The effect of Juzen-taiho-to/TJ-48 on the expression of killer-cell immunoglobulin-like receptors (CD158a/b) on peripheral lymphocytes in vitro experiment.
Juzen-taiho-to (TJ-48), a mixture of extracts from 10 medicinal herbs, has been used traditionally to treat patients with anemia, anorexia or fatigue. It is well known that the treatment of TJ-48 result in the decrease of patient's complaints, as well as the increase of NK cytolytic activity (NK activity) although its augmentation is not clear in the other kampo formula from the clinical viewpoint.
To investigate its biological activities, such as the augmentation of NK activity, we analyzed the effects of TJ-48 on the expression of killer-cell immunoglobulin-like receptors (KIRs) in vitro experiment. The peripheral lymphocytes were incubated in medium alone, or medium containing TJ-48 or interleukin-2 (IL-2) plus TJ-48 at several concentrations for 48 h. After each incubation, cells were collected and their KIRs were detected by flow cytometry using monoclonal antibodies CD158a and CD158b. TJ-48 increased the populations of CD16 + CD158a + and CD16 + CD158b + cells in a dose-dependent manner. In contrast, CD16-CD158a/b+ cells did not increase. Additionally, the extract of TJ-48 enhanced the increase of KIRs expression induced by IL-2.
These actions contribute to the augmentation of NK cytolytic activity by TJ-48, and might explain, in part, its antitumor effects which has been observed in vivo.
[c] 2005 Elsevier GmbH. All rights reserved.
Keywords: Juzen-taiho-to; Killer-cell immunoglobulin-like receptors (kirs); Interleukin-2; Antitumor effect; Kampo formulation; Flow cytometric analysis
Juzen-taiho-to (TJ-48), a type of traditional Chinese medicine, has traditionally been used to treat patients with anemia, anorexia or fatigue (Terasawa, 1993). Previous studies have shown that Juzen-taiho-to has various biological activities: enhancement of phagocytosis (Maruyama et al., 1998), cytokine induction (Haranaka et al., 1985; Kubota et al., 1992) and antibody production (Hamada et al., 1988), and an inhibitory effect on tumor progression or metastasis (Ohnishi et al., 1996, 1998). Recently, we reported that Juzen-taiho-to enhanced the expression of inducible nitric oxide synthase in an LPS-activated murine macrophage cell line (Kawamata et al., 2000), and we concluded that this action, in part, explains the inhibitory effect of Juzen-taiho-to on the progressive growth of neoplastic cells. However, the biodefence against neoplastic cells or microbe infection involves several cell lineages, such as natural killer cells (NK cells) and macrophage. Especially, NK cells play a crucial role against neoplastic cells. The antitumor effects of Juzen-taiho-to are partially attributable to the augmentation of NK activity, because Juzen-taiho-to enhances NK cytolytic activity (Takahashi and Nakazawa, 1995; Horie et al., 1992).
Since the inhibitory receptors (killer-cell immunoglobulin-like receptors: KIRs) on NK cells were molecularly cloned (Wagtmann et al., 1995), analysis of KIR expression has contributed to determining the mechanism of NK cytolytic activity (Vivier and Daeron, 1997). We have recently demonstrated that the expression of CD158a/b (Colonna, 1997; Morreta et al., 1997) on lymphocytes is upregulated by interleukin-2 (IL-2) but not by interferon-gamma (IFN-g) or IL-4 (Kogure et al., 1999), and this effect is reduced in patients with rheumatoid arthritis (RA) (Kogure et al., 2001). It is considered that the upregulation of KIRs by IL-2 results in enhanced ability to sort target cells such as neoplastic cells from normal cells according to major histocompatibility complex (MHC) class I expression. Therefore, the analysis of the regulation of the KIRs expression may lead to novel insights concerning the function of NK cells. However, the effects of Juzen-taiho-to, which augments NK activity, on KIRs expression have not been studied.
Here, we demonstrate the enhancement of the KIRs expression in vitro by Juzen-taiho-to.
Materials and methods
Spray-dried hot-water extract of TJ-48 was kindly provided by Tsumura (Tokyo, Japan). TJ-48 is composed of 10 herbs (Table 1). The extract was dissolved in distilled water at a concentration of 10 mg/ml, passed through filters up to 0.3 [micro]m in pore size for sterilization, and diluted at appropriate concentrations with RPMI1640 supplemented with 10% fetal calf serum (Biological Industries, Israel).
Fluorescein isothiocyanate (FITC)-conjugated anti-human CD16, phycoerythrin (PE)-conjugated anti-human CD158a (EB6) and PE-conjugated anti-human CD158b (GL183) were purchased from Immunotech, Marseille, France. Recombinant human IL-2 was obtained from Pharmabiotechnology, Hannover, Germany.
