Abstract
Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (Treg) cell function. In this study, we show for the first time that the capacity of murine CD4+CD25+ Treg cells to suppress in vitro proliferation by CD4+CD25− T responder (Tresp) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcεR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited Treg cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored Treg cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of Treg cells with loratadine, but not famotidine, rescued Treg cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited Treg cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both Treg cells and Tresp cells expressed H1 receptors. Exposure to histamine caused Treg cells to express lower levels of CD25 and the Treg cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited Treg cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in Treg cell suppressor function, thereby enhancing the development of protective immunity.
In recent years, the importance of several different T regulatory (Treg)3 cell subsets in the establishment and maintenance of immune tolerance have become increasingly apparent. Perhaps the best studied of these are “natural” Treg 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+ Treg cells constitutively express CD25 (IL-2R α-chain) (3) and the Foxp3 transcription factor, which plays a critical role in Treg cell development and effector function (4, 5, 6). Endogenous Treg 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). Treg cells also express the ectonucleotidases CD39 and CD73, which allows Treg cells to generate adenosine and thereby mediate immune suppression (11). Although Treg cell suppressor function appears to be strictly contact-dependent in vitro, immunosuppressive cytokines such as IL-10 and TGF-β contribute to Treg suppressor activity in vivo (12). The ability of Treg 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 Treg 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 Treg cells, it seems likely that mast cells and Treg cells might affect each others’ function. However, only limited information is available on the interactions that take place between mast cells and Treg cells. In a mouse model of sepsis, adoptive transfer of Treg 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 Treg cells is essential for the establishment of tolerance to alloantigens (16). In addition, a recent study (17) showed that Treg 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 Treg cells possess the capacity to recruit mast cells and regulate their activation, the effect(s) that mast cells have on Treg 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+ Treg cells by signaling through the H1 receptor. In addition, the histamine-induced decrease in Treg cell suppressor function was associated with reduced expression of CD25 and the Treg 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 Treg cell activity.
Materials and Methods
Mice
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.
Isolation and culture of T cells
CD4+CD25+ Treg cells were isolated from mouse spleen cell preparations by negative selection for CD4+ cells and positive selection for CD25+ cells using a MACS mouse Treg cell isolation kit (Miltenyi Biotec). The CD4+CD25− T cell population was retained for use as T responder (Tresp) 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+ Treg cell fraction and CD4+CD25− Tresp 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 differentiation and IgE priming
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).
T cell proliferation assays
CD4+CD25− Tresp cells (1 × 105) and/or CD4+CD25+ Treg 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− Tresp 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+ Treg cells and/or BMMC. After 72 h, quadruplicate cultures were pooled, and Tresp cell proliferation was analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).
IL-2 assay
CD4+CD25− Tresp cells (1 × 105) and/or CD4+CD25+ Treg 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).
Flow cytometry
CD4+CD25+ Treg 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.
Real-time quantitative RT-PCR
Total RNA was extracted from equal numbers of Tresp cells and Treg 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.
Western blotting
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.
Statistical analysis
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.
Results
BMMC inhibit CD4+CD25+ Treg cell suppressor function
To determine the capacity of mast cells to modulate the function of CD4+CD25+ Treg cells, CD4+CD25− Tresp cells were stimulated with anti-CD3/anti-CD28 mAb-coated beads alone or in combination with CD4+CD25+ Treg cells (5:1 ratio) and/or anti-TNP IgE-primed BMMC and TNP-BSA. As expected, Treg cells inhibited Tresp cell proliferation (p < 0.001), as determined by [3H]TdR incorporation (Fig. 1,A). TNP-activated BMMC did not affect Tresp cell proliferation. Importantly, there was a marked and significant (p < 0.001) reduction in Treg cell-mediated suppression of Tresp cell proliferation in the presence of TNP-activated BMMC. Neither Tresp cell proliferation nor Treg cell-mediated suppression of Tresp 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 Tresp cell proliferation and were not caused by increased proliferation of Treg cells and/or BMMC. Tresp cells were therefore labeled with Oregon Green 488 dye, which allowed us to specifically measure Tresp cell division by flow cytometry. As shown in Fig. 1 B, anti-CD3/anti-CD28 mAb-coated bead-stimulated Tresp cells that were cocultured with Treg cells showed a smaller percentage of proliferating Tresp cells and fewer rounds of cell division over a 72-h period in comparison to Tresp cells activated in the absence of Treg cells. Furthermore, the addition of anti-TNP IgE-primed BMMC and TNP-BSA to Treg cell-Tresp cell cocultures restored Tresp cell proliferation to levels that were equivalent to that seen when Tresp cells were activated in the absence of Treg cells. TNP-activated BMMC had no effect on Tresp cell division in the absence of Treg cells. Unactivated BMMC did not affect Tresp cell division in the absence or presence of Treg cells (data not shown). Taken together, these data show that activated BMMC down-regulated the suppressor function of CD4+CD25+ Treg cells.
