We have previously demonstrated that dietary histamine is accumulated in the spleens of l-histidine decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis. To characterize the clearance system for dietary histamine in mice, we investigated the cell type and mechanism responsible for histamine uptake in the spleens of HDC-deficient mice. Immunohistochemical analyses using an antihistamine antibody indicated that a portion of the CD14+ cells in the spleen is involved in histamine storage. Peritoneal macrophages obtained from Balb/c mice and a mouse macrophage cell line, RAW264.7, had potential for histamine uptake, which was characterized by a low affinity and high capacity for histamine. The histamine uptake by RAW264.7 cells was observed at physiological temperature and was potently inhibited by pyrilamine, chlorpromazine, quinidine, and chloroquine, moderately inhibited by Nα-methylhistamine, dopamine, and serotonin, and not affected by tetraethylammonium and 1-methyl-4-phenylpyridinium. Intracellular histamine was not metabolized in RAW264.7 cells and was released at physiological temperature in the absence of extracellular histamine. These results suggest that histamine uptake by macrophages may be involved in the clearance of histamine in the local histamine-enriched environment.
- cation transporter
histamine has been found to exert its roles in a wide variety of physiological and pathological processes, such as inflammation, allergy, gastric acid secretion, and neurotransmission (1, 3, 18, 25). Because histamine is a potent mediator in these responses, it is very important to maintain local homeostasis by eliminating this histamine from the microenvironment. Expression of histamine-metabolizing enzymes, such as diamine oxidase (DAO, histaminase) and histamine N-methyl transferase (HMT), in some tissues contributes to the clearance of systemic histamine (13, 26). Another possible mechanism of histamine elimination is cellular uptake. However, no plasma membrane transporter specific for histamine has been identified and the characteristics of cellular histamine uptake are largely unknown. Recently, a family of organic cation transporters has been cloned (7), and some of them have been reported to be capable of histamine uptake (6). Little attention, however, was paid to this histamine uptake due to its relatively lower affinity compared with the other organic cations. Because the enzymes involved in histamine metabolism, such as DAO and HMT, and the putative transporters involved in histamine uptake have been found in limited types of tissues, it is possible that another system is involved in the local clearance of histamine.
We recently established an l-histidine decarboxylase (HDC)-deficient mouse strain, in which de novo synthesis of histamine is undetectable (12). However, a small but detectable amount of histamine was observed in some tissues of these HDC-deficient mice. Regarding the origin of this histamine, commercially available mouse diets were found to contain a trace amount of histamine, and some kinds of enterobacteria are known to produce histamine (16). Furthermore, we have recently observed that the histamine content in several tissues, such as brain, skin, stomach, and spleen, were significantly increased in the HDC-deficient mice when they were maintained on a histamine-enriched diet (11). These findings indicate that the HDC-deficient mouse strain is a good model for analyses of dietary histamine uptake without the possible interfering effects of endogenous histamine synthesis. Our purpose in this study is to identify and characterize the cellular uptake system for histamine.
MATERIALS AND METHODS
Materials. The following materials were purchased from the sources indicated: an antihistamine antibody from Sigma (St. Louis, MO), an anti-CD14 antibody from Pharmingen (San Diego, CA), an anti-heat shock cognate 70 (Hsc70) antibody from StressGen (Victoria, Canada), an Alexa 546-conjugated anti-rat IgG antibody, an Alexa 488-conjugated anti-rabbit IgG antibody, and an Alexa 488-conjugated anti-rat IgG antibody from Molecular Probes (Eugene, OR), a rhodamine-conjugated anti-rabbit IgG antibody from Leinco Technology, (Ballwin, MO), [3H]histamine (23.3 Ci/mmol) from DuPont-New England Nuclear (Boston, MA), and dimaprit, Nα-methylhistamine, thioperamide, and clozapine from Tocris Cookson (Bristol, UK). All other chemicals were commercial products of reagent grade.