Peripheral blood mononuclear cells (PBMC) obtained from eight healthy subjects were separated from heparinized blood by Lymphoprep (Nyegaard, Oslo, Norway) gradient centrifugation (Ross and Winchester, 1986). Aliqots of the PBMC were incubated in culture dishes in a humidified 5% C[O.sub.2]/95% air atmosphere at 37[degrees]C for 60 min. After the incubation, non-adherent cells were collected. These cell suspensions were washed twice in phosphate-buffered saline (PBS).
The cell cultures were incubated in medium only or medium-containing TJ-48 at the concentration (1-30 [micro]g/ml) indicated in the text in a humidified 5% C[O.sub.2]/95% air atmosphere at 37[degrees]C for 48 h. In the experiment testing Juzen-taiho-to after treatment with IL-2, the cells were first cultured in medium with IL-2 alone for 24 h, and then Juzen-taiho-to was added into the medium. For the next 24 h, the cells were cultured in medium containing IL-2 and Juzen-taiho-to. After each incubation, cells were collected and their surface antigens were analyzed using flow cytometry (Epics XL, Beckman Coulter, France). Each experiment was carried out in duplicate. The concentration of TJ-48 and the duration of culture has been determined according to the results of preliminary experiments.
Surface phenotyping was carried out using a two-color immunofluorescence staining technique, with isotype-specific mouse anti-human antibody conjugated with either FITC or PE (Frohhn et al., 1997). Each sample of stained cells was suspended in 0.5 ml of PBS and analyzed by flow cytometry. Lymphocyte subsets were identified by gating analysis and fluorescence profiles were obtained for 10,000 cells of each sample. Negative controls for each experiment were performed with FITC- and PE-labeled mouse immunoglobulin-G (Ig-G).
Data are expressed as mean (SD) values. All data were collected in a computer database and analyzed using the StatView-J 4.02 program (Abacus Concept, Berkeley, CA, USA). The Mann-Whitney u-test was performed for the data set of each surface antigen. For all statistical tests, differences were regarded as statistically significant if p was less than 0.05.
The upregulation of KIRs by Juzen-taiho-to
First, we investigated the effects of Juzen-taiho-to alone on the expression of CD158a/b on peripheral lymphocytes. Fig. 1 shows the effect of Juzen-taiho-to on the expression of CD16 and CD158b in a representative subject. The population of CD16 + CD158b + cells was increased by the treatment with Juzen-taiho-to, but the population of CD16-negative CD158b-positive cells was not increased by this treatment. The summarized data are shown in Fig. 2. Juzen-taiho-to upregulated the expression of CD158a/b, most of which was expressed on CD16-positive cells These effects of Juzen-taiho-to were dose-dependent in the concentration range of 1-30 [micro]g/ml (Fig. 2).
[FIGURE 1 OMITTED]
The effects of IL-2 and Juzen-taiho-to
IL-2 upregulates the expression of CD158a/b on peripheral lymphocytes, so we further analyzed the interaction of IL-2 and Juzen-taiho-to. Thirty micrograms per millilitre of Juzen-taiho-to upregulated the expression of both CD158a and CD158b to the same degree as treatment with IL-2 (100 U/ml), as shown in Fig. 3.
Lymphocytes which were cultured in medium containing IL-2 for 24 h and then in medium containing IL-2 and Juzen-taiho-to for the next 24 h expressed higher levels of the CD158a/b molecules than lymphocytes cultured in medium containing IL-2 alone (Fig. 3).
Juzen-taiho-to, which is now covered by the National Health Insurance Scheme in Japan, has been used traditionally to treat patients with anemia, anorexia or fatigue (Terasawa, 1993). Several reserch groups have demonstrated the therapeutic benefit of Juzen-taiho-to in preventing progressive growth and subsequent tumor metastasis in a murine experimental model (Hamada et al., 1988; Ohnishi et al., 1996). It has been reported that these antitumor effects are based on the activation of macrophage (Maruyama et al., 1998; Kawamata et al., 2000), cytokine induction (Haranaka et al., 1985; Kubota et al., 1992), and augmentation of NK cell activity (Takahashi and Nakazawa, 1995; Horie et al., 1992). However, the effect of Juzen-taiho-to alone or with cytokines on the expression of KIRs:CD158a/b, which is involved in NK cell function, has not been studied. In this study, we demonstrated that Juzentaiho-to upregulates the expression of KIRs, suggesting that it activates NK cells.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
The upregulation of KIRs expression does not directly induce a decrease of NK cytolytic activity, namely, when activated NK cells lyse neoplastic cells, they must specifically distinguish non-MHC class I cells from normal cells under the delicate balance required for specific killing. Importantly, KIRs recognized by CD158a/b consist of two kinds of receptors (Colonna, 1997; Morreta et al., 1997). CD158a reacts with KIR2DL1 (inhibitory receptor: IR) and KIRDS1 (activating receptor: AR), CD158b reacts with KIR2DL2 (IR), KIR2DL3 and KIR2DS2 (AR). CD158a/b monoclonal antibodies (mAb) cannot distinguish among these receptors, and the ligands that react with AR have been identified as ligands that react with IR (Vales-Gomez et al., 1998). Indeed, high stimulation through KIRs transmitted inhibitory signals to NK cells, whereas low stimulation transmitted activating signals (Warren et al., 2001). Warren et al. speculated that there is a fail-safe mechanism for activating NK cells if other NK cell-activating receptors or target cell ligands to activate those receptors are not present when HLA concentrations on target cells are below that capable of inhibiting NK cell function. To this end, the expression of KIRs may be upregulated on the activated NK cells (Kogure et al., 1999). In our study, IL-2, which augments the NK cytolytic activity (Kogure et al., 2002), also upregulated the expression of KIRs (Fig. 3). The upregulation of KIRs by Juzen-taiho-to might contribute to an increase of NK activity, and further explain, in part, its antitumor effects which are observed in vivo (Ohnishi et al., 1996; Horie et al., 1992).