Inhibition of CD4+CD25+ Treg cell suppressor function by activated BMMC. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, anti-TNP-IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were also added to the cultures. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Oregon Green 488-labeled CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with unlabeled CD4+CD25+ Treg cells (2 × 104). Where indicated, anti-TNP IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were added to the cultures. After 72 h of incubation, Tresp cell proliferation was determined by flow cytometry. Data are from a representative experiment. Cumulative data from three independent experiments are shown as percent dividing Tresp cells ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Inhibition of CD4+CD25+ Treg cell suppressor function by activated BMMC. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, anti-TNP-IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were also added to the cultures. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Oregon Green 488-labeled CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with unlabeled CD4+CD25+ Treg cells (2 × 104). Where indicated, anti-TNP IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were added to the cultures. After 72 h of incubation, Tresp cell proliferation was determined by flow cytometry. Data are from a representative experiment. Cumulative data from three independent experiments are shown as percent dividing Tresp cells ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Supernatant from activated BMMC cultures abrogates Treg cell suppressor function
To determine whether the inhibitory effect of mast cells on CD4+CD25+ Treg cell function was due to a soluble factor, we next examined the effect of activated BMMC culture supernatant on CD4+CD25+ Treg cell function. Cell-free culture supernatants were harvested after exposing anti-TNP IgE-primed BMMC to TNP-BSA for 24 h. CD4+CD25− Tresp cells were then stimulated with anti-CD3/anti-CD28 mAb-coated beads either alone or in combination with CD4+CD25+ Treg cells (5:1 ratio) in the absence or presence of supernatant from cultures of activated BMMC. As shown in Fig. 2,A, Treg 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 Tresp cell proliferation. Similarly, the addition of activated BMMC culture supernatant to cocultures of Oregon Green 488 dye-labeled Tresp cells and unlabeled Treg cells resulted in reduced Treg cell-mediated suppression of Tresp cell division (Fig. 2 B). Collectively, these data demonstrate that the inhibitory effect of BMMC on Treg cell function was mediated by a BMMC-derived soluble factor.
Inhibition of CD4+CD25+ Treg cell suppressor function by activated BMMC culture supernatant. A, Anti-TNP IgE-primed BMMC were cultured for 24 h in the presence of TNP-BSA (10 ng/ml), after which cell-free culture supernatants were harvested. CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, activated BMMC culture supernatant was added to the cultures at a concentration that was reflective of 2 × 104 BMMC. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Oregon Green 488-labeled CD4+CD25+ Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, activated BMMC culture supernatant was added to the cultures at a concentration that was reflective of 2 × 104 BMMC. After 72 h of incubation, Tresp cell proliferation was determined by flow cytometry. Data are from a representative experiment. Cumulative data from three independent experiments are shown as percent dividing Tresp cells ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Inhibition of CD4+CD25+ Treg cell suppressor function by activated BMMC culture supernatant. A, Anti-TNP IgE-primed BMMC were cultured for 24 h in the presence of TNP-BSA (10 ng/ml), after which cell-free culture supernatants were harvested. CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, activated BMMC culture supernatant was added to the cultures at a concentration that was reflective of 2 × 104 BMMC. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Oregon Green 488-labeled CD4+CD25+ Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104). Where indicated, activated BMMC culture supernatant was added to the cultures at a concentration that was reflective of 2 × 104 BMMC. After 72 h of incubation, Tresp cell proliferation was determined by flow cytometry. Data are from a representative experiment. Cumulative data from three independent experiments are shown as percent dividing Tresp cells ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Histamine inhibits CD4+CD25+ Treg cell suppressor function
