Introduction

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Histamine is a well known bioactive amine in the granules of mast cells and basophils. In addition to its roles in inflammation, histamine has been suggested to be an immunomodulator distinctively via the stimulation of H₂-receptors (Plaut and Lichtenstein, 1982; Khan et al., 1989; Hellstrand et al., 1994; Laberge et al., 1995). The immunomodulatory effects of histamine include the regulation of cytotoxic T-cell activity (Plaut and Lichtenstein, 1982; Khan et al., 1989), the enhancement of NK cell activity (Hellstrand et al., 1994), the induction of secretion of lymphocyte chemoattractant factor from CD8+ T cells (Laberge et al., 1995), and the regulation of cytokine production in peripheral blood mononuclear cells (PBMC) (Carlsson et al., 1985; Dohlsten et al., 1987; Elenkov et al., 1998; van der Pouw Kraan et al., 1998). Conversely, in vivo administration of proinflammatory cytokines, including IL-1 and TNF- as well as LPS produced the induction of histidine decarboxylase, a histamine-synthesizing enzyme, in tissues in mice (Endo, 1982, 1989). Because the inducible histidine decarboxylase appeared to be present in macrophages (Takamatsu et al., 1996) and T lymphocytes (Aoi et al., 1989), it is quite likely that the newly synthesized histamine again modulate the immune response.

IL-18 is functionally similar to IL-12 in mediating Th1 response and inducing NK cell activity. IL-18 with IL-12 synergistically produced IFN- in T lymphocytes and monocytic cells (Munder et al., 1998; Okamura et al., 1998; Yoshimoto et al., 1998; Dinarello, 1999) in which IL-12 has been shown to up-regulate -subunit of IL-18 receptor complex (Yoshimoto et al., 1998). Therefore, IL-18 like IL-12 was expected to be a genuine Th1 cytokine in the earlier works (Dinarello, 1999). However, recent studies have demonstrated that IL-18 stimulates cultured bone marrow cells to release IL-4 and histamine (Yoshimoto et al., 1999) and that IL-18 increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma (Wild et al., 2000). These findings suggest that the dominant effects of IL-18 on Th1/Th2 balance may be dependent on the coexisting cytokine and the state of activation of subsets of immune cells. In fact, Yoshimoto et al. (1999) found that IL-18 was either antiallergic or Th2-inducing, depending on the presence or absence of IL-12 in cultured mast cells and basophils.

In the previous study, we demonstrated that histamine induced IL-18 secretion from human PBMC in vitro (Kohka et al., 2000), which in turn stimulated the production of IFN- and inhibited the production of IL-10 and IL-2. Although the IL-18-induced production of IFN- in human PBMC was synergistic with endogenous IL-12, the histamine-induced production of IFN- was not associated with any increase in IL-12 production (Kohka et al., 2000). Thus, histamine is a unique autacoid triggering IL-18-initiating cytokine cascade without inducing IL-12 in human PBMC (Kohka et al., 2000). However, there is controversy on the effects of histamine on IFN- production in human PBMC among earlier works (Carlsson et al., 1985; Dohlsten et al., 1987) and our own results (Kohka et al., 2000). Moreover, although IL-10 production was concentration dependently inhibited by histamine by using nonstimulated PBMC in our previous study (Kohka et al., 2000), it was reported that histamine increased IL-10 production in human whole blood culture stimulated by LPS (Elenkov et al., 1998; van der Pouw Kraan et al., 1998), suggesting the differential effects of histamine under the conditions with varied monocyte stimulation.