Animals. All the experiments were performed according to the Guideline for Animal Experiments of Kyoto University. Generation of an HDC-deficient mouse strain was described previously (12). The HDC+/+ and HDC–/– mice, originally generated with a mixed genetic background of 129/Sv and CD1, were backcrossed to the Balb/c strain by 6–8 generations. Experiments were performed using 8- to 12-wk-old female HDC+/+ and HDC–/– mice, 8-wk-old female Balb/c mice, 8-wk-old WBB6F1-W/W+ (W/W+), and –W/WV (W/WV) mice. All strains except the HDC-deficient mice were obtained from Japan SLC (Hamamatsu, Japan). The low-histamine diet (ver. 3), which contains ∼1 nmol/g histamine, was purchased from Nihon Nosan Kogyo K. K. (Yokohama, Japan). The histamine-enriched diet was prepared by adding 80 μmol/g histamine to the low-histamine diet. In histamine-ingestion experiments, mice were freely fed with the low-histamine diet before experiments and then switched to the histamine-enriched diet for 7 days. Significant increases in plasma histamine levels were confirmed under these conditions (low-histamine diet, 0.04 ± 0.01 μM; histamine-enriched diet, 11.3 ± 1.25 μM in HDC–/– mice). For the purpose of collecting tissues and peritoneal cells, these mice were killed by cervical dislocation.
Determination of histamine content. Spleens were minced to obtain single-cell suspensions. Spleen cells were homogenized in 50 mM potassium phosphate, pH 6.8, containing 0.1% Triton X-100. The homogenates were centrifuged at 17,000 g for 30 min at 4°C, and the supernatant was used for the measurement of histamine content as previously described (23). The histamine formed was separated on a cation exchange column, WCX-1 (Shimadzu, Kyoto, Japan), by /high-performance liquid chromatography (HPLC) and then measured by the o-phtalaldehyde method (19).
Immunofluorescence study. Spleens from Balb/c mice were collected and treated with Bouin's fixative (Muto Pure Chemicals, Tokyo, Japan) for 24 h at 4°C. Sections (8 μm in thickness) were cut on a Jung Frigocut 3000E cryostat. The sections were incubated with an antihistamine antibody (1: 100) and an anti-CD14 antibody (1:100) for 1 day at 4°C. The sections were then stained with an Alexa 488-conjugated anti-rabbit IgG antibody (1:100) and a rhodamine-conjugated anti-rat IgG antibody (1:100). The fluorescent images were analyzed using a confocal microscope (MRC-1024, Bio-Rad Laboratories, Hercules, CA).
Preparation of peritoneal macrophages. Cells in the peritoneal cavity of Balb/c mice were harvested by lavage of the cavities with 3 ml of sterile phosphate-buffered saline (PBS). Lavage fluids were centrifuged at 200 g for 5 min at 4°C, and the pellet was washed in 2 ml of PBS. The peritoneal cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin for 1 h at 37°C to allow adhesion. A viability of >98% of the cells was confirmed by the trypan blue exclusion test.
Cell culture. A mouse macrophage cell line, RAW264.7, was grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, in 5% CO2 at 37°C in a fully humidified atmosphere. Exponentially growing cells were used in all experiments.
Measurement of [3H]histamine uptake. Peritoneal macrophages obtained from Balb/c mice or RAW264.7 cells were incubated in modified Eagle's medium containing 10 mM HEPES-NaOH, pH 7.3, or Krebs-Ringer-HEPES (KRH) buffer (10 mM HEPES-NaOH, pH 7.3, containing 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 1.0 mM glucose) for 10 min at 37°C. In sodium-free conditions, NaCl in the KRH buffer was replaced with LiCl. The cells were then incubated with [3H]histamine (2.33 μCi/ml) in the presence of cold histamine for the indicated periods at 37°C. The cells were rinsed twice in ice-cold PBS and lysed with PBS containing 1% Triton X-100. The radio-activities of the resultant soluble fractions were measured by a liquid scintillation counter. For the selective permeabilization of the plasma membranes, the cells were treated with 20 μg/ml digitonin for 10 min at 25°C instead of Triton X-100. Under these conditions, >95% of lactate dehydrogenase (a cytosolic enzyme) activity and no immunoreactive bands with an antiprotein disulfide isomerase (luminal protein of the endoplasmic reticulum) on immunoblot analyses was recovered in the leak-out fractions.
