Introduction

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Histamine is an important endogenous amine that exerts numerous functions in central and peripheral tissues. These physiological processes are mediated through at least four receptors, H₁ to H₄, which are all members of the seven membrane-spanning G protein-coupled receptor (GPCR) family (Hough, 2001). H₁ receptors are widely expressed throughout the body, with high expression levels being found in the brain, smooth muscle cells, endothelial cells, adrenal medulla, and heart. By controlling smooth muscle and endothelial cell contraction (thereby increasing vascular permeability) and by stimulating nitric oxide formation, H₁ receptors modulate inflammatory and allergic responses; antihistamines have been in clinical use for allergy treatment for decades (reviewed in Walsh et al., 2001). H₂ receptor activation causes cAMP accumulation through activation of a stimulatory G protein in gastric cells stimulating gastric acid secretion (Black et al., 1972). H₂ receptors are involved in the regulation of cytokine and chemokine production and differentiation and maturation of a variety of cells in cardiac tissue, smooth muscle, and cells of the immune system (Elenkov et al., 1998; Poluektova and Khan, 1998; Kohka et al., 2000; Caron et al., 2001; Jutel et al., 2001). H₃ receptors are predominantly found in the brain, where they function as presynaptic autoreceptors on histamine-containing neurons and are believed to control the release of many brain mediators including histamine itself (Hough, 1999). Thus, several target indications for H₃ receptor-interacting compounds have been suggested: Alzheimer's disease, sleep disorders, cognition and memory disorders, obesity, attention deficits, and others (Leurs et al., 1998). Phylogenic and homology analyses have revealed that H₃ receptors are surprisingly different, not only from H₁ and H₂ (Lovenberg et al., 1999; Leurs et al., 2000) but also from most of the known GPCRs. In the search of additional receptors more closely related to H₃, various groups recently cloned and pharmacologically characterized a novel histamine receptor, H₄ (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). H₄ has about 40% homology to H₃ (58% in the transmembrane region), has a similar genomic structure, and like H₃, seems to be functionally coupled to G protein G_i/o, thereby inhibiting forskolin-stimulated cAMP formation (Lovenberg et al., 1999). In contrast to these structural similarities, the expression pattern of H₄ dramatically differs from the expression profile of H₃. Hardly any evidence for H₄ expression in the brain and nervous tissues has been described. H₄ shows highest expression in the bone marrow and in leukocytes, moderate expression in spleen, thymus, lung, small intestine, colon, and heart (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). This expression pattern and the presence of several putative regulatory elements mediating tumor necrosis factor- and IL-6-stimulated transcription detected in the human H₄ gene promoter (Coge et al., 2001) suggest significant roles for H₄ in the immune system, but the present biological knowledge about H₄ is very limited.

Histamine is known to trigger IL-16 production in CD8⁺ cells (Laberge et al., 1995) and in epithelial cells (Arima et al., 1999); however, the receptor(s) mediating this response have not been fully characterized in the human system. IL-16 is further produced by many other cell types, including mast cells, eosinophils, B cells, dendritic cells, and epithelial cells (reviewed in Cruikshank et al., 2000). IL-16 has been found in the bronchoalveolar fluid of allergen- or histamine-challenged asthmatics (Cruikshank et al., 1995; Mashikian et al., 1998; Krug et al., 2000) and is increasingly expressed in the bronchial mucosa of atopic asthmatics (Laberge et al., 1997) by eosinophils and mast cells (Laberge et al., 1999). Through binding to its receptor (CD4), IL-16 is believed to play an important role in the recruitment of CD4⁺ T cells into the lungs of asthmatic patients. A crucial role of IL-16 in asthma is further supported by the protection of animals from allergic asthma after pretreatment with an IL-16-blocking peptide (de Bie et al., 1999) or a neutralizing anti-IL-16 antibody (Hessel et al., 1998).

By using the pharmacological tools currently available for H₃/H₄ receptors, we describe a first immunologically relevant function of H₄. Together with H₂, H₄ is involved in the control of IL-16 release from human lymphocytes.

	Materials and Methods

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Chemicals. Histamine, dimaprit, clobenpropit, -methyl-histamine, cimetidine, thioperamide, and pyrilamine were purchased from Sigma-Aldrich (St. Louis, MO). Pertussis toxin was purchased from Calbiochem (La Jolla, CA). Fetal calf serum was obtained from JRH Bioscience (Lenexa, KS). RPMI 1640 was purchased from Invitrogen (Carlsbad, CA). Sodium azide was purchased from Nacalai Tesque (Kyoto, Japan). All bulk chemicals not further specified were purchased from Wako Pure Chemicals (Osaka, Japan).