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+ Treg cell suppressor function, CD4+CD25− Tresp 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+ Treg cells (5:1 ratio). As shown in Fig. 3,A, Treg cell-mediated suppression of Tresp cell proliferation decreased with increasing doses of histamine, whereas histamine had no effect on Tresp cell proliferation in the absence of Treg cells. Histamine (10 μM) did not affect the proliferation or viability of Treg cells (data not shown). Since histamine could interfere with Treg cell suppressor function either by directly inhibiting the Treg cells or by rendering Tresp cells refractory to Treg cell-mediated suppression, we pretreated Tresp cells or Treg cells with histamine (10 μM) for 30 min and then washed extensively before assaying cell proliferation by [3H]TdR incorporation. Tresp 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+ Treg cells (Fig. 3,B). In contrast, Treg cells that were pretreated with histamine failed to inhibit the proliferation of untreated or histamine-pretreated Tresp cells. These data indicate that histamine directly inhibited Treg cell suppressor function rather than rendering Tresp cells refractory to Treg cell-mediated suppression. We also investigated the effect of histamine on Treg cell-mediated inhibition of IL-2 production by anti-CD3/anti-CD28 mAb-coated bead-stimulated Tresp cells. Although histamine had no effect on IL-2 production by Tresp cells in the absence of Treg cells, Treg cell-mediated suppression of IL-2 production by Tresp cells was abrogated in the presence of histamine (p < 0.001; Fig. 3 C).
Inhibition of Treg cell suppressor function by histamine. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the indicated doses of histamine. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, CD4+CD25− Tresp cells (1 × 105) were exposed to medium or 10 μM histamine for 30 min, washed, and then stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with untreated CD4+CD25+ Treg cells or CD4+CD25+ Treg cells (both at 2 × 104) that were pretreated with 10 μM histamine for 30 min and then washed. Exogenous histamine (10 μM) was also added to some cultures. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. C, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or 10 μM histamine. After 24 h of incubation, cell-free culture supernatants were harvested, and IL-2 levels were determined by ELISA. Data from an experiment that is representative of three independent experiments are shown as mean pg/ml ± SD of quadruplicate samples. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Inhibition of Treg cell suppressor function by histamine. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the indicated doses of histamine. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, CD4+CD25− Tresp cells (1 × 105) were exposed to medium or 10 μM histamine for 30 min, washed, and then stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with untreated CD4+CD25+ Treg cells or CD4+CD25+ Treg cells (both at 2 × 104) that were pretreated with 10 μM histamine for 30 min and then washed. Exogenous histamine (10 μM) was also added to some cultures. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. C, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or 10 μM histamine. After 24 h of incubation, cell-free culture supernatants were harvested, and IL-2 levels were determined by ELISA. Data from an experiment that is representative of three independent experiments are shown as mean pg/ml ± SD of quadruplicate samples. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
H1 receptor antagonism prevents BMMC-mediated inhibition of Treg cell suppressor function
To determine whether histamine was responsible for BMMC-mediated inhibition of CD4+CD25+ Treg cell suppressor function, we treated cocultures of CD4+CD25− Tresp cells, CD4+CD25+ Treg 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 Treg 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 Tresp cell proliferation in the absence of Treg cells. Similarly, treatment with loratadine, but not famotidine, significantly (p < 0.001) reduced the inhibitory effect of activated BMMC culture supernatant on Treg cell suppressor function (Fig. 4 B). Taken together, these data suggest that histamine was responsible for the inhibitory effect of activated BMMC on Treg cell suppressor function and that BMMC-derived histamine acts on Treg cells via the H1 receptor.