The immune response depends on the cell to cell adhesive interaction as well as soluble cytokine network system. In the previous study, we demonstrated that IL-18 up-regulated the expression of ICAM-1 on monocyte population in PBMC culture by using flow cytometry (Yoshida et al., 2001). The interaction of ICAM-1 with LFA-1 on T or NK cells generated the costimulatory signal, leading to the enhanced production of IL-12, IFN-, and TNF-. This means that cytokine cascade initiated by IL-18 has a close relationship to the functional up-regulation of adhesion molecule. In the present study, we investigated and analyzed the effects of histamine on IL-18-triggered cytokine responses, including IL-12, TNF-, IFN-, and IL-10 production, focused on the ICAM-1 expression on monocytes. We found that histamine concentration dependently inhibited the IL-18-induced up-regulation of ICAM-1 expression on monocytes through the stimulation of H₂-receptors and consequently inhibited the IL-18-induced IFN- production by way of the strong inhibition of IL-12 production. Thus, it was concluded that histamine through the stimulation of same H₂-receptors exerted differential effects on IL-18-initiating cytokine cascade, depending on the cytokine environment and the state of monocyte activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents and Drugs. Recombinant human IL-18 and anti-IL-18 monoclonal antibody (mAb) were purchased from Medical & Biological Laboratories (Nagoya, Japan). Recombinant human IL-12 was purchased from R & D Systems (Minneapolis, MN). Histamine dihydrochloride was purchased from Nakalai Tesque Inc. (Kyoto, Japan). Dimaprit dihydrochloride, 4-methylhistamine dihydrochloride, and 2-(2-pyridyl)ethylamine dihydrochloride were the gifts from Drs. W.A.M. Duncan and D.-J. Durant (The Research Institute, Smith Kline & French Laboratories, Welwyn Garden City, Hertfordshire, UK). (R)--Methylhistamine dihydrochloride [(R)--MH] was the gift from Dr. J.-C. Schwartz (Unite de Neurobiologie, Center Paul Broca de l'INSERM, Paris, France). d-Chlorpheniramine maleate, ranitidine, and famotidine were provided by Yoshitomi Pharmaceutical Co. Ltd. (Tokyo, Japan), Glaxo Japan (Tokyo, Japan), and Yamanouchi Pharmaceutical Co. Ltd. (Tokyo, Japan), respectively. Thioperamide hydrochloride was provided by Eisai Co. Ltd. (Tokyo, Japan). For flow cytometric analysis, FITC-conjugated mouse IgG1 mAb against ICAM-1 (anti-CD54 mAb, 6.5B5, which recognizes the D1 domain) (Kohka et al., 1998) was purchased from DAKO (Glostrup, Denmark). FITC-conjugated MOPC 21, an IgG1 class-matched control, was purchased from Sigma Chemical (St. Louis, MO). FITC-conjugated anti-CD11a (MHM24) and anti-CD18 (MHM23) mAb and phycoerythrin-conjugated anti-CD3 (T cell), anti-CD14 (monocyte), and anti-CD19 (B cell) (HD37) mAb were purchased from DAKO. For blocking cell aggregation or inhibiting IL-18-induced cytokine production, anti-CD11a (MHM 24), anti-CD18 (MHM23), and anti-CD54 (6.5B5) Abs were purchased from DAKO.

Isolation and Culture of PBMC. Normal human PBMC were obtained from human volunteers after oral informed consent. Twenty to 50 ml of peripheral blood was withdrawn from the vein of the forearm. PBMC were isolated from buffy coat of 10 healthy volunteers by centrifugation on Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden) then washed three times in RPMI 1640 medium (Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 20 µg/ml kanamycin, and 100 µg/ml streptomycin and penicillin (Sigma Chemical). PBMC were suspended at a final concentration of 1 × 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum.

Flow Cytometric Analysis. PBMC (1 × 10⁶ cells/ml) were cultured with IL-18, histamine, H1-, H2-, H3-receptor agonists, and/or H1-, H2-, H3-receptor antagonists at 37°C in a 5% CO₂/air mixture under different conditions indicated. The culture cells (5 × 10⁵ cells/sample) were washed once with washing buffer (phosphate-buffered saline supplemented with 2.5% normal horse serum, 0.1% NaN₃, and 0.01 M HEPES, pH 7.3). Then the cells were incubated with 1 µg of FITC-conjugated anti-ICAM-1 Ab, anti-CD11a Ab, anti-CD18 Ab, anti-CD29 Ab, anti-CD44 Ab, and anti-CD62L Ab, or else MOPC21 or phycoerythrin-conjugated anti-CD3 Ab, anti-CD14 Ab, and anti-CD19 Ab for 20 min at 4°C. After washing, the cells were fixed with 2% paraformaldehyde and analyzed with a FACScan Calibur (BD Biosciences, San Jose, CA), and data were processed using the CELL QUEST (BD Biosciences) program. The data were expressed as the relative fluorescent intensities against class-matched control (MOPC 21). The results are the mean ± S.E.M. of five donors.