Blood Donors. Blood of both females and males were used for the studies. None of the donors was on any medication for at least 3 weeks before blood donation. All experiments were approved by the local ethical committee and performed in accordance with the Declaration of Helsinki.

Cell Purification and Cell Cultures. Peripheral blood mononuclear cells (PBMC) were prepared from heparinized human blood using Ficoll HyPaque (Amersham Biosciences UK, Ltd., Buckinghamshire, UK) according to the manufacturer's recommended protocol. Enriched monocytes were obtained from whole PBMC by incubation with RPMI 1640 + 10% fetal calf serum at 4°C for 30 min at constant rotation. Nonaggregated cells were removed, and the monocyte enriched pellet was resuspended in RPMI 1640 and plated at a density of 2 × 10⁶ cells/well in six-well plates with 10 ng/ml rhIL-4 (kindly provided by Drs. H-D. Hoerlein and J. Peters, Bayer AG, Wuppertal, Germany) and 25 ng/ml granulocyte/macrophage-colony-stimulating factor (Peprotech, Rocky Hill, NJ). After 7 days of culture, immature dendritic cells were collected and counted. Mature dendritic cells were obtained by incubation of immature dendritic cells for 2 more days in the presence of lipopolysaccharide (10 ng/ml; Sigma-Aldrich). FACS analysis revealed a purity of 90%, as assessed by the percentage of CD11c⁺ cells.

Flow Cytometry. Cells [2 × 10⁵ cells/tube in phosphate-buffered saline supplemented with 0.5% fetal calf serum and 0.1% sodium azide (FACS buffer)] were incubated with FACS buffer, isotype control antibody, or specific antibodies as specified for 30 min at 4°C in the dark. Samples were then washed once by centrifugation and resuspended in FACS buffer. Flow cytometric analysis was conducted using FACScan (Becton, Dickinson and Company, Mountain View, CA), and data were analyzed using Cellquest software.

Primers and PCR Conditions for mRNA Determination. All primers for quantitative RT-PCR were purchased from Nihon Idenshi Kenkyujo (Sendai, Japan). RNA was prepared by using the TRIzol reagent (Invitrogen no. 15596) strictly following the manufacture's instructions and treated with DNA-free (no. 1906; Ambion, Austin, TX) to remove any genomic DNA contamination. First-strand cDNA synthesis was performed by SUPERSCRIPT first-strand synthesis system for RT-PCR (Invitrogen no. 11904-018) using random primers. Copy DNA from human monocytes and from brain was purchased from CLONTECH (Palo Alto, CA).

IL-16 Determination. CD8⁺ cells were seeded in RPMI 1640 supplemented with 5% fetal calf serum on 96-well plates at a density of 2 × 10⁵ cells/well in 200 µl with or without histamine (Sigma-Aldrich). At the time points indicated, supernatants were removed and frozen at 30°C until further use. Total IL-16 concentrations were determined using a commercially available ELISA kit (Endogen, Woburn, MA) according to the manufacturer's instructions.

Migration Assay. IL-16-containing cell culture supernatants were collected and stored at 30°C until further use. Migration was assessed by seeding 2 × 10⁵ highly purified CD4⁺ cells over a removable 3-µm membrane of a Transwell migration chamber (Corning Costar, Corning, NY). Five hundred microliters of test supernatant at various dilutions was added to the lower well. The cell-containing membrane was placed over the test supernatants, and cells were allowed to migrate at 37°C for 2 h. For inhibition studies, a control mouse anti-human IgG antibody or anti-IL-16 antibody (clone 4.1; Cruikshank et al., 1995) was added to the lower well and incubated for 15 min at 37°C before the addition of cells. The amount of anti-IL-16 antibody added was able to block migration of CD4⁺ cells induced by 70 ng/ml rhu-IL-16, as determined in preliminary experiments. After a migration period of 2 h, cells were incubated on ice for 20 min to remove cells adhering to the membrane. The cells in the lower wells were collected and counted using FACS Cellquest software (Becton, Dickinson and Company). The migration index was calculated by dividing the absolute number of migrated cells by the number of cells spontaneously migrated in control wells.

Statistics. Unless otherwise stated, data are expressed as means ± S.D. of at least three independent experiments. Statistical significance was determined using the unpaired Student's t test if applicable or with the Welch test if variances were nonhomogeneous using commercially available statistic software (GraphPad Software, Inc., San Diego, CA).

