Abstract

While production of cytokines such as IL-12 by activated dendritic cells supports development of Th1 type immunity, a source of early IL-4 that is responsible for Th2 immunity is not well understood. We now show that coculture of basophils could promote a robust Th2 differentiation upon stimulation of naive CD4 T cells primarily via IL-4. Th2 promotion by basophils was also observed even when naive CD4 T cells were stimulated in a Th1-promoting condition or when fully differentiated Th1 phenotype effector CD4 T cells were restimulated. IL-4–deficient basophils failed to induce Th2 differentiation but suppressed Th1 differentiation. It was subsequently revealed that the IL-4–deficient basophils must engage cell-to-cell contact to exert the inhibitory effect on Th1 differentiation. Stimulation of naive CD4 T cells within an in vivo environment of increased basophil generation supported development of Th2 type immunity. Taken together, our results suggest that basophils may provide an important link for the development of Th2 immunity.

Introduction

The cytokine environment plays a key role in determining processes of naive CD4 T-cell differentiation.1  IL-12, primarily produced by activated antigen-presenting cells, promotes development of Th1 type effector CD4 T cells. On the other hand, IL-4 is important for Th2 differentiation of naive CD4 T cells to occur. Many efforts to identify the cell types that produce initial IL-4 have been made in various experimental settings. Naive CD4 T cells,2  mast cells,3  eosinophils,4  basophils,5  and NKT cells6  were shown to produce IL-4 in certain circumstances. However, their contributions for the development of Th2 type immune responses have not been formally tested.

Basophils are the least common granulocytes found in the circulation. Numbers of basophils substantially increase in certain circumstances, including intestinal parasite Nippostrongylus brasiliensis (Nb) infection,5  allergic pulmonary inflammation,7  and chronic skin allergic inflammation,8  conditions that are highly associated with Th2 type immunity. Immunomodulatory roles of basophils have been also proposed. Basophils from healthy individuals produced IL-4 upon stimulation of Schistosoma egg antigen, suggesting that basophils may represent early sources of the IL-4 needed for subsequent development of the Schistosoma-specific Th2 type immune responses.9  Basophils were shown to induce IgE synthesis by B cells in vitro in an IL-4–dependent, CD40L-dependent manner.10,11  It was recently demonstrated that basophils acquire capacity to constitutively express IL-4 transcripts during lineage differentiation.12  Magnitudes of the IL-4 production from basophils have been shown to be greater than that of Th2 type CD4 T cells.13  Taken together, basophils seem to have a great potential to participate and modulate development of Th2 type immune responses. Yet, animal models to test the importance of basophils in the context of Th2 type immunity have not been established.

Here we report that stimulation of naive CD4 T cells in the presence of basophils is sufficient to promote the development of IL-4–producing Th2 type effector CD4 T cells without exogenous IL-4. Basophil-mediated Th2 differentiation effect was primarily mediated by the IL-4 produced by basophils, as the Th2 differentiation was not found when IL-4–deficient basophils were used instead. Interestingly, IL-4–deficient basophils were still capable of inhibiting differentiation of naive CD4 T cells into Th1 phenotype cells. Separating the IL-4–deficient basophils by a Transwell experiment abrogated such inhibitory effect, suggesting that cell-to-cell contact appears to be necessary for the IL-4–independent suppression of Th1 differentiation. Finally, naive CD4 T cells stimulated in vivo within basophil-enriched environments preferentially differentiated into Th2 type cells. Taken together, our results demonstrate that basophils may have a novel function to enhance development of Th2 type immunity as well as to suppress Th1 type immunity by secreting IL-4 and/or engaging cell-to-cell contact.

Materials and methods

Mice

5C.C7 TCR Tg Rag2−/− mice express T-cell receptor specific for pigeon cytochrome C (PCC) peptide (amino acid residues 81 to 104 of PCC) in the context of I-Ek. OT-II TCR Tg mice express T-cell receptor specific for ovalbumin (OVA) peptide (amino acid residues 323 to 339 of OVA) in the context of I-Ab. 5C.C7 TCR Tg, OT-II TCR Tg, B10.A Rag2−/−, and C57BL/6 G4/G4 mice14  were obtained from Dr William E. Paul (National Institutes of Health). C57BL/6 mice were purchased from Jackson (Bar Harbor, ME). All experimental procedures were conducted according to the guidelines of the Cleveland Clinic Foundation institutional animal care committee.

