Mast Cells Down-Regulate CD4⁺CD25⁺ T Regulatory Cell Suppressor Function via Histamine H1 Receptor Interaction¹

In recent years, the importance of several different T regulatory (T_reg)³ cell subsets in the establishment and maintenance of immune tolerance have become increasingly apparent. Perhaps the best studied of these are “natural” T_reg cells that develop in the thymus and are important endogenous regulators of immune responses to self- and foreign Ags (1, 2). The vast majority of naturally occurring CD4⁺ T_reg cells constitutively express CD25 (IL-2R α-chain) (3) and the Foxp3 transcription factor, which plays a critical role in T_reg cell development and effector function (4, 5, 6). Endogenous T_reg cells can potently suppress the in vitro activation of other T cell subsets through contact-dependent processes that have been suggested to involve CTLA-4 (7), membrane-bound TGF-β (8), and/or the release of granzyme B, without or with a requirement for perforin (9, 10). T_reg cells also express the ectonucleotidases CD39 and CD73, which allows T_reg cells to generate adenosine and thereby mediate immune suppression (11). Although T_reg cell suppressor function appears to be strictly contact-dependent in vitro, immunosuppressive cytokines such as IL-10 and TGF-β contribute to T_reg suppressor activity in vivo (12). The ability of T_reg cells to prevent the activation of autoreactive T cells and limit nonspecific bystander damage by modulating immune responses to foreign Ags is of obvious benefit; nevertheless, the initiation of protective immune responses toward pathogens must involve processes that overcome or at least diminish the immunosuppressive activity of endogenous T_reg cells.

Although mast cells are traditionally viewed in the context of allergy and asthma, the ability of mast cells to become activated by diverse stimuli and to produce a wide variety of mediators makes them important players in many other physiological processes (13). Mast cells are an important component of the innate immune system, acting as sentinel cells that detect infecting microorganisms via pattern recognition molecules such as TLR (14). The wide variety of cytokines, chemokines, and other mediators, including histamine, that are produced and released by mast cells following their activation are believed to promote the recruitment of other immune effector cells and modulate their activity. Since mast cells are first-line responders to microbial infections and are present in environments that may also contain endogenous T_reg cells, it seems likely that mast cells and T_reg cells might affect each others’ function. However, only limited information is available on the interactions that take place between mast cells and T_reg cells. In a mouse model of sepsis, adoptive transfer of T_reg cells correlates with an increase in mast cell numbers in the peritoneum (15), whereas mast cell recruitment to skin allografts in response to IL-9 produced by T_reg cells is essential for the establishment of tolerance to alloantigens (16). In addition, a recent study (17) showed that T_reg cells down-regulate FcεRI expression by mast cells in vitro through a contact-dependent mechanism and also down-regulate IgE-mediated leukotriene C₄ production by mast cells. Although these studies indicate that T_reg cells possess the capacity to recruit mast cells and regulate their activation, the effect(s) that mast cells have on T_reg cell suppressor function has not yet been investigated.

In this study, we show for the first time that histamine released by activated murine bone marrow-derived mast cells (BMMC) inhibited the suppressor function of CD4⁺CD25⁺ T_reg cells by signaling through the H1 receptor. In addition, the histamine-induced decrease in T_reg cell suppressor function was associated with reduced expression of CD25 and the T_reg cell-specific transcription factor Foxp3. Activation-induced release of histamine by mast cells may promote the development of protective immune responses to infecting microorganisms by transiently down-regulating endogenous T_reg cell activity.

For all experiments, 6- to 8-wk-old female C57BL/6 mice (Charles River Laboratories Canada) were used. Animals were cared for and housed in the Carleton Animal Care Facility (Dalhousie University) in accordance with Canadian Council on Animal Care guidelines.