Cytokine Assays. PBMC (1 × 10⁶ cells/ml) were incubated with IL-18, histamine, H₁-, H₂-, H₃-receptor agonists, and/or H₁-, H₂-, H₃-receptor antagonists for 24 h at 37°C in a humidified atmosphere of 5% CO₂ in air. Any kinds of reagents were added to the media at the start of incubation. After culture, the cell suspensions were transferred into Eppendorf tubes and centrifuged. The cell-free supernatant fractions were assay for IL-18, IL-12, TNF-, IFN-, and IL-10 protein. The cytokines were measured using ELISAs with the multiple Abs sandwich principle (for IL-18, Medical & Biological Laboratories Co., Ltd., and for other cytokines, Quantikine; R & D Systems). ELISA for IL-12 detected p70 protein. The detection limits of the ELISAs for IL-18, IL-12, TNF-, IFN-, and IL-10 were 10 pg/ml. The results were expressed as the mean ± S.E.M. of five donors.

Aggregation Assay. PBMC (described under Isolation and Culture of PBMC) were seeded in each well of six flat-bottomed well plates for 24 h at 37°C in a 5% CO₂/air mixture. After treatment in the presence of IL-18 (100 ng/ml), histamine (10⁴ M), IL-18 + histamine, IL-18 + anti-ICAM-1 Ab, IL-18 + anti-LFA-1 Ab or IL-18 + histamine + IL-12 (100 ng/ml) at 37°C for 24 h in 5% CO₂/air mixture, over 300 cells in each well were counted. The degree of cell aggregation was scored by means of the aggregation rate (%) = (the number of aggregated cells/total number of cells counted) × 100. To evaluate the blocking activity of the mAbs, cells were preincubated with 20 µg/ml mAb for 30 min before treatment of IL-18. Photographs were taken with an Olympus inverted microscope (Olympus, Tokyo, Japan).

Statistical Examination. The statistical significances were evaluated using analysis of variance, followed by Student's two-tailed t test. A probability value less than 0.05 was considered to be statistically significant.

	Results

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Effect of Histamine on IL-18-Induced ICAM-1 Expression on Human Monocytes. The effect of IL-18 (100 ng/ml) and/or histamine (10⁴ M) on the changes in the expression of human leukocyte antigens (ICAM-1, CD11a, CD18, CD29, CD44, or CD62L) was examined by double-stained flow cytometry with anti-CD14, anti-CD3, and anti-CD19 Abs 24 h after the incubation of PBMC. As shown in Fig. 1, IL-18 (100 ng/ml) produced the up-regulation of ICAM-1 specifically on monocytes but not on T or B cells. Histamine (10⁴ M) inhibited the ICAM-1 expression induced by IL-18 (100 ng/ml). The same concentration of histamine alone did not influence the ICAM-1 expression on monocytes 24 h after incubation of PBMC. The expression of CD11a, CD18, CD29, CD44, and CD62L was not changed by IL-18 (100 ng/ml) or histamine (10⁴ M) in monocyte, T cell, and B cell population (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Effects of histamine on IL-18-induced ICAM-1 expression on the surface of PBMC. A, expression of ICAM-1 on the surface of PBMC. PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) and histamine (104 M) for 24 h. After the treatment, PBMC (5 × 105 cells/sample) were double-stained with cell-specific antigen (CD14, CD3, or CD 19) and ICAM-1. The experiments were repeated 10 times separately using different donors. Typical results are shown. B, histogram of the expression of ICAM-1 on the surface of monocytes stimulated with IL-18 (100 ng/ml) and histamine (104 M). PBMC (5 × 105 cells/sample) were stained with anti-ICAM-1 Ab or class-matched control (CMC).

Dose-Response Relationship for Effect of Histamine on IL-18-Induced ICAM-1 Expression on Human Monocytes. As shown in Fig. 2, we investigated the effects of different concentrations of histamine on IL-18 (100 ng/ml)-induced ICAM-1 expression. Histamine (10⁷-10⁴ M) concentration dependently inhibited the expression of ICAM-1 induced by IL-18 (100 ng/ml) when ICAM-1 expression was determined at 24 h after the start of culture (Fig. 2). The IC₅₀ value of histamine for the inhibition of the IL-18-induced expression of ICAM-1 was 1.0 µM. The expression of ICAM-1 induced by IL-18 (100 ng/ml) was completely inhibited by 10⁴ M histamine.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship for the effect of histamine on ICAM-1 expression. PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) and different concentrations (0, 107, 106, 105, and 104 M) of histamine for 24 h. After the treatment, PBMC (5 × 105 cells/sample) were stained with anti-ICAM-1 Ab or class-matched control (CMC) and the expression of ICAM-1 in monocytes was analyzed. Filled () and open () circles represent the results obtained with anti-ICAM-1 Ab after the incubation in the presence and absence of IL-18, respectively. White squares () are the fluorescence intensity obtained with class-matched control after the incubation with IL-18 (CMC). The results are the means ± S.E.M. of five donors. *, P < 0.05 and **, P < 0.01 compared with the corresponding value in the absence of histamine. When an error bar was within a symbol, the bar was omitted.