	Results

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Histamine Induced IL-16 Release from CD8⁺ Cells. As previously reported (Laberge et al., 1995), histamine led to a concentration- and time-dependent release of IL-16 into the supernatant of freshly prepared CD8⁺ T cells from peripheral blood. IL-16 release reached a maximum at 1 µM histamine after 24 h. The basal release observed at this time point (51 ± 11 pg/2.5 × 10⁵ cells) was increased approximately 6-fold in the presence of 1 µM histamine (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Concentration- and time-dependent release of IL-16 from histamine-stimulated human CD8+ cell cultures. CD8+ cells were seeded at 2 × 105 cells/well in 200 µl of medium with or without histamine. At the time points indicated, supernatants were removed and frozen until IL-16 determination by ELISA. Data represent mean ± S.D. of independent experiments preformed with cells from four different donors. , 0 µM histamine; , 0.01 µM histamine; , 0.1 µM histamine; , 1.0 µM histamine; , 10.0 µM histamine.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Suppression of IL-16 release by histamine receptor antagonists. CD8+ cells were seeded at 2 × 105 cells/well in 200 µl of medium in the absence (0.1% DMSO) or presence of various concentrations of receptor antagonists [, pyrilamine (H1); , cimetidine (H2); , thioperamide (H3/4)] 30 min before stimulation with histamine (1 µM). After 24 h, supernatants were removed and frozen until IL-16 determination by ELISA. Drug effect was calculated based on the amount of histamine-induced IL-16 in the absence of antagonists (control values, 100%). Data represent mean ± S.D. of four to six independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Stimulation of IL-16 release by histamine receptor agonists. CD8+ cells were seeded at 2 × 105 cells/well in 200 µl of medium and either immediately stimulated with various concentrations of histamine receptor agonists (A) or incubated with pertussis toxin (100 ng/ml) overnight before histamine receptor agonists were added at a final concentration of 10 µM (B). After 24 h, supernatants were removed and frozen until determination of IL-16 concentrations by ELISA. The effect of pertussis toxin was calculated based on the amount of agonist-induced IL-16 in the absence of pertussis toxin (set as 100%). Data represent mean ± S.D. of four to six (A) or three (B) independent experiments, respectively. , p < 0.05; , p < 0.01 versus control values without pertussis toxin; -methyl-H., -methyl histamine.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Lymphocyte migration activity of histamine and clobenpropit-induced IL-16. CD4+ migration was assessed in cell-free supernatants derived from CD8+ cells previously stimulated by histamine (1 µM; A and B) or clobenpropit (10 µM; B) for 24 h, as described under Materials and Methods. Supernatants were either diluted with cell culture medium (A) or pretreated with a neutralizing anti-IL-16 antibody, a control mouse-anti human IgG antibody (control antibody), or directly incubated with CD4+ cells (B). Antibody effects were calculated in percent inhibition versus controls (set as 100%). Data in A represent mean values of duplicate incubations and are shown for one representative experiment of five (see Results for details). Migratory activity is given as the migration index (see Materials and Methods for details). Data in B are given as mean ± S.D. from three to four individual experiments. , P < 0.05 versus control values.

Histamine H₄ Receptor Expression Studies. Messenger RNA studies using primer pairs selective for human H₁ to H₄ receptor genes supported our pharmacological studies in CD8⁺-enriched cells; a high expression signal was noted for H₄ in all donors tested (n = 3), followed by moderate levels of H₂ and H₁. No evidence at all was noted for H₃ expression, an observation strongly supporting the suggestion that thioperamide exerted its inhibitory actions by antagonizing H₄ (Fig. 5).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Histamine receptor subtype distribution in human CD8+ lymphocytes. After enrichment by negative selection, CD8+ cells were spun down by centrifugation, and histamine receptor expression was assessed by quantitative RT-PCR using the Lightcycler method, as described under Materials and Methods. Three samples representing cells from different individuals are shown based on normalization of the signals to 2 microglobulin.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Histamine receptor mRNA expression in human lung tissue, various lung cell populations, and immune cells. Gene-specific primers were used for amplification (see Materials and Methods for details). As a representative summary of measurements performed in two to seven analogous samples of the respective cell types/tissue investigated, the distribution of H1 to H4 mRNA is shown for human total lung, trachea, fibroblasts (NHLF), epithelial cells (NHBE), bronchial smooth muscle cells (BSMC), and microvascular endothelial cells (HMVEC-L). Human umbilical vein endothelial cells (HUVEC) are shown in comparison (A). H4 and H3 receptor expression is shown for highly purified CD8+ lymphocytes (individual samples from three different donors), CD4+, monocytes (CD14), tonsil B cells and the human mast cell line HMC-1 (B), and for immature dendritic cells (iDC), mature dendritic cells (DC), and eosinophils (Eos; C). GAPDH served as a standard, numbers given correspond to defined copy numbers of the corresponding genes investigated. A human brain sample served as a positive control for H3 and as a negative control for H4 (C).