Accumulation of dietary histamine in spleen cells. We previously reported that a high level of histamine was accumulated in the spleens of HDC-deficient mice, which have a mixed genetic background (129/Sv × CD1), given a histamine-enriched diet for 7 days (11). High levels of accumulated histamine were reproduced in the spleens of both HDC+/+ and HDC–/– mice, which were backcrossed to the Balb/c strain by six to eight generations, under the same dietary conditions (Table 1). The increase in histamine content in these mice was comparable to the parental Balb/c mice. Because W/WV mice, which lack tissue mast cells (17), exhibited a similar level of increase in histamine content to the W/W+ mice and Balb/c mice, it is likely that tissue mast cells are not involved in this histamine uptake.
To identify the cell type responsible for histamine accumulation in the spleen, an immunofluorescence study using an antihistamine antibody was performed. Histamine-immunoreactive cells were observed in the red pulp of the spleens of Balb/c, HDC+/+, and HDC–/– mice given a histamine-enriched diet (data not shown). Double staining using an antihistamine antibody and an anti-CD14 antibody revealed that a portion of the CD14+ cells in the spleens of Balb/c mice was immunoreactive to an antihistamine antibody (Fig. 1).
Uptake of histamine by peritoneal macrophages and a macrophage cell line, RAW264.7, during in vitro incubation. Because CD14+ spleen cells were immunoreactive to the antihistamine antibody, we then measured histamine uptake by peritoneal macrophages of Balb/c mice. A significant uptake of histamine was observed at 37°C, although not at 4°C (Fig. 2A). The time course of histamine uptake was unchanged under Na+-free conditions (Fig. 2B). The same experiments were performed using a mouse macrophage cell line, RAW264.7. The rate of histamine uptake in RAW264.7 cells was about 5 times higher than that in the peritoneal macrophages (Fig. 2C), and about 50% of this uptake was suppressed under Na+-free conditions (Fig. 2D). Greater than 95% of the intracellular histamine was recovered in the soluble fraction of these cells and not in the membrane fraction (data not shown).
Dose-dependent accumulation of histamine in RAW264.7 cells. The dose-dependent accumulation of histamine in RAW264.7 cells was examined in the presence of various concentrations of histamine. Histamine uptake reached a plateau level at doses of >10 mM (Fig. 3A). The Km value for histamine was calculated to be 6.0 mM in RAW264.7 cells and 5.6 mM in peritoneal macrophages (data not shown). RAW264.7 cells were found to accumulate a maximum of ∼50 nmol of histamine/106 cells by2hof incubation at 37°C in the presence of 10 mM histamine, which is comparable to the levels of histamine stored in granules of mast cells (data not shown).
Release of histamine from RAW264.7 cells in the absence of extracellular histamine. To determine whether the accumulated histamine could be liberated from the cells, the cells were incubated in the presence of 6 mM histamine for 2 h, washed twice in histamine-free medium, and incubated for the indicated periods in the absence of extracellular histamine. Intracellular histamine leaked out into the medium within 6 h at 37°C, but not at 4°C (Fig. 3B). A good correlation was found between the decrease in histamine content in the cells and the increase in the medium. Furthermore, no metabolites of histamine were detected after 6 h of incubation on the basis of HPLC analyses (data not shown).
Distribution of accumulated histamine in RAW264.7 cells. We then investigated the subcellular distribution of accumulated histamine by an immunofluorescence study using the antihistamine antibody. Immunoreactive signals were observed in the cells incubated in the presence of histamine, but not in the absence of histamine (Fig. 4). The immunofluorescent signals observed with the antihistamine antibody largely colocalized with that observed with an antibody against Hsc70, which is a cytosolic molecular chaperone. Digitonin treatment, which is often used for selective permeabilization of plasma membranes, resulted in a rapid and complete release of accumulated histamine (1% Triton X-100; 100 ± 0.687%, 20 μg/ml Digitonin; 101 ± 8.38%). These results strongly indicate that histamine is accumulated in the cytosolic compartment of RAW2674.7 cells.
Effects of inhibitors of cytoskeletal activity on histamine uptake. Because macrophages are known to be active with regard to fluid-phase endocytosis, it is possible. that this histamine uptake reflects a pinocytotic or endocytotic process. We performed uptake experiments using a series of inhibitors for cytoskeletal activity, such as colchicine, nocodazole, and cytochalasin D, to verify this hypothesis (Table 2). RAW264.7 cells were pretreated with each inhibitor for 30 min, and histamine uptake was measured in the presence of the inhibitor. No inhibitors were found to effectively block histamine uptake by RAW264.7 cells, excluding the possibility of the involvement of cytoskeletal reorganization and, hence, a pinocytotic or endocytotic process in histamine uptake.