CD4⁺CD25⁺ T_reg cells were isolated from mouse spleen cell preparations by negative selection for CD4⁺ cells and positive selection for CD25⁺ cells using a MACS mouse T_reg cell isolation kit (Miltenyi Biotec). The CD4⁺CD25⁻ T cell population was retained for use as T responder (T_resp) cells in suppression assays. The purity of the MACS-isolated T cell fractions was confirmed by two-color flow cytometric analysis using anti-CD25-PE mAb (Miltenyi Biotec) and anti-CD4-FITC mAb (eBioscience). Purity of the CD4⁺CD25⁺ T_reg cell fraction and CD4⁺CD25⁻ T_resp cell fraction was typically 90 and 95%, respectively. For all experiments, T cells were cultured at 37°C/5% CO₂/95% humidity in RPMI 1640 medium (Sigma-Aldrich Canada) supplemented with 5% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 5 mM HEPES (all from Invitrogen).

BMMC were obtained by culturing bone marrow cells from murine femurs and tibias in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10% WEHI-3B conditioned medium, 50 U/ml penicillin, 50 μg/ml streptomycin, 50 μM 2-ME (Sigma-Aldrich Canada), and 200 nM PGE₂ (Sigma-Aldrich Canada) for 4–6 wk. BMMC purity of >98% was achieved, as determined by staining of fixed cytocentrifuged BMMC preparations with the mast cell-specific dye toluidine blue (18). BMMC were primed overnight with 1 μg/ml anti-TNP IgE obtained from cultures of IGEL b4 hybridoma cells (American Type Culture Collection).

CD4⁺CD25⁻ T_resp cells (1 × 10⁵) and/or CD4⁺CD25⁺ T_reg cells (2 × 10⁴) were cultured in quadruplicate wells of a 96-well microtiter plate with either anti-CD3/anti-CD28 mAb-coated T cell expander beads (5 × 10⁴; Invitrogen) and/or anti-TNP IgE-primed BMMC (2 × 10⁴) and 10 ng/ml TNP-BSA (Sigma-Aldrich Canada) for 48 h. In some experiments, anti-TNP IgE-primed BMMC were cultured for 24 h in the presence of 10 ng/ml TNP-BSA to generate activated BMMC culture supernatants, which were then harvested and added to T cell cultures at a concentration that was reflective of 2 × 10⁴ BMMC. Histamine, loratadine, famotidine, 2-pyridylethylamine dihydrochloride (2-PEA) (all from Sigma-Aldrich Canada), or amthamine dihydrobromide (ADHB) (Tocris Biosciences) were added to T cell cultures as indicated. Cultures were pulsed with 0.25 μCi of [³H]TdR (MP Biomedicals) for the last 6 h of culture. Alternatively, CD4⁺CD25⁻ T_resp cells were labeled with 2 μM Oregon Green 488 dye (Invitrogen) for 15 min at room temperature and seeded in 96-well microtiter plates as previously described without or with unlabeled CD4⁺CD25⁺ T_reg cells and/or BMMC. After 72 h, quadruplicate cultures were pooled, and T_resp cell proliferation was analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

CD4⁺CD25⁻ T_resp cells (1 × 10⁵) and/or CD4⁺CD25⁺ T_reg cells (2 × 10⁴) were cultured in quadruplicate wells of a 96-well microtiter plate with either anti-CD3/anti-CD28 mAb-coated T cell expander beads (5 × 10⁴) and/or anti-TNP IgE-primed BMMC (2 × 10⁴) and 10 ng/ml TNP-BSA for 24 h. Culture supernatants were then harvested, and IL-2 levels in quadruplicate samples were determined using an IL-2 OptEIA ELISA kit (BD Biosciences).

CD4⁺CD25⁺ T_reg cells were stained for 30 min at 4°C with 5 μg/ml anti-CD25-FITC mAb (Cedarlane Laboratories) or fixed, permeabilized, and stained with 1 μg/ml anti-Foxp3-FITC mAb (eBioscience), according to the manufacturers’ specifications. Control cells were stained with the appropriate FITC-conjugated isotype control Ab. Cells were analyzed using a FACSCalibur flow cytometer and CellQuest software.