Isolation of basophils

Mice were subcutaneously implanted with a miniosmotic pump (Alzet; Durect, Cupertino, CA) containing 5 μg IL-3 (Peprotech, Rocky Hill, NJ). At day 7 after implantation, bone marrow cells and liver mononuclear cells were harvested as previously reported.5  Following incubation with FcγR blocking antibody (2.4G2), cells were then labeled with anti-FcϵRI–FITC (MAR-1; eBioscience, San Diego, CA) and incubated with anti-FITC microbeads (Miltenyi Biotec, Auburn, CA). FcϵRI-expressing basophils were enriched by positive magnetic separation (Miltenyi Biotec) and then subsequently purified by cell sorting using a FACSVantage SE (Becton Dickinson, San Jose, CA). Purity after sorting was more than 95%. In some experiments, bone marrow cells were cultured with IL-3, and bone marrow-derived basophils and mast cells were isolated by cell sorting.

Purification of CD4 cells and dendritic cells

Lymph node CD4 T cells were purified as previously reported.5  In brief, single-cell suspension was incubated with FITC-conjugated anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-NK1.1 (PK136), anti-HSA (M1/69), anti–I-Ak/I-Ab (11-5.2 or AF6-120.1), and anti-CD16 (24G2) monoclonal antibody (mAb), followed by anti-FITC microbeads. All antibodies were purchased from BD PharMingen (San Diego, CA). Magnetic separation was performed using an LS magnetic column (Miltenyi Biotec). Negative fraction was collected. Purity of the CD4 T cells was usually more than 95%. In some experiments, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Molecular Probe, Eugene, OR) to track cell division. For isolation of splenic dendritic cells, spleen was incubated with 1 mg/mL collagenase D (Invitrogen, Carlsbad, CA) and 0.4 mg/mL DNase I (Roche, Indianapolis, IN) at 37°C for 25 minutes, followed by centrifugation using Nycodenz (Sigma, St Louis, MO) gradient. Low-density cells were collected and incubated with anti-CD11c (N418) microbeads (Miltenyi Biotec). Magnetic separation was performed using an MS column. Purity of the CD11c+ dendritic cells was usually more than 90%.

In vitro stimulation of CD4 T cells

Purified 5C.C7 CD4 cells (2 × 105) were stimulated with 1 μM PCC peptide (a kind gift from Dr William E. Paul) and splenic dendritic cells (4 × 104) in the presence of different numbers of basophils for 72 hours. Cells were harvested and restimulated on immobilized anti-CD3/CD28 antibodies for intracellular cytokine staining. Th1 type CD4 T cells were generated by stimulating CD4 T cells with the peptide antigen in the presence of 10 ng/mL IL-12 (Peprotech) plus 10 μg/mL anti–IL-4 antibody (11B11).

CD4 T-cell activation in vivo

Purified 5C.C7 CD4 T cells (2 × 106) were intravenously transferred into B10.A Rag−/− mice. The next day, mice were intravenously administered with 50 μg PCC protein (Sigma). In some experiments, mice were pretreated with 5 μg IL-3 by a miniosmotic pump for 7 days prior to T-cell transfer. At day 7 after immunization, cells from lymph nodes, spleen, and liver were harvested and restimulated for cytokine production.

Intracellular cytokine staining

Activated CD4 T cells were harvested and restimulated with platebound anti-CD3 (3 μg/mL) and anti-CD28 (3 μg/mL) for 6 hours in the presence of 2 μM monensin (Calbiochem, San Diego, CA) during the last 2 hours. Cells were subsequently fixed with 4% paraformaldehyde, permeabilized in buffer (PBS containing 0.1% BSA, 0.5% Triton X), and stained with fluorescein isothiocyanate (FITC)–anti-CD4 (RM4-5), phycoerythrin (PE)–IL-4 (11B11), and allophycocyanin (APC)–IFNγ (XMG1.2). Expression of intracellular cytokines was determined using FACSCalibur (Becton Dickinson) and analyzed by FlowJo software (Treestar, Ashland, OR).