	Discussion

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The unique expression profile reported in the first publications on the novel histamine receptor H₄ (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001) immediately suggested that a variety of immunological functions can be expected as a consequence of activation of this receptor subtype. One such function (i.e., the regulation of histamine-triggered IL-16 release from human CD8⁺ cells) is described in the present article. H₄ expression analyses (PCR measurements), and pharmacological studies using selective antagonists, agonists, and inhibitors of signal transduction (pertussis toxin) leave no doubt on the involvement of H₄ to the control of IL-16 release in human lymphocytes. A significant role of H₂ is also clearly shown, which is in line with similar data reported earlier (Berman et al., 1984). The agonist and antagonist concentrations used are selective for the respective receptor subtype, i.e., cimetidine does not bind to H₄ even at concentrations >10 µM (Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001) and thioperamide does not bind to H₁ or H₂ in the concentration ranges tested (Arrang et al., 1987). Also, the concentration range in which thioperamide was efficacious is in accordance with those described previously by others in cellular systems (Oda et al., 2000). For further interpretation, literature-reported histamine receptor-binding data of the tool compounds used in the present study have been summarized in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Reported histamine receptor affinity values of histamine ligands used (ranges given in nanomolar) Note that these values have been obtained under different experimental conditions (various labeled ligands used, binding conditions, etc.) and reflect data across various laboratories. For further details see Hough (2001) and the references cited therein.

Clearly, H₄ and H₂ seem to be involved in the control of IL-16 release from human CD8⁺ T cells to a similar extent. A block of either receptor resulted in a 70 to 75% inhibition of cytokine levels (Fig. 2) and selective inhibition of the H₄ signaling by pertussis toxin pretreatment abrogated histamine-induced IL-16 release by approximately 50% (Fig. 3B). Combined inhibition of both H₂ and H₄, however, did not completely abrogate IL-16 release (data not shown), pointing toward a minor contribution of H₁ (10-15%) or to the involvement of an additional histamine receptor not yet known. Taking their opposite adenylyl cyclase coupling into consideration, an additive effect or synergism between H₂ (leading to cAMP elevation) and H₄ (leading to decrease of cAMP) was not expected. Rather, histamine seems to regulate IL-16 liberation from CD8⁺ cells by acting in parallel on various of its receptors, an observation made for this biogenic amine in several of its immune functions (Kimata et al., 1996; Sirios et al., 2000; Caron et al., 2001).

The following experimental evidence strongly argues for a direct action of histamine on H₄ on CD8⁺ lymphocytes. First and most importantly, highly purified CD8⁺ cells (positive MACS selection, purity >98%) released similar amounts of bioactive IL-16 upon stimulation by histamine or the H₄ agonist clobenpropit compared with the 85 to 90% pure CD8⁺-enriched population (Fig. 3; data not shown).

Secondly, mRNA message for H₄ was also detectable, although donor variations were more pronounced and expression levels were lower as compared with samples derived from the enriched CD8⁺ cultures (Figs. 5 and 6B; some data not shown). The reasons for the discrepancy in mRNA levels are unclear. Possible explanations are differences in the purification protocol and the fact that H₄ receptor mRNA expression seems to be quickly regulated by cell stimulation (Coge et al., 2001). In addition, significant donor-to-donor variations have also been reported for H₄ mRNA expression levels in blood neutrophils (Oda et al., 2000).

Finally, such highly purified CD8⁺ cells indeed seemed to express H₄ receptors, as suggested by flow cytometry analyses. The signal obtained in CD8⁺ cells after incubation with fluorescently labeled histamine was significantly competed by excess amounts of unlabeled histamine, cimetidine, thioperamide, and clobenpropit. However, a proper quantitative analyses could not be done, possibly due to fast receptor down-regulation after histamine binding (see supplemental data set). Similarly, Western blot analyses of CD8⁺ cell lysates showed a reactive band at the expected molecular mass of 43 kDa, but data interpretation was hampered by the lack of specificity of the only commercially available anti-H₄ antibody (data not shown).

The presence (Coge et al., 2001; Morse et al., 2001; Zhu et al., 2001) or absence (Oda et al., 2000; Liu eta al., 2001) of H₄ mRNA in lung tissue, and lung cells were under debate. Our data clearly show that a variety of human primary lung cells express H₄. Whether H₄ also contributes to the regulation of IL-16 production in lung epithelial cells is currently under investigation. In accordance with a putative role of H₄ in inflammatory processes in the lung, several cytokine-modulated regulatory elements have been identified on the H₄ gene, and modulation of H₄ expression by IL-5, -10, and -13 in immune cells has recently been reported (Coge et al., 2001; Liu et al., 2001).

