Organic cation transporter-3 mediates exogenous histamine uptake by primary mast cells

Graphical abstract

Abstract

Background

Mast cells exhibit uptake of histamine from the extracellular microenvironment, which can affect the extent of histamine release following their activation. How mast cells take up exogenous histamine, however, is not fully understood. Organic cation transporter-3 (OCT3), which is encoded by Slc22a3, is a bidirectional plasma membrane transporter for small cation molecules (including histamine), which is expressed in many types of cells, including mast cells. However, its precise roles in mast cells remain uncertain.

Objective

This study examined whether OCT3 plays a role in histamine uptake in mast cells.

Methods

Bone marrow–derived mast cells (BMMCs) obtained from OCT3-deficient (Slc22a3-knockout [Slc22a3-KO]) or wild-type (WT) mice were analyzed by microscopic examination, quantitative PCR, and flow cytometry to assess morphology and differentiation. Intracellular monoamine levels were evaluated by using HPLC and histamine enzyme immunoassay. The FcεRI-mediated activation of BMMCs was evaluated by β-hexosaminidase assay, ELISA, Western blot, and calcium flux assay. Passive cutaneous anaphylaxis reaction was used for in vivo analysis.

Results

The WT and Slc22a3-KO mouse BMMCs were comparable in morphology and differentiation. The Slc22a3-KO mouse BMMCs exhibited low intracellular histamine levels associated with low uptake of extracellular histamine and showed a reduction of histamine release following FcεRI activation when compared with WT BMMCs. Compared with WT mice Slc22a3-KO mice exhibited a reduction in passive cutaneous anaphylaxis reaction.

Conclusion

These findings suggest that OCT3 mediates exogenous histamine uptake by primary mast cells and that deficiency of OCT3 reduces intracellular histamine levels and downregulates IgE-mediated histamine release in mast cells.

Introduction

Mast cells play a crucial role in allergic diseases such as allergic rhinitis by releasing histamine following their activation. Mast cells produce histamine by histidine decarboxylase (HDC) in the cytosol; they transport it from the cytosol to granules, store it in the granule, and release it following activation.1, 2, 3 Importantly, mast cells can also exhibit uptake of histamine from the extracellular microenvironment. The histamine that is taken up follows the same transport as endogenous histamine synthesized by HDC and becomes the resource of released histamine from activated mast cells. Consequently, exogenous histamine can enhance mast cell histamine release.¹ Thus, it is important to understand how mast cells take up histamine in the extracellular microenvironment to control mast cell histamine release. However, the mechanisms have not been fully elucidated.

Organic cation transporter-3 (OCT3), which is encoded by Slc22a3 and is a member of the OCT family, is a bidirectional and polyspecific plasma membrane transporter for small cation molecules, including monoamines such as histamine, serotonin, dopamine, epinephrine, norepinephrine, and certain drugs.² Slc22a3 is ubiquitously expressed in many types of cells and tissues, such as brain, lung, and others (including mast cells and basophils).^3,4 In murine basophils, Oct3 is implicated in histamine uptake from the extracellular microenvironment.⁵ However, the functional roles of OCT3 in mast cells remain unclear.

The present study aimed to elucidate the roles of OCT3 in exogenous histamine uptake by mast cells. For this purpose, we compared bone marrow–derived cultured mast cells (BMMCs) obtained from wild-type (WT) mice and OCT3-deficient (Slc22a3-knockout [Slc22a3-KO]) mice.

Results and discussion

Differentiation status and morphologic features are comparable between WT and Slc22a3-KO mouse BMMCs

We generated BMMCs from WT and Slc22a3-KO mice and used them after 5 to 7 weeks of culture in RPMI-1640 medium supplemented with 10% FCS and 10 ng/mL of IL-3, as previously described.⁶ The histamine content of RPMI-1640 with 10% FCS was 247.15 ± 4.42 nM (n = 5), as measured by histamine enzyme immunoassay. All animal experiments were approved by the institutional review board of the University of Yamanashi, following the National Research Council's Guide for the Care and Use of Laboratory Animals (eighth edition).