Results

Generation of basophils by in vivo IL-3 administration

Although basophils have been implicated to play immunomodulatory functions in certain circumstances, experimental evidence to support the hypothesis is missing. Because basophils are the least common leukocytes found in the circulation, obtaining sufficient numbers of basophils has been a technical challenge. It was previously shown that in vitro culture of bone marrow cells with IL-3 leads to differentiation of progenitors into basophils.15  However, IL-3 stimulation primarily promotes generation of mast cells (FcϵRI+ c-kit+), and basophils (FcϵRI+ c-kit) generated by IL-3 in vitro are relatively small (Figure 1A). We instead sought an alternative strategy to generate basophils in vivo by administering IL-3 into mice via a miniosmotic pump. IL-3 administration induced a dramatic increase of a population that resembles basophils based on surface phenotypes (Figure 1B). These cells coexpressed high-affinity IgE receptor, FcϵRI, and CD49b (DX5), which are the phenotypes that we and others have recently identified from mice infected with Nb5  and from mice induced for chronic skin allergic inflammation.8  The most dramatic increases were found in the bone marrow, indicating enhanced generation of basophils from bone marrow progenitors by IL-3. Peripheral accumulation of basophils was also found in the liver and to a lesser degree in the spleen. However, we found no basophils in the lymph nodes. Interestingly, we did not observe generation of mast cells by in vivo IL-3 treatment. Phenotypically, FcϵRI+ CD49b+ basophils expressed a high level of Thy1.2 and 2B4 as recently reported (Figure 1C).8  These basophils did not express c-kit (data not shown). Upon stimulation, they produced a great amount of IL-4, IL-13, and histamine (data not shown). Basophils were further isolated by cell sorting and analyzed by Wright-Giemsa staining (Figure 1D). Light microscopic examination showed lobulated ringlike nuclei nuclei with less numerous cytoplasmic granules as previously reported.16  This resembles basophils observed in Nb-infected mice and in mice with chronic skin allergic inflammation.5,8  We isolated basophils by cell sorting and further characterized their functional potentials for the rest of the study.

Figure 1

In vivo generation of basophils from bone marrow progenitors by IL-3 treatment. (A) In vitro generation of bone marrow–derived basophils. Bone marrow cells were cultured with 10 ng/mL of IL-3 for 7 days. At the end of culture, cells were harvested and stained for FcϵRI and c-kit. FcϵRI/c-kit double positive cells represent mast cells, while FcϵRI single positive cells represent basophils. Relative proportions are indicated. (B) In vivo generation of basophils by IL-3. C57BL/6 wild-type mice were implanted with a miniosmotic pump containing 5 μg IL-3. Seven days after pump implantation, cells from bone marrow, liver, spleen, and lymph nodes were harvested and stained for CD49b and FcϵRI. Proportions of CD49b+ FcϵRI+ cells (basophils) are indicated. No basophils were found after PBS pump implantation (data not shown). (C) Phenotypic analysis of IL-3–generated basophils. Basophils obtained by IL-3 pump implantation were analyzed for the expression of Thy1.2 and 2B4 (filled histogram plots), surface molecules known to be expressed on basophils. Open histogram plots represent isotype control antibody staining. (D) Cytospin samples of basophils isolated from indicated tissues by cell sorting were stained for Wright-Giemsa staining. Images were acquired using an Olympus BX51 microscope equipped with a 40×/0.75 numerical aperture objective lens (Olympus, Center Valley, PA). Images were taken using an Olympus DP70 digital camera with DP controller software. The images were subsequently processed with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA).