Total RNA was extracted from equal numbers of T_resp cells and T_reg cells using TRIzol reagent and reverse transcribed using Superscript II RNase H reverse transcriptase (both from Invitrogen), according to the manufacturer’s instructions. Histamine H1 receptor mRNA was quantified by TaqMan MGB Probe and TaqMan Master Mix (both from Applied Biosystems) on the ABI Prism 7000 sequence detection system (95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min). GAPDH mRNA was analyzed using the TaqMan Rodent GAPDH Control Reagents (Applied Biosystems). Data were analyzed using the relative standard curve method, according to the manufacturer’s protocol. The result was expressed as a ratio of histamine H1 receptor to GAPDH mRNA. In addition, PCR products were resolved on 2% agarose gel and visualized under UV light after staining with ethidium bromide.

T cells (2.5 × 10⁶) were pelleted and lysed with ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM Na₂HPO₄, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 5 mM EDTA, and 5 mM EGTA) containing freshly added protease and phosphatase inhibitors (5 μg/ml leupeptin, 5 μg/ml pepstatin, 10 μM aprotinin, 1 mM PMSF, 10 mM NaF, 1 mM DTT, and 100 μM Na₃VO₄). Protein was quantified using Bio-Rad Protein Assay reagent (Bio-Rad) and diluted with SDS-PAGE sample buffer (200 mM Tris-HCl (pH 6.8), 30% glycerol, 6% SDS, 15% 2-ME, and 0.01% bromophenol blue). Samples were then boiled for 5 min and stored at −80°C. Frozen cell lysates were thawed on ice; proteins were resolved by SDS-PAGE (20 μg protein/lane) and electrotransferred onto nitrocellulose membranes. Membranes were blocked in TBST (20 mM Tris-HCl (pH 7.6), 200 mM NaCl, and 0.05% Tween 20) containing 5% fat-free milk overnight at 4°C, then washed with TBST and incubated with anti-histamine H1 receptor mAb (clone H-300; Santa Cruz Biotechnology) diluted 1/200 in blocking solution overnight at 4°C. Membranes were washed with fresh TBST and incubated with HRP-conjugated goat anti-rabbit Ab (Santa Cruz Biotechnology) diluted 1/1000 in blocking solution for 1 h at room temperature. Membranes were washed with TBST, reacted with ECL reagents (GE Healthcare) for 1 min, and exposed to x-ray film. To confirm equal protein loading, membranes were incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-ME) at 37°C for 30 min to remove Abs, then washed with fresh TBST and sequentially probed with anti-actin mAb (clone I-19; Santa Cruz Biotechnology) and HRP-conjugated bovine anti-goat IgG Ab (Santa Cruz Biotechnology), both at 1/1000 dilution.

Data were analyzed using the Instat statistics program (GraphPad Software). Statistical comparisons were performed using Student’s t test or ANOVA with the Tukey-Kramer multiple comparisons posttest.