Effects of Histamine Receptor Antagonists and Selective Histamine Receptor Agonists on IL-18-Induced ICAM-1 Expression. To determine the histamine receptor subtypes involved in the effects of histamine on ICAM-1 expression, one of three classes of receptor antagonists, d-chlorpheniramine (H₁-receptor antagonist), famotidine (H₂-receptor antagonist), or thioperamide (H₃-receptor antagonist) was added to the culture medium at the concentration of 10⁶ or 10⁴ M with histamine (10⁴ M). Apparently, famotidine, 10⁶ or 10⁴ M, concentration dependently antagonized the inhibitory effects of histamine on ICAM-1 (Fig. 3A). On the other hand, the same concentrations of d-chlorpheniramine and thioperamide did not produce any antagonizing action on histamine effect. Another H₂-receptor antagonist, ranitidine, exerted substantially similar effect to famotidine (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Effects of histamine receptor antagonists and selective histamine receptor agonists on IL-18-induced ICAM-1 expression in PBMC. A, PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) and histamine (104 M) in the presence of 106 or 104 M different class of histamine receptor antagonist for 24 h; d-chlorpheniramine (H1-antagonist), famotidine (H2-antagonist), or thioperamide (H3-antagonist). After the treatment, PBMC (5 × 105 cells/sample) were stained with anti-ICAM-1 Ab or class-matched control (CMC) and the expression of ICAM-1 in monocytes was analyzed. The results are the means ± S.E.M. of five donors. **, P < 0.01 compared with the value in the presence of IL-18 and histamine. B, PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) in the presence of 106 or 104 M histamine or different class of histamine receptor agonist; 2-PEA (H1-agonist), dimaprit (H2-agonist), 4-MH (H2-agonist), or (R)--MH (H3-agonist) for 24 h. After the treatment, PBMC (5 × 105 cells/sample) were stained with anti-ICAM-1 Ab or CMC and the expression of ICAM-1 in monocytes was analyzed. The results are the means ± S.E.M. of five donors. **, P < 0.01 compared with the value in the presence of IL-18 alone.

Effect of Histamine on IL-18-Induced Cytokine Response in Human PBMC. We investigated the effect of histamine on IL-18 (0.1-100 ng/ml)-induced production of IL-12, IFN-, TNF-, and IL-10 by ELISA (Fig. 4). In the condition without IL-18, the production of IL-12, IFN-, and TNF- was under detection level, and the level of IL-10 was about 600 pg/ml, spontaneously, which was similar to the results observed previously (Kohka et al., 2000; Yoshida et al., 2001). Histamine concentration dependently inhibited the releases of IL-12 from PBMC induced by three different concentrations of IL-18 (10 and 100 ng/ml) when determined 24 h after the start of culture (Fig. 4A). Histamine also concentration dependently inhibited the TNF- and IFN- production (Fig. 4, B and C). The IC₅₀ values for inhibitory effects of histamine on IL-18-induced production of IL-12, TNF-, and IFN- were estimated to be 4.0, 3.0, and 4.0 µM, respectively. On the other hand, histamine reversed the inhibition of IL-10 production by IL-18 (Fig. 4D).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Dose-response relationship for the effect of histamine on IL-18-induced cytokine response. PBMC (1 × 106 cells/ml) were incubated with increasing concentrations of histamine (0, 107, 106, 105, and 104 M) in the presence of different concentration of IL-18 (0.1, ; 1, ; 10, ; and 100 ng/ml, ) for 24 h and the levels of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined by ELISA as described under Materials and Methods. The results are the means ± S.E.M. of five donors. *, P < 0.01 compared with the corresponding value in the absence of histamine in each dose-response curve. When an error bar was within a symbol, the bar was omitted.