Because the functional roles of OCT3 in mast cells remain unclear, we first compared mast cell differentiation and morphology between WT mouse BMMCs and Slc22a3-KO mouse BMMCs. The kinetics of BMMC differentiation assessed by KIT and FcεRIα surface expression showed little difference between WT and Slc22a3-KO mouse BMMCs (Fig 1, A and B). WT mouse BMMCs, but not Slc22a3-KO mouse BMMCs, expressed Oct3, whereas both genotypes of BMMCs showed little expression of other OCT members (ie, Oct1 and Oct2) and plasma membrane monoamine transporter (Pmat), which is another histamine transporter (see Fig E1 in the Online Repository at www.jaci-global.org). In addition, WT mouse BMMCs and Slc22a3-KO mouse BMMCs showed similar morphology, as judged by toluidine blue, esterase staining, Diff-Quick staining, and electron microscopy examination (Fig 1, C and D). Furthermore, the levels of expression of the mast cell differentiation marker genes, such as Mcpt5, Mcpt6, Gata2, Mitf, and Stat5, were comparable between the WT mouse BMMCs and Slc22a3-KO mouse BMMCs (Fig 1, E). These findings suggest few differences in the differentiation status and morphologic features between WT mouse BMMCs and Slc22a3-KO mouse BMMCs.

Fig 1.

Comparable differentiation and morphology between WT mouse BMMCs and Slc22a3-KO mouse BMMCs. Flow cytometry indicates the surface expression of KIT and FcεRIα weekly (A) and by the fifth week (B). Representative images of toluidine blue, esterase, Diff-Quik staining (C), and electron microscopy (D). Gene expression of Mcpt5, Mcpt6, Gata2, Mitf, and Stat5a (E). Values represent means ± SDs. ∗P <.05. ns, Nonsignificant.

OCT3 is important for exogenous histamine uptake by mast cells

We then quantified intracellular concentrations of several monoamines, including histamine, serotonin, dopamine, epinephrine, and norepinephrine (known as OCT3 substrates) by using HPLC-MS. Among these monoamines, histamine was the only one that showed a significant (by approximately 50%) reduction in Slc22a3-KO mouse BMMCs versus in WT mouse BMMCs (Fig 2, A). In contrast, intracellular serotonin levels were comparable between the WT mouse BMMCs and Slc22a3-KO mouse BMMCs (Fig 2, A). The concentrations of dopamine, epinephrine, and norepinephrine were below the range of detection (data not shown). In addition, we differentiated BMMCs into connective tissue–like mast cells (CTMCs) in vitro, as previously described⁷ (see Fig E2, A in the Online Repository at www.jaci-global.org). Intracellular histamine levels increased in CTMCs versus in BMMCs, but the levels in Slc22a3-KO mouse CTMCs were still lower than those in WT mouse CTMCs (see Fig E2, B), which is consistent with the findings in BMMCs.

Fig 2.

Intracellular histamine and serotonin levels in BMMCs (A). Gene expression of Hdc (B), and Hnmt (C) in BMMCs. Hnmt expression in mouse astrocytes (C) was used as a positive control. Extracellular histamine levels of BMMC culture after 24 hours incubated in FCS-free medium (D). Intracellular histamine in BMMCs after 16 hours of incubation with 100 μM histamine (E). Values represent means ± SDs. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, Nonsignificant.