To determine the capacity of mast cells to modulate the function of CD4⁺CD25⁺ T_reg cells, CD4⁺CD25⁻ T_resp cells were stimulated with anti-CD3/anti-CD28 mAb-coated beads alone or in combination with CD4⁺CD25⁺ T_reg cells (5:1 ratio) and/or anti-TNP IgE-primed BMMC and TNP-BSA. As expected, T_reg cells inhibited T_resp cell proliferation (p < 0.001), as determined by [³H]TdR incorporation (Fig. 1,A). TNP-activated BMMC did not affect T_resp cell proliferation. Importantly, there was a marked and significant (p < 0.001) reduction in T_reg cell-mediated suppression of T_resp cell proliferation in the presence of TNP-activated BMMC. Neither T_resp cell proliferation nor T_reg cell-mediated suppression of T_resp cell proliferation was altered in the presence of unactivated BMMC (data not shown). Because as many as three different cell populations were present in our assay system, it was important to confirm that changes in [³H]TdR incorporation were due to increased T_resp cell proliferation and were not caused by increased proliferation of T_reg cells and/or BMMC. T_resp cells were therefore labeled with Oregon Green 488 dye, which allowed us to specifically measure T_resp cell division by flow cytometry. As shown in Fig. 1 B, anti-CD3/anti-CD28 mAb-coated bead-stimulated T_resp cells that were cocultured with T_reg cells showed a smaller percentage of proliferating T_resp cells and fewer rounds of cell division over a 72-h period in comparison to T_resp cells activated in the absence of T_reg cells. Furthermore, the addition of anti-TNP IgE-primed BMMC and TNP-BSA to T_reg cell-T_resp cell cocultures restored T_resp cell proliferation to levels that were equivalent to that seen when T_resp cells were activated in the absence of T_reg cells. TNP-activated BMMC had no effect on T_resp cell division in the absence of T_reg cells. Unactivated BMMC did not affect T_resp cell division in the absence or presence of T_reg cells (data not shown). Taken together, these data show that activated BMMC down-regulated the suppressor function of CD4⁺CD25⁺ T_reg cells.

To determine whether the inhibitory effect of mast cells on CD4⁺CD25⁺ T_reg cell function was due to a soluble factor, we next examined the effect of activated BMMC culture supernatant on CD4⁺CD25⁺ T_reg cell function. Cell-free culture supernatants were harvested after exposing anti-TNP IgE-primed BMMC to TNP-BSA for 24 h. CD4⁺CD25⁻ T_resp cells were then stimulated with anti-CD3/anti-CD28 mAb-coated beads either alone or in combination with CD4⁺CD25⁺ T_reg cells (5:1 ratio) in the absence or presence of supernatant from cultures of activated BMMC. As shown in Fig. 2,A, T_reg cell suppressor function in a [³H]TdR incorporation assay was abrogated in the presence of activated BMMC culture supernatant (p < 0.001). BMMC culture supernatant by itself did not affect T_resp cell proliferation. Similarly, the addition of activated BMMC culture supernatant to cocultures of Oregon Green 488 dye-labeled T_resp cells and unlabeled T_reg cells resulted in reduced T_reg cell-mediated suppression of T_resp cell division (Fig. 2 B). Collectively, these data demonstrate that the inhibitory effect of BMMC on T_reg cell function was mediated by a BMMC-derived soluble factor.

FcεR cross-linking causes mast cells to release large amounts of histamine, which is a soluble factor with numerous effects on the immune system (19). To determine whether histamine was able to inhibit CD4⁺CD25⁺ T_reg cell suppressor function, CD4⁺CD25⁻ T_resp cells were stimulated with anti-CD3/anti-CD28 mAb-coated beads in the presence of increasing doses of histamine (0–10 μM), either alone or in coculture with CD4⁺CD25⁺ T_reg cells (5:1 ratio). As shown in Fig. 3,A, T_reg cell-mediated suppression of T_resp cell proliferation decreased with increasing doses of histamine, whereas histamine had no effect on T_resp cell proliferation in the absence of T_reg cells. Histamine (10 μM) did not affect the proliferation or viability of T_reg cells (data not shown). Since histamine could interfere with T_reg cell suppressor function either by directly inhibiting the T_reg cells or by rendering T_resp cells refractory to T_reg cell-mediated suppression, we pretreated T_resp cells or T_reg cells with histamine (10 μM) for 30 min and then washed extensively before assaying cell proliferation by [³H]TdR incorporation. T_resp cells that were pretreated with histamine proliferated normally following stimulation with anti-CD3/anti-CD28 mAb-coated beads and were suppressed by untreated CD4⁺CD25⁺ T_reg cells (Fig. 3,B). In contrast, T_reg cells that were pretreated with histamine failed to inhibit the proliferation of untreated or histamine-pretreated T_resp cells. These data indicate that histamine directly inhibited T_reg cell suppressor function rather than rendering T_resp cells refractory to T_reg cell-mediated suppression. We also investigated the effect of histamine on T_reg cell-mediated inhibition of IL-2 production by anti-CD3/anti-CD28 mAb-coated bead-stimulated T_resp cells. Although histamine had no effect on IL-2 production by T_resp cells in the absence of T_reg cells, T_reg cell-mediated suppression of IL-2 production by T_resp cells was abrogated in the presence of histamine (p < 0.001; Fig. 3 C).