On the other hand, in our study Juzen-taiho-to did not upregulate the population of CD16-negative CD158a/b-positive cells; it upregulated only CD16-positive CD158a/b-positive cells. In contrast, IL-2 also upregulates the expression of KIRs on the CD16 non-expressing cells (Kogure et al., 2001). The immunological significance of this difference is still obscure; however, the majority of CD16-negative CD158a/b-positive cells are probably T lymphocytes. It is speculated that the role of KIR expression on T cells is related to the ability to escape attack by cytotoxic T cells which react with autoantigens. Juzen-taiho-to might act mainly on NK cells to avoid attack of normal cells by NK cells when the NK cells become activated.
IL-2 upregulates the expression of KIRs (Kogure et al., 1999). Therefore, we further analyzed the interaction of IL-2 and Juzen-taiho-to. Thirty micrograms per millilitre of Juzen-taiho-to upregulated the expression of CD158a/b to the same degree as treatment with IL-2 (100 U/ml), as shown in Fig. 3. When lymphocytes were cultured in medium containing IL-2 alone for 24 h and in medium containing IL-2 and Juzen-taiho-to for the next 24 h, the KIRs were expressed at higher levels than in lymphocytes cultured in medium containing either IL-2 or Juzen-taiho-to alone (Fig. 3). These findings suggest that Juzen-taiho-to increases the upregulation of KIRs by IL-2. It has been observed that the oral administration of Juzen-taiho-to enhances the ability to produce IL-2, as well as NK activity (Horie et al., 1992). Thus, biodefences may be enhanced by the interaction between IL-2 and Juzen-taiho-to in the regulation of KIRs expression. However, it is unknown whether Juzen-taiho-to also indirectly enhances the effects of IL-2, because Juzen-taiho-to alone also upregulated the expression of KIRs. It is still unclear whether IL-2 and Juzen-taiho-to upregulate KIRs via the same pathway.
Alternatively, there is another problem in this experiment. Although Juzen-taiho-to is a crude drug and it is usually administered as it is in the field of Japanese Oriental Medicine, the major bioactive constituents which contribute to the effects of Juzen-taiho-to have not been revealed. To solve this problem further pharmacological and chemical studies will be required.
In conclusion, we demonstrated that Juzen-taiho-to upregulated the expression of KIRs. These effects contribute to the augmentation of NK cytolytic activity, and might explain, in part, its antitumor effects which were observed in vivo (Hamada et al., 1988; Ohnishi et al., 1996; Ohnishi et al., 1998; Horie et al., 1992).
Table 1. Composition of Juzen-taiho-to Herbs Ratio Astragali radix 3.0 Cinnamomi cortex 3.0 Rehmannia radix 3.0 Paeonia radix 3.0 Cnidii rhizoma 3.0 Atractyloidis lanceae rhizoma 3.0 Angelicae radix 3.0 Ginseng radix 3.0 Hoelen 3.0 Glycyrrhizae radix 1.5
The authors wish to thank T.S. Oda and N. Kuribayashi for excellent assistance.
Received 22 November 2003; accepted 23 May 2004
Abbreviations: CD, clusters of differentiation; KIR, killer-cell immunoglobulin-like receptor; MHC, major histocompatibility complex; NK, natural killer; IL, interleukin; IFN, interferon
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T. Kogure (a,*), K. Ltoh (a), T. Tatsumi (a), N. Sekiya (b), S. Sakai (b), Y. Shimada (b), J. Tamura (c), K. Terasawa (b)
(a) Department of Integrated Japanese Oriental Medicine, School of Medicine, Gunma University, Maebashi, Gunma, Japan
(b) Department of Japanese Oriental Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
(c) Department of General Medicine, School of Medicine, Gunma University, Maebashi, Gunma, Japan
*Corresponding author. Tel./fax: + 81 27 220 866.
E-mail address: email@example.com (T. Kogure).
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|Author:||Kogure, T.; Ltoh, K.; Tatsumi, T.; Sekiya, N.; Sakai, S.; Shimada, Y.; Tamura, J.; Terasawa, K.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||May 1, 2005|
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