Effects of Histamine Receptor Antagonists and Selective Histamine Receptor Agonists on IL-18-Induced Cytokine Response in PBMC. To examine the involvement of subtypes of histamine receptors in the effects of histamine on cytokine response, H₁-, H₂-, and H₃-receptor antagonist (d-chlorpheniramine, famotidine, and thioperamide) were added to the culture medium at the concentration of 10⁶ or 10⁴ M with histamine (10⁴ M) (Fig. 5). Famotidine concentration dependently antagonized the inhibitory (IL-12, TNF-, IFN-) or stimulatory (IL-10) effects of histamine (Fig. 5). On the other hand, the same concentrations of d-chlorpheniramine and thioperamide did not produce any antagonizing action on histamine effect. Another H₂-receptor antagonist ranitidine exerted a substantially similar effect to famotidine (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of histamine receptor antagonists on the modulatory effects of histamine on IL-18-induced cytokine response in PBMC. PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) and histamine (104 M) in the presence of 106 or 104 M different class of histamine receptor antagonist; d-chlorpheniramine (H1-antagonist), famotidine (H2-antagonist), or thioperamide (H3-antagonist). At the end of the culture, the concentrations of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined using an ELISA assay kit as under Materials and Methods. The results are the means ± S.E.M. of five donors. **, P < 0.01 compared with the value in the presence of IL-18 and histamine.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effects of selective histamine receptor agonists on IL-18-induced cytokine response in PBMC. PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) in the presence of 106 or 104 M histamine or different class of histamine receptor agonists; 2-PEA (H1-agonist), dimaprit (H2-agonist), 4-MH (H2-agonist), or (R)--MH (H3-agonist). At the end of the culture, the concentrations of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined using an ELISA assay kit as described under Materials and Methods. The results are the means ± S.E.M. of five donors. *, P < 0.05 and **, P < 0.01 compared with the value in the presence of IL-18 alone.

Function of ICAM-1 as Determined by Aggregation Assay. We examined the effect of IL-18 (100 ng/ml), histamine (10⁴ M), IL-18 + histamine, IL-18 + anti-ICAM-1 Ab (100 µg/ml), IL-18 + anti-LFA-1 Ab (100 µg/ml), and IL-18 + histamine + IL-12 (100 ng/ml) on the aggregation of PBMC. The effects of IL-18 on aggregation are showed in Fig. 7. Histamine, anti-IL-18 Ab, and anti-ICAM-1 Ab blocked IL-18-induced aggregation (Fig. 7). The addition of IL-12 to the culture in the presence of IL-18 and histamine reversed the inhibitory effect of histamine on IL-18-induced aggregation of PBMC.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Involvement of ICAM-1 expression in the aggregation of PBMC induced by IL-18. PBMC (1 × 106 cells/ml) were incubated with IL-18 (100 ng/ml) in the absence or presence of histamine (104 M), anti-ICAM-1 Ab (10 µg/ml), anti-LFA-1 Ab (100 µg/ml), and/or IL-12 (100 ng/ml). The aggregation was evaluated as described under Materials and Methods. The results are the means ± S.E.M. of five donors. **, P < 0.01 compared with the value in untreated control. ##, P < 0.01 compared with the value treated with IL-18 alone.

Effect of Addition of IL-12 on Modulatory Effects of Histamine on IL-18-Induced Production of IFN- and IL-10 in Human PBMC. The addition of increasing concentrations of IL-12 to the culture medium at the start of incubation antagonized either the inhibitory effect of histamine (10⁴ M) on IL-18-induced production of IFN- or the stimulator on IL-10 production (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effect of IL-12 on the modulatory effects of histamine on IL-18-induced production of IFN- and IL-10 in PBMC. PBMC (1 × 106 cells/ml) were cultured with IL-18 (100 ng/ml) and histamine (104 M) in the absence or presence of increasing concentrations of IL-12 for 24 h. At the end of the culture, the concentrations of IFN- and IL-10 in the conditioned media were determined using an ELISA kit as described under Materials and Methods. The results are the means ± S.E.M. of five donors. **, P < 0.01 compared with the value in the presence of IL-18 and histamine.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the previous study (Yoshida et al., 2001), we demonstrated that IL-18 time and concentration dependently up-regulated the expression of ICAM-1 specifically on monocytes. IL-18 also induced the aggregation of PBMC dependent on the interaction of ICAM-1/LFA-1. The up-regulation of ICAM-1 on monocytes leads T and NK cells to adhere to monocytes, and the resultant interaction between monocytes and T or NK cells via ICAM-1/LFA-1 produces additive signaling together with the primary IL-18 receptor stimulation for the production of IL-12, TNF-, and IFN- (Yoshida et al., 2001). Conversely, the inhibition of ICAM-1/LFA-1 interaction by Abs against ICAM-1 and LFA-1 significantly inhibited the IL-18-initiated cytokine production as well as cell aggregation. Based on these findings, it was concluded that up-regulation of ICAM-1 plays an important role for the IL-18-initiated cytokine production in human PBMC (Yoshida et al., 2001).