To determine whether histamine was responsible for BMMC-mediated inhibition of CD4⁺CD25⁺ T_reg cell suppressor function, we treated cocultures of CD4⁺CD25⁻ T_resp cells, CD4⁺CD25⁺ T_reg cells, and anti-TNP IgE-primed BMMC with the histamine H1 receptor antagonist loratadine (20) or the histamine H2 receptor antagonist famotidine (21) before the addition of TNP-BSA and anti-CD3/anti-CD28 mAb-coated beads. Fig. 4,A shows significant (p < 0.001) rescue of T_reg suppressor function in the presence of activated BMMC when loratadine was also present; however, there was no rescue by famotidine. Neither loratadine nor famotidine affected T_resp cell proliferation in the absence of T_reg cells. Similarly, treatment with loratadine, but not famotidine, significantly (p < 0.001) reduced the inhibitory effect of activated BMMC culture supernatant on T_reg cell suppressor function (Fig. 4 B). Taken together, these data suggest that histamine was responsible for the inhibitory effect of activated BMMC on T_reg cell suppressor function and that BMMC-derived histamine acts on T_reg cells via the H1 receptor.

To confirm that histamine inhibited T_reg cell function by acting through H1 receptors, we showed that exposure to increasing doses (2.5–10 μM) of loratadine resulted in a dose-dependent rescue of T_reg cell suppressor function in the presence of histamine (Fig. 5,A). In contrast, the highest dose of famotidine (10 μM) had only a slight effect on T_reg cell suppressor function in the presence of histamine. We also examined the effects of 2-PEA, a highly selective H1 receptor agonist (22), and ADHB, a highly selective H2 receptor agonist (23), on T_reg cell-mediated inhibition of T_resp cell proliferation in response to anti-CD3/anti-CD28 mAb-coated bead stimulation. Exposure to increasing doses of 2-PEA to T_reg cell-T_resp cell cocultures resulted in a dose-dependent decrease in T_reg cell-mediated suppression, whereas ADHB, even at the highest dose used, had little effect on T_reg cell function (Fig. 5 B). Collectively, these data indicate that the inhibitory effect of histamine on T_reg cell suppressor function was mediated exclusively through H1 receptors.

Although mouse T cells are known to express H1 receptors (24), H1 receptor expression by CD4⁺CD25⁺ T_reg cells has not yet been examined and compared with CD4⁺CD25⁻ T_resp cells. Real-time quantitative RT-PCR showed that H1 receptor mRNA expression by T_resp cells exceeded that of T_reg cells by ∼2-fold (p < 0.01; Fig. 6,A); however, similar levels of H1 receptor protein were expressed by T_resp cells and T_reg cells (p > 0.05; Fig. 6 B).

CD4⁺CD25⁺ T_reg cells are characterized by the constitutive expression of the high-affinity IL-2R α-chain, CD25 (3). Moreover, IL-2 is important for the development, maintenance, and activation of T_reg cells (25, 26, 27). We therefore examined the effect of histamine on CD25 expression by T_reg cells. After 24 h of culture in the absence or presence of 10 μM histamine, T_reg cells were stained for CD25 and analyzed by flow cytometry. In comparison to untreated T_reg cells, histamine-treated T_reg cells showed a marked decrease in CD25 expression (mean fluorescence intensity (MFI) = 105 ± 24 vs 27 ± 13, p < 0.01; Fig. 7,A). We also determined the effect of histamine on T_reg cell expression of the forkhead box/winged helix transcription factor Foxp3, which is important for T_reg cell development and programming T_reg cell suppressor function (4, 5, 6). Fig. 7 B shows that 24 h of exposure to 10 μM histamine caused a marked decrease in Foxp3 expression in comparison to untreated T_reg cells (MFI = 20 ± 9 vs 62 ± 8, p < 0.01). T_reg cell expression of CD25 and Foxp3 was therefore down-regulated by histamine. The inhibitory effect of histamine on CD25 and Foxp3 expression by T_reg cells was not evident at earlier time points (2 or 10 h), nor was it transient since washing out histamine from T_reg cell cultures after 30 min of exposure did not prevent CD25 and Foxp3 down-regulation at the 24-h time point (data not shown).