Figure 1

In vivo generation of basophils from bone marrow progenitors by IL-3 treatment. (A) In vitro generation of bone marrow–derived basophils. Bone marrow cells were cultured with 10 ng/mL of IL-3 for 7 days. At the end of culture, cells were harvested and stained for FcϵRI and c-kit. FcϵRI/c-kit double positive cells represent mast cells, while FcϵRI single positive cells represent basophils. Relative proportions are indicated. (B) In vivo generation of basophils by IL-3. C57BL/6 wild-type mice were implanted with a miniosmotic pump containing 5 μg IL-3. Seven days after pump implantation, cells from bone marrow, liver, spleen, and lymph nodes were harvested and stained for CD49b and FcϵRI. Proportions of CD49b+ FcϵRI+ cells (basophils) are indicated. No basophils were found after PBS pump implantation (data not shown). (C) Phenotypic analysis of IL-3–generated basophils. Basophils obtained by IL-3 pump implantation were analyzed for the expression of Thy1.2 and 2B4 (filled histogram plots), surface molecules known to be expressed on basophils. Open histogram plots represent isotype control antibody staining. (D) Cytospin samples of basophils isolated from indicated tissues by cell sorting were stained for Wright-Giemsa staining. Images were acquired using an Olympus BX51 microscope equipped with a 40×/0.75 numerical aperture objective lens (Olympus, Center Valley, PA). Images were taken using an Olympus DP70 digital camera with DP controller software. The images were subsequently processed with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA).

Basophils support Th2 differentiation upon stimulation of naive CD4 T cells

Basophils are efficient producers of Th2 type cytokines such as IL-4 and IL-13, and the induction of basophil responses has been closely associated with Th2 type immunity. To test if basophils are capable of supporting Th2 type immune responses, we set up in an vitro coculture experiment in which 5C.C7 TCR transgenic CD4 T cells specific for cytochrome C peptide in the context of I-Ek were stimulated with peptide plus splenic CD11c+ dendritic cells in the absence of exogenous cytokines for 3 days. About 40% of the activated 5C.C7 CD4 T cells produced IFNγ and no IL-4 upon restimulation (Figure 2A). Addition of 2 × 104 FACS-sorted basophils in the culture shifted T-cell differentiation patterns. Thus, most of the activated T cells became IL-4–producing Th2 type cells upon restimulation, and their IFNγ production was dramatically reduced (Figure 2A). Activated CD4 T cells did not stain for IL-4 unless they were restimulated on immobilized anti-CD3/CD28 antibodies, indicating that IL-4 positivity is not due to a simple binding of IL-4 made by basophils to surface IL-4 receptors on CD4 T cells (Figure 2A). These CD4 T cells also produced other Th2 type cytokines, such as IL-13 and IL-10 when determined by enzyme-linked immunosorbent assay (ELISA) (data not shown). Coculture of basophils did not alter the T-cell proliferation pattern, as the same degrees of CFSE dilution were observed (Figure 2B).

Figure 2

In vitro stimulation of naive CD4 T cells with basophils promotes induction of Th2 type response. A total of 5 × 105 5C.C7 CD4 T cells specific for PCC peptide were cultured with 1 μM peptide and 5 × 104 splenic CD11c+ dendritic cells in the presence or absence of basophils for 3 days. Cells were restimulated on platebound antibodies for intracellular cytokine expression. (A) A total of 2 × 104 basophils isolated from the liver of IL-3–treated mice in vivo were added in the cultures. Proportions of IL-4– and IFNγ–producing cells are shown. (B) CFSE profiles of CD4 T cells cocultured with or without basophils were determined. (C) Different numbers of basophils (none, 200, 2000, and 20 000) obtained from indicated tissues (liver, spleen, or bone marrow) were added in the coculture. Graphs indicate percentage of cytokine-producing CD4 T cells. The means ± SD of 2 independent experiments are shown. (D) Indicated numbers of in vitro–generated bone marrow–derived basophils and mast cells were cocultured, and cytokine production was determined following restimulation. (E) Naive 5C.C7 CD4 T cells were stimulated described for panel A in the presence of IL-12 with or without 2 × 104 basophils, and cytokine production was determined. (F) Th1 cells generated in vitro (by stimulation with 10 ng/mL IL-12, 10 μg/mL anti–IL-4) were restimulated with antigen in the presence or absence of 2 × 104 basophils. All the experiments were repeated more than 3 times with similar results.