Characterization of histamine uptake. To evaluate the substrate specificity in this histamine uptake by RAW264.7 cells, competitive analyses for histamine uptake in the presence of various concentrations of organic cations and compounds structurally related to histamine were performed. The histamine uptake into RAW264.7 cells at 6 mM was inhibited by both dopamine and serotonin (apparent Ki value, 25 mM and 20 mM, respectively). However, such inhibition was not observed in the case of histidine or GABA (Fig. 5A). Histamine uptake was markedly inhibited by pyrilamine or chlorpromazine (apparent Ki value, 0.60 and 0.20 mM), although terfenadine did not exhibit such inhibitory effects. An atypical antipsychotic drug, clozapine, which has recently been reported to be a weak agonist of the H4 receptor (10), had no effects on histamine uptake at least up to 0.3 mM (Fig. 5B). The H2 receptor ligand, dimaprit, famotidine, and cimetidine, did not exhibit inhibitory effects on histamine uptake at doses up to 3 mM. Nα-methylhistamine showed the same inhibitory potential as histamine, whereas thioperamide did not inhibit histamine uptake at doses of up to 1 mM (Fig. 5C). Although both tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP+) have been reported to be good substrates for Na+-independent organic cation transporters (7), these compounds did not inhibit histamine uptake in RAW264.7 cells (Fig. 5D). Quinidine significantly inhibited histamine uptake (apparent Ki value, 0.25 mM), whereas guanidine and l-carnitine did not. Chloroquine dose dependently suppressed histamine uptake. Histamine uptake was also suppressed (70.0 ± 4.51% of control) after intracellular acidification by preloading the cells for 15 min with 30 mM NH4Cl, according to the procedure reported previously (2).
Various kinds of cells, such as mast cells, basophils, enterochromaffin-like cells, and histamine neurons, are known to produce and store histamine. We have found that a significant amount of histamine was accumulated in the spleens of various strains of mice, including the HDC-deficient mice, when they were maintained on a histamine-enriched diet for 7 days. Because the HDC-deficient mice showed no detectable de novo synthesis of histamine, and a very low enzymatic activity of HDC has been reported in the spleens of wild-type mice, de novo synthesis of histamine is unlikely to be involved in the accumulation of dietary histamine in the spleens. The largest pool of histamine in peripheral tissues apart from the stomach is known to be mast cells. We previously reported that mouse bone marrow-derived cultured mast cells have the potential to take up histamine, and this uptake is also characterized by its low affinity for histamine (apparent Km value, 1.6 mM) (11). However, because the increase in histamine accumulation was also observed in W/WV mice, which lack tissue mast cells (17), mast cells are likely not to be involved in accumulation in the spleen. An immunohistochemical study using an antihistamine antibody indicated that a portion of the CD14+ cells may be involved in histamine storage, although histamine uptake by mouse whole splenocytes was undetectable in our assay system (data not shown). This result indicates that only a small population of spleen cells may be able to take up histamine, although the subpopulations of CD14+ cells in the spleen still remain largely unknown. Activated whole spleen cells with lipopolysaccharide were also found to be incapable of histamine uptake, as well as unstimulated spleen cells (data not shown).
Corbel et al. (4) demonstrated that mouse bone marrow cells were capable of histamine uptake and that splenocytes, thymocytes, and peritoneal cells were not. Regarding the histamine uptake by peritoneal cells, there seems to be a discrepancy between Corbel et al. and us, which may be a result of a difference in the cell populations used; Corbel et al. used whole peritoneal cells, whereas we separated peritoneal macrophages by their adhesion in this study. Mouse peritoneal macrophages and a macrophage cell line, RAW264.7, both of which are known to be CD14+ cells, were found to be capable of histamine uptake. The uptake of histamine in peritoneal macrophages was found to be solely Na+ independent, whereas that in RAW264.7 cells was significantly decreased in the absence of extracellular Na+. However, the apparent Km values for histamine were not different in the presence and in the absence of extracellular Na+ (8.2 vs. 8.3 mM, data not shown). Although the rate of histamine uptake by peritoneal macrophages was lower than that by RAW264.7 cells, it was not influenced by the presence of extracellular Na+. We performed further analyses using RAW264.7 cells to exclude the possibility that a small population of contaminating cells may interfere with the characterization of histamine uptake.