As shown in Fig. 2, histamine concentration dependently inhibited the ICAM-1 up-regulation induced by IL-18. This inhibitory effect of histamine on ICAM-1 expression on monocytes was expected to mimic the effects of anti-ICAM-1 and LFA-1 Abs on cytokine production observed in the previous study (Yoshida et al., 2001). In fact, histamine concentration dependently suppressed the production of IL-12, IFN- induced by IL-18 in the present study (Fig. 4). Therefore, it is quite likely that the inhibitory effects of histamine on IL-18-induced production of IL-12 were due to the inhibition of ICAM-1 expression on monocytes. The reduction of IFN- production appeared to be secondary to the reduced IL-12 production (Fig. 8). The requirement of relatively higher concentration of exogenous IL-12 for reversing the inhibitory effect of histamine on IL-18-induced IFN- production may reflect the IL-12 concentration needed for the functional antagonism of histamine action on ICAM-1 expression. In fact, nanomolar order of IL-12 was required for the expression of ICAM-1 on monocytes under this condition (data not shown). The inhibition of IL-12 production by histamine was also reported by two groups (Elenkov et al., 1998; van der Pouw Kraan et al., 1998) in which they stimulated whole blood cell culture with LPS or Staphylococcus aureus Cowan I. Although the conditions for the stimulation of monocytes were different in the present study and their experiments, it seems likely that the mode of action of histamine on IL-12 production may be ascribed to a common mechanism, the inhibition of ICAM-1 expression on monocytes. This issue remains to be determined.

Histamine is known to activate four kinds of G protein-coupled receptors, H₁-, H₂-, H₃-, and H₄-receptors (Hill et al., 1997; Oda et al., 2000; Nakamura et al., 2001). The experiments to characterize the receptor subtypes involved in the effects of histamine on IL-18-induced cytokine production as well as ICAM-1 expression revealed that those were typical H₂-receptors but not H₁- and H₃-receptors. Because recently cloned H₄-receptors have totally different pharmacological profile from that of H₂ (Oda et al., 2000; Nakamura et al., 2001), it was concluded that only H₂-receptors were involved in histamine action.

Recently, we demonstrated that histamine concentration dependently stimulated the production of IL-18 and IFN-, and inhibited those of IL-2 and IL-10 in human PBMC under the condition without adding any stimulus for monocytes or lymphocytes (Kohka et al., 2000). All these responses were also mediated solely by histamine H₂-receptors. Because the IL-18 levels in the culture medium induced by histamine alone were sufficient to induce IL-12, it was suggested that histamine strongly inhibited the production of IL-12 distinctively (Kohka et al., 2000). We confirmed the notion from the present study in that histamine strongly inhibited the IL-12 production induced by IL-18 through the stimulation of H₂-receptors (Figs. 5 and 6). The inhibition of IL-12 production by histamine made a complex pattern of cytokine activation in the presence of IL-18 and histamine from the start of culture. Those were composed of histamine concentration-dependent inhibition of IFN- production and concentration-dependent stimulation of IL-10 production (Fig. 4); the completely inverse effects compared with those under the condition in which histamine was present well before the production of IL-18 (Kohka et al., 2000). Under the condition in which IL-18 and histamine were present from the start of culture, IL-12 production may occur to some extent depending on the concentration of histamine as shown in Fig. 4. In the presence of IL-18 and IL-12, IFN- production was dependent on both IL-18 and IL-12 (Kohka et al., 2000). The dependence of IL-18-induced IFN- production on endogenous IL-12 produced in PBMC demonstrated in the previous study (Kohka et al., 2000) as well as the fact that exogenously added IL-12 reversed the inhibitory effect of histamine on IL-18-induced IFN- production strongly indicated that the concentration-dependent inhibition of IL-12 production by histamine was the cause of the inhibition of IL-18-induced IFN- production by histamine (Fig. 8). IFN- was reported to inhibit IL-10 production by monocytes (Chomarat et al., 1993; Donnelly et al., 1995); therefore, it is conceivable that stimulation of IL-10 production by histamine in the presence of IL-18 was secondary to the inhibition of IFN- production.