Rescue of Treg cell suppressor function by the H1 receptor antagonist loratidine in the presence of activated BMMC or activated BMMC culture supernatant. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the H1 receptor antagonist loratidine (10 μM) or the H2 receptor antagonist famotidine (10 μM). Where indicated, anti-TNP IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were also added to the cultures 30 min later. Cultures were incubated for 48 h and pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Anti-TNP IgE-primed BMMC were cultured for 24 h in the presence of TNP-BSA (10 ng/ml), at which point cell-free culture supernatant was harvested. CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 Ab-coated beads (5 × 104) alone or in coculture with CD4+CD25+ Treg cells and/or loratadine or famotidine. Where indicated, activated BMMC culture supernatant was added 30 min later to the cocultures at a concentration that was reflective of 2 × 104 BMMC. Cultures were incubated for 48 h and pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Rescue of Treg cell suppressor function by the H1 receptor antagonist loratidine in the presence of activated BMMC or activated BMMC culture supernatant. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the H1 receptor antagonist loratidine (10 μM) or the H2 receptor antagonist famotidine (10 μM). Where indicated, anti-TNP IgE-primed BMMC (2 × 104) and TNP-BSA (10 ng/ml) were also added to the cultures 30 min later. Cultures were incubated for 48 h and pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, Anti-TNP IgE-primed BMMC were cultured for 24 h in the presence of TNP-BSA (10 ng/ml), at which point cell-free culture supernatant was harvested. CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 Ab-coated beads (5 × 104) alone or in coculture with CD4+CD25+ Treg cells and/or loratadine or famotidine. Where indicated, activated BMMC culture supernatant was added 30 min later to the cocultures at a concentration that was reflective of 2 × 104 BMMC. Cultures were incubated for 48 h and pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
To confirm that histamine inhibited Treg 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 Treg 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 Treg 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 Treg cell-mediated inhibition of Tresp cell proliferation in response to anti-CD3/anti-CD28 mAb-coated bead stimulation. Exposure to increasing doses of 2-PEA to Treg cell-Tresp cell cocultures resulted in a dose-dependent decrease in Treg cell-mediated suppression, whereas ADHB, even at the highest dose used, had little effect on Treg cell function (Fig. 5 B). Collectively, these data indicate that the inhibitory effect of histamine on Treg cell suppressor function was mediated exclusively through H1 receptors.
Histamine signals through H1 receptors to inhibit Treg cell suppressor function. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or 10 μM histamine. The indicated doses of the H1 receptor antagonist loratadine or the H2 receptor antagonist famotidine were added 30 min before histamine treatment. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the indicated doses of histamine, the H1 receptor agonist 2-PEA, or the H2 receptor agonist ADHB. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Histamine signals through H1 receptors to inhibit Treg cell suppressor function. A, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or 10 μM histamine. The indicated doses of the H1 receptor antagonist loratadine or the H2 receptor antagonist famotidine were added 30 min before histamine treatment. After 48 h of incubation, cultures were pulsed with [3H]TdR for the final 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. B, CD4+CD25− Tresp cells (1 × 105) were stimulated with anti-CD3/anti-CD28 mAb-coated beads (5 × 104) alone or in combination with CD4+CD25+ Treg cells (2 × 104) and/or the indicated doses of histamine, the H1 receptor agonist 2-PEA, or the H2 receptor agonist ADHB. After 48 h of incubation, cultures were pulsed with [3H]TdR for the last 6 h of culture. Data from an experiment that is representative of three independent experiments are shown as mean cpm ± SD of quadruplicate cultures. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
H1 receptor expression by Treg cells and Tresp cells
Although mouse T cells are known to express H1 receptors (24), H1 receptor expression by CD4+CD25+ Treg cells has not yet been examined and compared with CD4+CD25− Tresp cells. Real-time quantitative RT-PCR showed that H1 receptor mRNA expression by Tresp cells exceeded that of Treg cells by ∼2-fold (p < 0.01; Fig. 6,A); however, similar levels of H1 receptor protein were expressed by Tresp cells and Treg cells (p > 0.05; Fig. 6 B).
Treg and Tresp cells express H1 receptor mRNA and protein. A, mRNA was extracted from equal numbers of CD4+CD25− Tresp cells and CD4+CD25+ Treg cells. H1 receptor expression relative to GAPDH was measured by quantitative real-time RT-PCR (lower panel) and visualized by ethidium bromide staining of the PCR product (upper panel). B, Cell lysates were prepared from 2.5 × 106 CD4+CD25− Tresp cells or an equal number of CD4+CD25+ Treg cells. Western blot analysis was performed to determine levels of H1 receptor protein (56 kDa; upper panel). Actin (42 kDa) expression was determined to confirm equal protein loading. Expression of H1 receptor relative to actin was quantified by densitometric analysis and expressed in arbitrary units (lower panel). Data represent the mean of three separate experiments ± SEM. Statistical significance was determined by Student’s t test.