Figure 2

In vitro stimulation of naive CD4 T cells with basophils promotes induction of Th2 type response. A total of 5 × 105 5C.C7 CD4 T cells specific for PCC peptide were cultured with 1 μM peptide and 5 × 104 splenic CD11c+ dendritic cells in the presence or absence of basophils for 3 days. Cells were restimulated on platebound antibodies for intracellular cytokine expression. (A) A total of 2 × 104 basophils isolated from the liver of IL-3–treated mice in vivo were added in the cultures. Proportions of IL-4– and IFNγ–producing cells are shown. (B) CFSE profiles of CD4 T cells cocultured with or without basophils were determined. (C) Different numbers of basophils (none, 200, 2000, and 20 000) obtained from indicated tissues (liver, spleen, or bone marrow) were added in the coculture. Graphs indicate percentage of cytokine-producing CD4 T cells. The means ± SD of 2 independent experiments are shown. (D) Indicated numbers of in vitro–generated bone marrow–derived basophils and mast cells were cocultured, and cytokine production was determined following restimulation. (E) Naive 5C.C7 CD4 T cells were stimulated described for panel A in the presence of IL-12 with or without 2 × 104 basophils, and cytokine production was determined. (F) Th1 cells generated in vitro (by stimulation with 10 ng/mL IL-12, 10 μg/mL anti–IL-4) were restimulated with antigen in the presence or absence of 2 × 104 basophils. All the experiments were repeated more than 3 times with similar results.

Magnitudes of Th2 differentiation were correlated with numbers of the basophils added in the culture (Figure 2C). As more basophils were added in the culture, the proportions of IL-4–producing cells increased and those of IFNγ producing cells simultaneously decreased. Basophils isolated from different tissues (liver, spleen, and bone marrow) of IL-3–treated mice were comparably efficient in promoting Th2 differentiation and suppressing Th1 differentiation (Figure 2C). For most of the experiments, we combined basophils from the bone marrow and the liver and used them for the coculture experiments.

In addition to basophils, mast cells have been implicated to play immunomodulatory roles in the development of Th2 type immunity.3  To directly test the possibility, mast cells and basophils were generated from bone marrow cells cultured with IL-3 as shown in Figure 1A. Mast cells (FcϵRI+ c-kit+) and basophils (FcϵRI+ c-kit) were then separately isolated by cell sorting and used in coculture experiments. As shown in Figure 2D, activated 5C.C7 CD4 T cells in the presence of bone marrow–derived mast cells did not differentiate into Th2 cells, as no IL-4 production was found. In contrast, basophils isolated from the same bone marrow IL-3 culture were capable of promoting Th2 differentiation, although to a lesser degree than those basophils generated in vivo (Figure 2D). These results strongly indicate that the induction of Th2 differentiation by in vitro coculture is a unique feature of basophils but not of mast cells.

Coculture of basophils was able to induce IL-4 production when naive CD4 T cells were stimulated in the presence of Th1-promoting cytokine, IL-12 (Figure 2E). When CD4 T cells were stimulated with IL-12 in the absence of basophils, more than 80% of the activated CD4 T cells produced IFNγ and virtually no cells produced IL-4. The presence of basophils suppressed Th1 differentiation and induced a significant level of IL-4–producing CD4 T cells. Furthermore, differentiated Th1 CD4 T cells restimulated in the presence of basophils also produced IL-4. Unlike naive T cells stimulated with IL-12 (Figure 2E), however, reduction of IFNγ production was not found (Figure 2F).

IL-4 produced by basophils is critical for the basophil-mediated Th2 differentiation

Because IL-4 is one of the primary cytokines produced by basophils, we next tested if basophil-driven IL-4 plays a key role during this process. First, we performed a Transwell experiment with basophils to see if any soluble factors produced by basophils are responsible for the Th2 differentiation. As shown in Figure 3A, separating basophils from the T cells and the DCs did not alter the T-cell differentiation pattern. Coculture of basophils induced about 40% of the CD4 T cells to produce IL-4. About 30% of the CD4 T cells still produced IL-4 when basophils were separated by the Transwell, suggesting that it is more likely mediated by a soluble factor(s) produced by basophils. Second, we generated IL-4–deficient basophils by treating C57BL/6 G4 homozygous (IL-4–deficient) mice with IL-3.14  Basophils were isolated, and a coculture experiment was performed using OT-II CD4 T cells specific for OVA peptide in the context of I-Ab. In vivo generation of G4 basophils by IL-3 was comparable as IL-4–sufficient wild-type mice, suggesting that IL-4 is not necessary during in vivo generation of basophils (data not shown). Coculture of IL-4–deficient G4 basophils failed to induce Th2 differentiation of the activated OT-II CD4 T cells, while wild-type basophils induced a comparable level (about 40%) of Th2 differentiation (Figure 3B). These results indicate that the IL-4 produced by basophils is primarily responsible for the induction of Th2 differentiation as well as suppression of Th1 suppression (Figure 3B).