IL-12 was identified as NK cell stimulatory factor in the conditioned media of human B-cell line RPMI 8866 (Kobayashi et al., 1989). IL-12 is a crucial inducer of cell-mediated immunity by promoting the development, proliferation, and function of Th1 cells (Gately et al., 1998). IL-12, a heterodimeric cytokine composed of p35 and p40 (Gubler et al., 1991; Wolf et al., 1991), induced proliferation, IFN- production, and cytolytic activity in NK and T cells (Gately et al., 1998; Sinigaglia et al., 1999). The production of IFN- in turn stimulates the production of IL-12, facilitating Th1 response, whereas IL-12 inhibits the humoral immunity, including IgE production (Gately et al., 1998). Thus, IL-12 plays a central role for the activation and amplification of cell-mediated immunity. IL-18 has been reported to be functionally similar to IL-12 in mediating Th1 response and NK cell activity (Okamura et al., 1998; Dinarello, 1999). IL-18 and IL-12 synergistically produced IFN- in T and monocytic cells (Munder et al., 1998; Okamura et al., 1998; Yoshimoto et al., 1998) in which IL-12 up-regulated the expression of IL-18 receptor (Yoshimoto et al., 1998). It is quite likely that coexistence of IL-18 and IL-12 cooperatively can induce strong Th1 response. On the contrary, the effects of histamine on IL-12 and IFN- production observed in the present study will function as a negative force on Th1 positive feedback system through the inhibition of the production of key cytokine IL-12. In other words, histamine may be capable of controlling the excessive Th1 response and be beneficial in diseases associated with pathological Th1 responses, such as multiple sclerosis or Crohn's disease (Gately et al., 1998).

Recent studies demonstrated that IL-18 induced the production of Th2 cytokine IL-13 in NK and T cells (Hoshino et al., 1999). Also, Yoshimoto et al. (1999) showed that IL-18 with IL-3 stimulated cultured basophils to produce IL-4 and IL-13 as well as histamine. An interesting feature regarding the Th2 cytokine induction by IL-18 in the latter report was that it occurred in the absence of IL-12. As discussed above, the presence of histamine tends to the formation of cytokine environment lacking IL-12, which may provide IL-18 with Th2 cytokine-inducing ability. Therefore, it is possible that IL-18 tends to produce Th2 cytokines at the sites with local allergic inflammation associated with histamine release in quantity. As a whole, histamine action promotes Th2 shift under the condition where a considerable amount of IL-18 is present.

It is noteworthy that LPS and some proinflammatory cytokines, including IL-1 and TNF-, induced histidine decarboxylase, a histamine-synthesizing enzyme, in mice (Endo, 1982, 1989). Because macrophages (Takamatsu et al., 1996) and T lymphocytes (Aoi et al., 1989) appear to produce histamine, histamine synthesized in and released from such cells may play roles to modulate the cytokine environment in the tissues, in addition to storage histamine in granules of mast cells/basophils.

Histidine decarboxylase activity has been reported to increase in and around tumor tissues (Bartholeyns and Bouclier, 1984), in which the histamine produced was estimated to be involved in tumor proliferation and angiogenesis. Dual effects of histamine on Th activity; triggering Th1 cytokine production when the state of IL-18-initiating cytokine cascade is low, and inhibition of IL-18-initiating cytokine cascade through the strong inhibitory effect on IL-12 production when the IL-18-induced activation is already present, imply a role of histamine in the host immune response to tumor cells. The clinical trials reporting the effectiveness of cimetidine, an H₂-receptor antagonist, for the pre- and postoperative treatment of gastrointestinal cancer (Morris and Adams, 1995) are very intriguing from the aspect of the antagonism against histamine action on local Th1 responses in/around tumor tissues. Further work is necessary on this line.