An important finding of this study is the lack of H₃ expression in most of the cell types investigated. This puts many observations made in the past with thioperamide, previously only known as an H₃ antagonist, into a different perspective, since nearly all relevant findings using thioperamide in the immune system may be attributed to H₄ rather that to H₃. Among those putatively H₄-mediated immune functions of histamine are the inhibition of tumor necrosis factor release from mast cells (Bissonnette, 1996), stimulation of IL-10 in monocytes (Sirios et al., 2000), Ca²⁺ mobilization in human eosinophils (Raible et al., 1994), and IgE production from IL-4 + anti-CD58/LFA3-antibody stimulated human B cells (Kimata et al., 1996). However, alveolar macrophages, a population not studied here, were reported to express H₃ mRNA (Sirios et al., 2000), and therefore a role of H₃ in the immune system of the lung cannot be excluded.

The IL-16 protein released following H₄ activation obviously was bioactive since migration of CD4⁺ cells induced by clobenpropit- or histamine-stimulated CD8⁺ cell supernatants was significantly blocked by preincubation with a neutralizing anti-IL-16 antibody. Furthermore, IL-16 seemed to be the major migration-inducing factor present in CD8⁺ supernatants following histamine receptor activation. IL-16 neutralization abrogated CD4⁺ cell migration by more than 60% (histamine) or more than 70% (clobenpropit), respectively (Fig. 4B), an observation that is in accordance with previously published data (Laberge et al., 1995). Moreover, the addition of a neutralizing anti-MCP-1 antibody was without effect (data not shown).

With regard to inflammation, the suppression of IL-16 release by histamine receptor antagonists may be of therapeutic relevance. Histamine has been discussed as a mediator of asthma for a long time (reviewed in Barnes et al., 1998) and one of the mediators induced by this biogenic amine is IL-16. Indeed, this CD4⁺ cell chemoattractant was found in lungs of asthma patients following challenge (Cruikshank et al., 1995; Laberge et al., 1995; Krug et al., 2000), and intervention strategies against IL-16 have been successful in experimental animal models mimicking asthma symptoms (Hessel et al., 1998; de Bie et al., 1999). However, cimetidine treatment of mice failed to significantly reduce IL-16 levels in the bronchoalveolar lavage fluid of Ag-challenged mice (de Bie et al., 1998). Since thioperamide has not been investigated in that study and detailed analyses on the regulation of IL-16 release by histamine in the mouse are not available, those data do not necessarily contradict our findings. In addition, the bronchoalveolar fluid of Ag-challenged mice contains few CD8⁺ cells (F. Gantner and K. B. Bacon, unpublished observation), and IL-16 levels in the plasma have not been investigated.

Based on present knowledge, it is difficult to speculate which anti-histamine approach would be the therapeutically most promising. Selective H₁ antagonists became a standard therapy for allergic rhinitis, but their value in asthma-treatment is more than questionable (Van Ganse et al., 1997), although inhibition of bronchoconstriction and smooth muscle proliferation theoretically could be expected. H₂-selective antagonists, tremendously successful as anti-ulcerative drugs, were initially considered as being contraindicated in asthmatics due to possible inhibition of bronchorelaxation. However, no complications have been observed over more than two decades of clinical use of H₂ blockers. Rather, a significant therapeutic benefit was seen in a study analyzing asthmatics who received H₂ antagonist medication to treat their gastritis (Field and Sutherland, 1998). Those beneficial effects are possibly due to H₂ involvement in the regulation of many immunological processes relevant for asthma, such as stimulation of maturation, polarization, and chemokine induction in dendritic cells (Caron et al., 2001), IgE production in B cells (Kimata et al., 1996), IL-5 production in Th2 cells (Poluekova and Khan, 1998), and, finally, IL-16 release (Cruikshank et al., 1995; this study).

Neither direct bronchodilatory nor anti-inflammatory effect may be expected from compounds targeting H₃ selectively since this receptor neither seems to be present in human lung cells nor in most leukocytes (this study). Nevertheless, the development of H₃ agonists is considered for asthma treatment, primarily based on the observation that H₃ seems to be involved in the regulation of cholinergic nerves in the human airways and in the release of neuropeptides from airway sensory nerves (Ichinose and Barnes, 1989). Based on its restricted expression profile and its crucial role in the regulation of IL-16 release, the development of H₄-selective antagonist may be another promising anti-asthmatic approach. However, due to the limited knowledge of H₄ biology a final conclusion cannot yet be drawn.