There has been a paucity of information to date regarding the effect that mast cells have on T_reg cell function. We show here, for the first time, that histamine released by FcεR-activated BMMC abrogated the suppressor function of CD4⁺CD25⁺ T_reg cells. Activated BMMC potently inhibited the suppressor function of CD4⁺CD25⁺ T_reg cells through a mechanism that involved H1 histamine receptor signaling, because the H1 receptor antagonist loratadine blocked the inhibitory effect of activated BMMC, activated BMMC culture supernatant, or histamine on T_reg cell function. Furthermore, the H1 receptor agonist 2-PEA mimicked histamine-mediated inhibition of T_reg cell function. In contrast, H2 receptor antagonism with famotidine did not substantially block activated BMMC, activated BMMC culture supernatant, or histamine-mediated inhibition of T_reg cell function, and the H2 receptor agonist ADHB had little effect on T_reg cell function. Although both CD4⁺CD25⁺ T_reg cells and CD4⁺CD25⁻ T_resp cells expressed H1 receptors, histamine acted on T_reg cells rather than on T_resp cells to render them refractory to T_reg cell-mediated suppression because the proliferation of T_resp cells that were pretreated with histamine and then washed was potently suppressed by untreated T_reg cells, whereas T_reg cells that were pretreated with histamine and then washed did not inhibit the proliferation of untreated T_resp cells.

T_reg cells are dependent on IL-2 production by nonregulatory T cells because T_reg cell-expressed Foxp3 interacts with and prevents NFAT from binding to the IL-2 promoter (28). Indeed, IL-2 is critical for the activation and function of CD4⁺CD25⁺ T_reg cells (12, 26). It has been suggested that constitutive high-level expression of CD25 may allow T_reg cells to inhibit the proliferation of T_resp cells by outcompeting T_resp cells for available IL-2 (27). Decreased CD25 expression by histamine-treated T_reg cells might therefore impact negatively on their ability to sequester T_resp cell-secreted IL-2. In line with this, IL-2 in culture supernatant was markedly decreased when T_resp cells were activated in the presence of T_reg cells but was restored to control levels following the addition of histamine to T_resp cell-T_reg cell cocultures. However, alternative interpretations for reduced IL-2 levels in the presence of CD4⁺CD25⁺ T_reg cells must be considered since CD4⁺CD25⁺ T_reg cells also suppress IL-2 mRNA synthesis by CD4⁺CD25⁻ T_resp cells (29), as well as interfering with IL-2R signal transduction (30). In this regard, it is noteworthy that the suppressor function of T_reg cells is programmed by the Foxp3 transcription factor (5, 6), which also controls constitutive expression of CD25 by T_reg cells (6, 31, 32). Moreover, Foxp3 levels correlate with the suppressor capacity of CD4⁺CD25⁺ T_reg cells (33). The virtual ablation of Foxp3 expression by T_reg cells in the presence of histamine might therefore account for the BMMC-mediated reduction in T_reg cell suppressor function. Interestingly, IL-2 is important for the maintenance of Foxp3 expression by T_reg cells (34). It follows then that reduced CD25 expression by T_reg cells in the presence of histamine may compromise their ability to use IL-2, thereby depriving T_reg cells of signals needed for the maintenance of Foxp3 expression. However, it is important to note that CD25-deficient T_reg cells that express IL-2R β- and γ-chains remain responsive to IL-2 (35). Moreover, IL-7 and IL-15 are sufficient to maintain Foxp3 expression by T_reg cells in the absence of IL-2 (36). Because CD25 expression is not always necessary for the induction of T_reg cell suppressor function, it is possible that histamine inhibited CD4⁺CD25⁺ T_reg cell function by direct suppression of Foxp3 expression rather than by down-regulating constitutive T_reg cell expression of CD25 with an attendant decrease in Foxp3. Interestingly, the inhibitory effect of histamine on CD25 and Foxp3 expression by CD4⁺CD25⁺ T_reg cells was not reversed when histamine was removed, which was consistent with the loss of suppressor function by T_reg cells that were pretreated with histamine and then washed before addition to culture.