To provide potential explanations for the decrease in intracellular histamine levels in Slc22a3-KO mouse BMMCs, we postulated 4 hypotheses regarding Slc22a3-KO mouse mast cells: (1) a reduction in histamine synthesis, (2) an increase in histamine degradation, (3) an elevation in spontaneous histamine release, and (4) a decrease in histamine uptake. We found that Slc22a3-KO mouse BMMCs exhibited increased expression of Hdc, the sole enzyme responsible for histamine biosynthesis (Fig 2, B). In addition, the gene that encodes histamine N-methyltransferase (Hnmt), the enzyme involved in intracellular histamine degradation, was not expressed in WT mouse BMMCs or in Slc22a3-KO mouse BMMCs (Fig 2, C). To examine spontaneous histamine release, we cultured WT mouse BMMCs and Slc22a3-KO mouse BMMCs in FCS-free medium because FCS degrades histamine.⁸ Culture medium was collected after 24 hours to measure the extracellular concentrations of histamine. Extracellular histamine levels were lower in the culture of Slc22a3-KO mouse BMMCs than in the culture of WT mouse BMMCs (Fig 2, D). This may be due to reduced intracellular histamine levels in Slc22a3-KO mouse BMMCs and/or the bidirectional transport function of OCT3 (ie, possible failure of outward transport of intracellular histamine in Slc22a3-KO mouse BMMCs). Therefore, the first 3 hypotheses may not be valid, although we cannot entirely rule out their possibility.

To assess the potential decrease in histamine uptake in Slc22a3-KO mouse mast cells, both WT mouse BMMCs and Slc22a3-KO mouse BMMCs were incubated for 16 hours in medium to which an excessive amount of histamine (100 μM) had been added, after which the intracellular histamine levels were determined as previously described.¹ The intracellular histamine levels were dramatically enhanced in the WT mouse BMMCs, whereas the increase in the Slc22a3-KO mouse BMMCs was subtle (Fig 2, E). These findings suggest that in the case of BMMCs, OCT3 is important for histamine uptake from culture medium.

Deficiency of OCT3 selectively downregulates IgE-mediated histamine release in mast cells

Because deficiency of OCT3 led to reduced intracellular histamine content in BMMCs, we asked whether the reduction could affect IgE- or compound 48/80 (C48/80)-mediated histamine release in mast cells. Histamine release from Slc22a3-KO mouse BMMCs stimulated with IgE was significantly (by approximately 50%) reduced versus that from WT mouse BMMCs (84.87 ± 12.54 vs 145.03 ± 18.28 ng/mL [n = 3]). Similar results were obtained when Slc22a3-KO mouse BMMCs and WT mouse BMMCs were treated with C48/80 (117.26 ± 2.40 vs 193.75 ± 37.67 ng/mL [n = 3]) (Fig 3, A). In contrast to histamine release, IgE-mediated β-hexosaminidase release and cytokine production (TNF-a, IL-6, and IL-13) were comparable between WT mouse BMMCs and Slc22a3-KO mouse BMMCs (Fig 3, B and C). These results suggest that deficiency of OCT3 leads to selective reduction of histamine release following activation in mast cells.

Fig 3.

IgE- and C48/80-mediated histamine release in BMMCs (A). β-hexosaminidase (B), IL-6, IL-3, and TNF-α (C) release following IgE/αIgE stimulation. Gene expression of Fcer1a, Ms4a2 (Fcerb), Fcer1g, Mrgprb2, Lyn, Syk, Stim1, and Orai1 in BMMCs (D). Extent of phosphorylation of Akt (E) and calcium influx (F) following IgE/αIgE stimulation. Values represent means ± SDs. ∗∗P < .01. ns, Nonsignificant.

We confirmed that the expression levels of molecules involved in IgE- or C48/80-mediated intracellular signaling in mast cells, such as Fcer1a, Ms4a2 (Fcerb), Fcer1g, Mrgprb2, Lyn, Syk, Stim1, and Orai1, were equivalent between WT mouse BMMCs and Slc22a3-KO mouse BMMCs (Fig 3, D). Furthermore, the extent of AKT phosphorylation and calcium influx after IgE-mediated activation were equal in WT mouse BMMCs and Slc22a3-KO mouse BMMCs (Fig 3, E and F). Thus, the decrease of IgE- or C48/80-mediated histamine release in Slc22a3-KO mouse BMMCs was likely due to the lower level of intracellular histamine content than to any alteration in the degranulation signaling pathway.