Figure 3

IL-4 produced by basophils is primarily responsible for basophil-induced Th2 differentiation. (A) Coculture experiments were performed in a Transwell experiment. Naive 5C.C7 CD4 T cells were stimulated with 2 × 104 basophils together or separated from basophils by Transwell. Production of IL-4 and IFNγ was determined. (B) Purified OT-II CD4 T cells specific for OVA peptide in the context of I-Ab were cultured with OVA peptide 323 to 339–loaded splenic CD11c+ dendritic cells in the absence or presence of basophils isolated from wild-type mice or IL-4–deficient G4/G4 mice for 72 hours. Cytokine production was measured. (C) During primary stimulation, 10 μg/mL anti–IL-3 or anti–IL-13 antibodies were added. All the experiments were repeated more than 3 times with similar results.

Figure 3

IL-4 produced by basophils is primarily responsible for basophil-induced Th2 differentiation. (A) Coculture experiments were performed in a Transwell experiment. Naive 5C.C7 CD4 T cells were stimulated with 2 × 104 basophils together or separated from basophils by Transwell. Production of IL-4 and IFNγ was determined. (B) Purified OT-II CD4 T cells specific for OVA peptide in the context of I-Ab were cultured with OVA peptide 323 to 339–loaded splenic CD11c+ dendritic cells in the absence or presence of basophils isolated from wild-type mice or IL-4–deficient G4/G4 mice for 72 hours. Cytokine production was measured. (C) During primary stimulation, 10 μg/mL anti–IL-3 or anti–IL-13 antibodies were added. All the experiments were repeated more than 3 times with similar results.

It was previously demonstrated that IL-3 produced by activated CD4 T cells induces activation of splenic non-B non-T cells to produce IL-4 during Schistosoma infection.17  Moreover, increased accumulation of basophils was not found in IL-3–deficient mice infected with intestinal parasites, further suggesting the critical role of IL-3 in the basophil production.18  Indeed, we have recently demonstrated that treatment of mice infected with Nb with anti–IL-3 antibody partially diminished the accumulation of basophils.5  To test if IL-3, probably produced by activated CD4 T cells, induces basophil activation and further Th2 differentiation, we added neutralizing anti–IL-3 antibody during stimulation. As shown in Figure 3C, addition of anti–IL-3 did not alter Th2 differentiation in the presence of basophils. Moreover, neutralization of IL-13, another major cytokine produced by basophils, did not change T-cell differentiation, suggesting that these cytokines are not required for basophils to induce Th2 differentiation in vitro.

IL-4–independent and contact-dependent inhibition of IFNγ production by basophils

From the coculture experiments with G4 basophils, we noticed that the proportions of CD4 T cells producing IFNγ were substantially diminished when G4 basophils were added, even though IL-4 production was still abrogated in the absence of basophil-derived IL-4 (Figure 3B). To test if the IL-4–independent mechanism by which Th1 differentiation is suppressed is operative in the course of basophil-mediated T-cell differentiation, we set up a coculture experiment by adding different numbers of IL-4–deficient G4 basophils into the coculture experiment. As shown in Figure 4A, addition of G4 basophils gradually reduced IFNγ-producing cells, while development of IL-4–producing CD4 T cells was still compromised in the absence of basophil-derived IL-4. Inhibition of IFNγ production by IL-4–deficient G4 basophils was not due to basophil-derived IL-13, as comparable inhibition was still observed in the presence of neutralizing anti–IL-13 antibody (data not shown). We further performed a Transwell experiment to test if cell-to-cell contact is involved. Separating G4 basophils from OT-II CD4 T cells and DCs restored IFNγ production from the CD4 T cells up to a comparable level of IFNγ production of CD4 T cells stimulated in the absence of basophils (Figure 4B). Similar to the results obtained in Figure 3A, significant inhibition (about 80% to 90%) of IFNγ production was found whether T cells were cocultured with or separated from wild-type basophils. While coculture of G4 basophils caused about 50% inhibition of IFNγ production, culture in Transwell abolished such inhibition, restoring IFNγ production (Figure 4B). These results demonstrate that IL-4–deficient basophils, although incapable of inducing Th2 differentiation, suppress IFNγ production by a cell-to-cell contact–dependent mechanism.

