suffering from stress disease to reduce the susceptibility to bacterial infection.