Consistent with the in vitro findings, compared with WT mice, Slc22a3-KO mice showed a diminished extent of IgE- and C48/80-mediated passive cutaneous anaphylaxis reactions (Fig 4, A and B). To exclude the possibility of Slc22a3-KO mice being less responsive to histamine than WT mice are, subcutaneous histamine injections were administered into the ears of mice of each of the 2 types for the purpose of examining the reactions between the 2 genotypes. Both genotypes exhibited similar responses to histamine (Fig 4, C).

Fig 4.

A, Mouse ears in IgE-induced passive cutaneous anaphylaxis (PCA) (left) and the relevant dye quantification (right). Mouse ears in C48/80-induced PCA (B [left]) and histamine-induced reactions (C [left]), and the relevant dye quantification (B and C [right]). Representative results of 3 independent experiments. Values represent means ± SDs. ∗∗P < .01; ∗∗∗ P < .001. ns, Nonsignificant.

Soetanto et al reported that PMAT and OCT1 mediated histamine uptake from the extracellular space in RBL-2H3 Sc98 rat mast cells.⁹ They suggested that OCT3 may not be involved in histamine uptake because the OCT3 inhibitor corticosterone did not suppress histamine uptake and OCT3 expression in the cell line was low. The difference between the findings of the study by Soetanto et al⁹ and the findings of the current study could be explained by the use of different cell types (cell line vs primary cells) and inhibition strategies (chemical inhibitors vs gene knockout).

Schneider et al reported that OCT3-deficient bone marrow–derived basophils (BMBAs) did not take up exogenous histamine,⁵ consistent with our findings. However, increased intracellular histamine levels in OCT3-deficient BMBAs were observed. Schneider et al⁵ suggested that this was due to the absence of endogenously synthesized histamine release from the bidirectional OCT3 transporter in BMBAs. We speculate that this discrepancy arises from differences in histamine-synthesizing activity between BMBAs and BMMCs. Hdc and Slc22a3 mRNA levels gradually decreased during IL-3–induced bone marrow cell differentiation (see Fig E3 in the Online Repository at www.jaci-global.org). Schneider et al used BMBAs cultured for 8 to 9 days when HDC expression was high, whereas we studied BMMCs cultured for 4 to 5 weeks, likely leading to lower histamine synthesis.

Interestingly, Schneider et al showed that OCT3-deficient BMBAs with high intracellular histamine levels exhibited less IL-3–induced IL-4 and IL-6 production than WT BMBAs did. The authors suggested that intracellular histamine negatively regulated transcription of the cytokines.⁵ On the other hand, we observed that OCT3-deficient BMMCs with low intracellular histamine levels exhibited production of IgE-mediated IL-6, IL-13, and TNF-a comparable to that exhibited by WT BMMCs (Fig 3, C). This discrepancy also remains to be investigated. In either case, however, OCT3 inhibition might have potential to control allergic reaction by suppressing basophil-derived IL-4 or by selectively reducing histamine release from mast cells.

In conclusion, we suggest that OCT3 plays a significant role in extracellular histamine uptake in mast cells, which in turn can influence both intracellular histamine levels and their subsequent release following activation. Because Oct3 deficiency can selectively decrease the IgE-mediated release of histamine without affecting other types of mediator release, modulating OCT3 expression and function may hold unique potential for therapeutic interventions in allergic diseases that involve mast cell histamine release.

Clinical implications.

Modulating OCT3 expression and function in mast cells could offer unique potential therapeutic interventions (ie, selective histamine reduction) for allergic diseases in which mast cell histamine release contributes to the pathophysiology.

Disclosure statement

Supported by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant-in-aid for scientific research 22K19427 [to A.N.]).

Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.