Figure 4

IL-4–independent and contact-dependent suppression of IFNγ production by basophil coculture. (A) OT-II CD4 T cells were stimulated in the presence of different numbers of basophils (none, 800, 4000, 20 000, or 100 000) obtained from the liver of IL-3–treated IL-4–deficient G4/G4 mice. The ratios of T cells and basophils are indicated on the x-axis. Cytokine production was determined after 3 days of culture. The graph shows the average percentages ± SD of relative cytokine-producing CD4 T cells from 2 independent experiments. (B) Basophils isolated from either wild-type (left panel) or IL-4–deficient G4/G4 (right panel) mice were added either together (CC) or separated (TW) in Transwell. Shown are the average percentages ± SD of relative proportions of IFNγ–producing cells measured by intracellular staining from 3 independent experiments.

Figure 4

IL-4–independent and contact-dependent suppression of IFNγ production by basophil coculture. (A) OT-II CD4 T cells were stimulated in the presence of different numbers of basophils (none, 800, 4000, 20 000, or 100 000) obtained from the liver of IL-3–treated IL-4–deficient G4/G4 mice. The ratios of T cells and basophils are indicated on the x-axis. Cytokine production was determined after 3 days of culture. The graph shows the average percentages ± SD of relative cytokine-producing CD4 T cells from 2 independent experiments. (B) Basophils isolated from either wild-type (left panel) or IL-4–deficient G4/G4 (right panel) mice were added either together (CC) or separated (TW) in Transwell. Shown are the average percentages ± SD of relative proportions of IFNγ–producing cells measured by intracellular staining from 3 independent experiments.

Enhanced production of basophils promotes Th2-cell differentiation in vivo

Finally, we tested whether enhanced production of basophils is capable of switching the CD4 T-cell differentiation process in vivo. To this aim, we treated Rag−/− mice with an IL-3 pump to enhance basophil generation in vivo. At 7 days after pump implantation, 2×106 5C.C7 CD4 T cells were intravenously transferred to the recipients, and mice were intravenously challenged with cytochrome C protein without adjuvant. Mice were killed 7 days after immunization. CD4 T cells from each lymphoid organ and liver were in vitro stimulated, and their cytokine productions were determined by intracellular cytokine staining. As shown in Figure 5, CD4 T cells primed in vivo without the IL-3 pump primarily differentiated into IFNγ-producing cells. About 18% and about 15% of the Vβ3+ (5C.C7) CD4 T cells from lymph nodes and spleen produced IFNγ, respectively. Higher proportions (46%) of liver CD4 T cells produced IFNγ. No IL-4–producing cells were found. On the other hand, there was a substantial reduction of the IFNγ-producing cells when recipient mice were pretreated with the IL-3 pump. Moreover, significant levels of IL-4–producing CD4 T cells were found in the spleen and the liver of these mice. These results demonstrate that the enhanced production of basophils appears to provide an environment that favors development of Th2 immune responses in vivo. Of note, stimulation of CD4 T cells in vitro in the presence of IL-3 did not cause Th2 differentiation, suggesting that it is not a direct effect of IL-3 (data not shown).

Figure 5

In vivo Th2 differentiation of naive CD4 T cells promoted by basophils. B10.A Rag−/− mice were implanted with an IL-3 pump. At 7 days after pump implantation, 2 × 106 5C.C7 CD4 T cells were intravenously transferred to the recipients. The mice were then intravenously immunized with 50 μg PCC protein. Cells from lymph nodes, spleen, and liver were harvested at day 7 after immunization and restimulated to measure cytokine production. Shown are intracellular IL-4 and IFNγ production of Vβ3+ CD4 T cells. Experiments were repeated 3 times with similar results.