A number of mast cell-derived mediators are known to regulate the activity of effector T cells. For instance, mast cell-produced TNF-α up-regulates vascular cell adhesion molecule expression by mouse endothelial cells, thereby promoting T cell accumulation in the inflamed footpads of mice treated with the mast cell-activating compound 48/80 (37). In addition, the proliferation of murine CD4⁺ T cells that are suboptimally activated with anti-CD3 mAb in the absence of CD28 costimulation is enhanced in the presence of FcεRI-activated BMMC (38). On the basis of our findings, it is conceivable that BMMC-mediated inhibition of T_reg cell suppressor function contributed to enhanced CD4⁺ T cell proliferation in the absence of costimulation because the authors did not remove CD4⁺CD25⁺ T_reg cells from their responder population. Histamine signaling through the H1 receptor is reported to enhance proliferative responses and IFN-γ production by CD4⁺ Th1 cells (39); however, increased CD4⁺ T cell proliferation and cytokine production may have been caused by histamine H1 receptor-mediated inhibition of endogenous CD4⁺CD25⁺ T_reg cells contained within the CD4⁺ responder T cell population. These observations underscore the need to consider the possible effect(s) of endogenous CD4⁺CD25⁺ T_reg cells when using CD4⁺ T cells that are heterogeneous in terms of their CD25 expression as a readout system.

Although T_reg cells constitute an important mechanism for regulating immune responses to both self- and foreign Ags (1, 2), optimal development of protective immune responses against invading microorganisms requires transient modulation of T_reg cell activity. For example, TLR2 and TLR8 ligands directly inhibit T_reg cell suppressor activity (40, 41), whereas signaling via TLR9 renders rat CD4⁺CD25⁻ T_resp cells refractory to T_reg cell-mediated suppression (42). In addition, dendritic cell secretion of the proinflammatory cytokine IL-6 partially blocks T_reg cell suppressor function (43). Our findings suggest a possible role for mast cells in regulating the immunosuppressive activity of endogenous CD4⁺CD25⁺ T_reg cells. We suggest that at an early stage of infection-induced inflammation mast cells are activated and degranulate in response to microbial products. In this regard, streptococcal exotoxin B stimulates human mast cells to release histamine (44), whereas FimH from Escherichia coli is a potent stimulator of mouse mast cell degranulation (45). Histamine signaling through the H1 receptor leads to a transient reduction in the suppressor function of endogenous CD4⁺CD25⁺ T_reg cells, which allows for optimal activation of effector CD4⁺ and CD8⁺ T cells. As the infectious agent is cleared, mast cell stimulation by microbial products is reduced, and local histamine levels decline. At this point, T_reg cell suppressor function is restored, and the immune response is down-regulated to minimize bystander damage to healthy tissues. Interestingly, intratracheal administration of the H4 receptor agonist 4-methylhistamine is associated with the accumulation of Foxp3⁺ T cells in the lung in a mouse model of airway hypersensitivity (46), suggesting that histamine may also act via H4 receptors to recruit T_reg cells to resolve acute inflammation.

The authors have no financial conflict of interest.