Recently, a family of nonneuronal organic cation transporters (OCT1, OCT2, OCT3, OCTN1, and OCTN2) has been cloned and characterized in mammalian expression systems (7). This family has been found to transport a variety of organic cations with high capacity in a Na+-independent manner. Gründemann et al. (6) reported that histamine is a possible substrate of rat OCT2 and human EMT (OCT3). However, histamine uptake by RAW264.7 cells was not inhibited by MPP+, although Gründemann et al. have demonstrated the competition between MPP+ and histamine in a mammalian expression system. The expression of some OCT family members has been reported in limited tissues (5, 8, 14, 21, 22, 27), and there have been no reports about their expression in macrophages. Yabuuchi et al. (28) demonstrated that quinidine and pyrilamine are substrates for OCTN1. We detected by RT-PCR analyses very low levels of mRNA expression of OCTN1, abundant expression of OCTN2, and no expression of OCTN3 in RAW264.7 cells (data not shown), indicating that OCTN1 and OCTN2 are possible candidates for histamine uptake. However, histamine uptake by RAW264.7 cells was not inhibited by TEA, which is recognized as a substrate by all of the functionally active OCT family members, including OCTN1 and OCTN2 (5, 8, 14, 21, 22, 27). These results suggest that histamine uptake by RAW264.7 cells is mediated by a carrier distinct from the known OCT family members.
It is unlikely that histamine uptake by RAW264.7 cells is mediated by the internalization of histamine receptors, because no specific ligands to the histamine receptors could inhibit this uptake and little binding of histamine to the membrane fractions of the cells was observed. Two H1 receptor ligands, pyrilamine and chlorpromazine, significantly inhibited histamine uptake, although another H1 antagonist, terfenadine, did not. Furthermore, inhibition of histamine uptake by the other monoamines, dopamine, and serotonin indicates that this uptake system may function as an organic cation uptake system, which is not specific for histamine.
Because macrophages have been reported to possess active pinocytotic and endocytotic uptake systems, it is possible that histamine uptake may be dependent on these kinds of fluid-phase endocytosis. However, the inhibitors for cytoskeletal activity, such as colchicine, nocodazole, and cytochalasin D, which are known to suppress the pinocytotic and endocytotic process in macrophages (15, 20), were unable to block histamine uptake by RAW264.7 cells. This observation indicates that fluid-phase endocytosis may not be involved in the uptake of histamine.
Vestal et al. (24) reported the active uptake of propranolol by rabbit alveolar macrophages, which share some characteristics with histamine uptake in RAW264.7 cells: both are temperature dependent, inhibited by ammonium chloride and chloroquine, and effectively competed by chlorpromazine. Because Vestal et al. did not investigate the inhibitory effects of the endogenous amines, including histamine on propranolol uptake, it is unclear whether histamine uptake by RAW264.7 cells utilizes this system.
Histamine uptake by RAW264.7 cells was characterized by its low affinity and high capacity for histamine. Therefore, the physiological relevance of this uptake system may be limited to the clearance of a large amount of histamine release in the microenvironment, such as that through the degranulation of mast cells. Mast cells are often observed adjacent to macrophages in several tissues, such as the peritoneal cavity and lung, and it is possible that local concentrations of histamine reach millimolar levels around mast cells upon activation. The role of histamine uptake by macrophages may be temporal elimination, because histamine accumulated in RAW264.7 cells was found to be intact and to be released into the medium in the absence of extracellular histamine. A drastic increase in the serum concentrations of histamine, which is usually observed in IgE-dependent systemic anaphylaxis, has been reported to result in altered respiratory frequency and hypothermia (9). The temporal storage of histamine in macrophages may function as a buffer system to prevent rapid and drastic changes in histamine concentrations during allergic responses, although further experimental evidence is required to elucidate the physiological roles of histamine uptake by macrophages.
In summary, we have found that dietary histamine is accumulated, especially in the spleen of mice. Immunohistochemical analyses suggest that a portion of the CD14+ cells in the spleen may be involved in this histamine storage. Furthermore, mouse peritoneal macrophages and a mouse macrophage cell line, RAW264.7, have the potential to take up histamine, which may be one of the clearance systems of histamine.
This study was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.
We thank A. Popiel for help in preparation of the manuscript.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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