Treg and Tresp cells express H1 receptor mRNA and protein. A, mRNA was extracted from equal numbers of CD4+CD25− Tresp cells and CD4+CD25+ Treg cells. H1 receptor expression relative to GAPDH was measured by quantitative real-time RT-PCR (lower panel) and visualized by ethidium bromide staining of the PCR product (upper panel). B, Cell lysates were prepared from 2.5 × 106 CD4+CD25− Tresp cells or an equal number of CD4+CD25+ Treg cells. Western blot analysis was performed to determine levels of H1 receptor protein (56 kDa; upper panel). Actin (42 kDa) expression was determined to confirm equal protein loading. Expression of H1 receptor relative to actin was quantified by densitometric analysis and expressed in arbitrary units (lower panel). Data represent the mean of three separate experiments ± SEM. Statistical significance was determined by Student’s t test.
Histamine down-regulates CD25 and Foxp3 expression by Treg cells
CD4+CD25+ Treg 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 Treg cells (25, 26, 27). We therefore examined the effect of histamine on CD25 expression by Treg cells. After 24 h of culture in the absence or presence of 10 μM histamine, Treg cells were stained for CD25 and analyzed by flow cytometry. In comparison to untreated Treg cells, histamine-treated Treg 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 Treg cell expression of the forkhead box/winged helix transcription factor Foxp3, which is important for Treg cell development and programming Treg 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 Treg cells (MFI = 20 ± 9 vs 62 ± 8, p < 0.01). Treg cell expression of CD25 and Foxp3 was therefore down-regulated by histamine. The inhibitory effect of histamine on CD25 and Foxp3 expression by Treg cells was not evident at earlier time points (2 or 10 h), nor was it transient since washing out histamine from Treg cell cultures after 30 min of exposure did not prevent CD25 and Foxp3 down-regulation at the 24-h time point (data not shown).
Histamine inhibits Treg cell expression of CD25 and Foxp3. A and B, CD4+CD25+ Treg cells were cultured for 24 h in the absence or presence of 10 μM histamine. Percent CD4+CD25+ Treg cell viability at the end of 24 h was 24 ± 8 and 23 ± 8 in the absence or presence of histamine, respectively. Treg cells were then stained with FITC-conjugated anti-CD25 mAb or FITC-conjugated isotype control Ab and CD25 expression (open peak) in comparison to background fluorescence (filled peak) was analyzed by flow cytometry (A). Alternatively, CD4+CD25+ Treg cells were fixed, permeabilized, and stained with FITC-conjugated anti-Foxp3 mAb or FITC-conjugated isotype control Ab and Foxp3 expression (open peak) in comparison to background fluorescence (filled peak) was analyzed by flow cytometry (B). Flow cytometry histograms are from a representative experiment. Cumulative data from three independent experiments are shown as MFI ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Histamine inhibits Treg cell expression of CD25 and Foxp3. A and B, CD4+CD25+ Treg cells were cultured for 24 h in the absence or presence of 10 μM histamine. Percent CD4+CD25+ Treg cell viability at the end of 24 h was 24 ± 8 and 23 ± 8 in the absence or presence of histamine, respectively. Treg cells were then stained with FITC-conjugated anti-CD25 mAb or FITC-conjugated isotype control Ab and CD25 expression (open peak) in comparison to background fluorescence (filled peak) was analyzed by flow cytometry (A). Alternatively, CD4+CD25+ Treg cells were fixed, permeabilized, and stained with FITC-conjugated anti-Foxp3 mAb or FITC-conjugated isotype control Ab and Foxp3 expression (open peak) in comparison to background fluorescence (filled peak) was analyzed by flow cytometry (B). Flow cytometry histograms are from a representative experiment. Cumulative data from three independent experiments are shown as MFI ± SEM. Statistical significance was determined by ANOVA with the Tukey-Kramer multiple comparisons posttest.
Discussion
There has been a paucity of information to date regarding the effect that mast cells have on Treg cell function. We show here, for the first time, that histamine released by FcεR-activated BMMC abrogated the suppressor function of CD4+CD25+ Treg cells. Activated BMMC potently inhibited the suppressor function of CD4+CD25+ Treg 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 Treg cell function. Furthermore, the H1 receptor agonist 2-PEA mimicked histamine-mediated inhibition of Treg cell function. In contrast, H2 receptor antagonism with famotidine did not substantially block activated BMMC, activated BMMC culture supernatant, or histamine-mediated inhibition of Treg cell function, and the H2 receptor agonist ADHB had little effect on Treg cell function. Although both CD4+CD25+ Treg cells and CD4+CD25− Tresp cells expressed H1 receptors, histamine acted on Treg cells rather than on Tresp cells to render them refractory to Treg cell-mediated suppression because the proliferation of Tresp cells that were pretreated with histamine and then washed was potently suppressed by untreated Treg cells, whereas Treg cells that were pretreated with histamine and then washed did not inhibit the proliferation of untreated Tresp cells.
Treg cells are dependent on IL-2 production by nonregulatory T cells because Treg 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+ Treg cells (12, 26). It has been suggested that constitutive high-level expression of CD25 may allow Treg cells to inhibit the proliferation of Tresp cells by outcompeting Tresp cells for available IL-2 (27). Decreased CD25 expression by histamine-treated Treg cells might therefore impact negatively on their ability to sequester Tresp cell-secreted IL-2. In line with this, IL-2 in culture supernatant was markedly decreased when Tresp cells were activated in the presence of Treg cells but was restored to control levels following the addition of histamine to Tresp cell-Treg cell cocultures. However, alternative interpretations for reduced IL-2 levels in the presence of CD4+CD25+ Treg cells must be considered since CD4+CD25+ Treg cells also suppress IL-2 mRNA synthesis by CD4+CD25− Tresp cells (29), as well as interfering with IL-2R signal transduction (30). In this regard, it is noteworthy that the suppressor function of Treg cells is programmed by the Foxp3 transcription factor (5, 6), which also controls constitutive expression of CD25 by Treg cells (6, 31, 32). Moreover, Foxp3 levels correlate with the suppressor capacity of CD4+CD25+ Treg cells (33). The virtual ablation of Foxp3 expression by Treg cells in the presence of histamine might therefore account for the BMMC-mediated reduction in Treg cell suppressor function. Interestingly, IL-2 is important for the maintenance of Foxp3 expression by Treg cells (34). It follows then that reduced CD25 expression by Treg cells in the presence of histamine may compromise their ability to use IL-2, thereby depriving Treg cells of signals needed for the maintenance of Foxp3 expression. However, it is important to note that CD25-deficient Treg 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 Treg cells in the absence of IL-2 (36). Because CD25 expression is not always necessary for the induction of Treg cell suppressor function, it is possible that histamine inhibited CD4+CD25+ Treg cell function by direct suppression of Foxp3 expression rather than by down-regulating constitutive Treg cell expression of CD25 with an attendant decrease in Foxp3. Interestingly, the inhibitory effect of histamine on CD25 and Foxp3 expression by CD4+CD25+ Treg cells was not reversed when histamine was removed, which was consistent with the loss of suppressor function by Treg 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 Treg cell suppressor function contributed to enhanced CD4+ T cell proliferation in the absence of costimulation because the authors did not remove CD4+CD25+ Treg 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+ Treg cells contained within the CD4+ responder T cell population. These observations underscore the need to consider the possible effect(s) of endogenous CD4+CD25+ Treg cells when using CD4+ T cells that are heterogeneous in terms of their CD25 expression as a readout system.
Although Treg 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 Treg cell activity. For example, TLR2 and TLR8 ligands directly inhibit Treg cell suppressor activity (40, 41), whereas signaling via TLR9 renders rat CD4+CD25− Tresp cells refractory to Treg cell-mediated suppression (42). In addition, dendritic cell secretion of the proinflammatory cytokine IL-6 partially blocks Treg cell suppressor function (43). Our findings suggest a possible role for mast cells in regulating the immunosuppressive activity of endogenous CD4+CD25+ Treg 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+ Treg 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, Treg 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 Treg cells to resolve acute inflammation.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by an operating grant to D.W.H. from the Natural Sciences and Engineering Research Council (NSERC) and an operating grant to T.-J.L. from the Canadian Institutes of Health Research. N.A.F. was supported by a trainee award from the Cancer Research Training Program with funding from the Dalhousie Cancer Research Program. S.J.F. was supported by an NSERC postgraduate award.
Abbreviations used in this paper: Treg, T regulatory; 2-PEA, 2-pyridylethylamine dihydrochloride; BMMC, bone marrow-derived mast cell; MFI, mean fluorescence intensity; Tresp, T responder; ADHB, amthamine dihydrobromide.