Figure 5

In vivo Th2 differentiation of naive CD4 T cells promoted by basophils. B10.A Rag−/− mice were implanted with an IL-3 pump. At 7 days after pump implantation, 2 × 106 5C.C7 CD4 T cells were intravenously transferred to the recipients. The mice were then intravenously immunized with 50 μg PCC protein. Cells from lymph nodes, spleen, and liver were harvested at day 7 after immunization and restimulated to measure cytokine production. Shown are intracellular IL-4 and IFNγ production of Vβ3+ CD4 T cells. Experiments were repeated 3 times with similar results.

Discussion

Defining an initial source of IL-4 necessary for in vivo Th2 development has been an area of active investigations. Data from the current study suggest that basophils can assist Th2 T-cell differentiation of activated naive CD4 T cells primarily by secreting IL-4 and in part by engaging cell-to-cell contact. In vivo, naive CD4 T cells stimulated in a basophil-enriched environment preferentially differentiate into Th2 phenotype effector cells. Therefore, we argue that basophils may be important cell types that have a potential to link to the development of Th2 type adaptive immunity.

Basophils are efficient producers of IL-4. Unlike T cells that require epigenetic modification of Il4 genes, basophils acquire a capacity to efficiently produce IL-4 and IL-13 during the developmental process in the bone marrow.12  Moreover, it was shown that basophils produce greater amount of IL-4 compared with differentiated Th2 CD4 T cells per cell basis.13  In support of this, our data demonstrate that IL-4 produced from basophils is primarily responsible for the development of Th2 type responses found in the coculture experiment. Differentiation into IFNγ-producing cells was, however, significantly reduced when T cells were cocultured with IL-4–deficient basophils. It is intriguing that cell-to-cell contact is critical for the IL-4–independent basophil-mediated inhibition of Th1 differentiation, suggesting yet another important mechanism exerted by the basophils in the course of T-cell differentiation. The contact-dependent mechanisms may be dispensable while basophil-derived IL-4 is abundant, although it may synergize with the IL-4–mediated effect. The contact-mediated effect becomes critical when IL-4 is unavailable. Molecules involved in the contact remain to be defined. In addition, it remains to be determined whether basophils establish a direct contact with T cells to deliver a regulatory signal to suppress IFNγ production or, alternatively, interact with DCs, which subsequently alter DC functions to activate T cells to produce IFNγ.

Naive CD4 T cells primed in vivo within a basophil-enriched environment displayed substantially diminished IFNγ as well as enhanced IL-4–producing phenotypes. Most interesting is that appearance of the IL-4–producing CD4 T cells showed a close correlation to the level of basophil accumulation in vivo (Figures 1B and 5). Because a high level of serum IL-4 can be found in the basophil-enriched mice that received an IL-3 pump (K.O. and B.M., unpublished results, June 2006), it appears that the level of basophil accumulation may have a direct effect on recruiting differentiated Th2 type effector CD4 T cells into the sites. In contrast, reduction of IFNγ production was rather independent of the level of basophil accumulation, as a comparable level of reduction of IFNγ production was found regardless of the site, including lymph nodes in which no basophil accumulation was found. It is possible that basophils may produce chemokines that attract Th2 type effector CD4 T cells. In support of this, it was recently shown that production of IL-4 and IL-13 by innate type cells was critical for recruiting Th2 CD4 T cells into an Nb infection site, the lung.4  However, its biologic significance remains to be defined. Experiments aimed at understanding a role of chemokines are underway.

The nature of the signals involved in basophil production in vivo remains to be determined. IL-3 is a potential candidate for the basophil generation. Basal level of blood basophils can be found in mice deficient in IL-3, although the parasite-induced increase in basophil numbers is severely impaired.18  Yet, prolonged exposure to IL-3 clearly increases the generation of basophils in vivo. Thus, IL-3 may be responsible for the production of basophils in response to certain stimuli such as parasitic infection. In support of this, we previously found that basophil accumulation after Nb infection was partially impaired upon IL-3 neutralization.5  Finding a source of IL-3 should lead us to understand a mechanism that underlies in vivo basophil generation. It was previously proposed that the IL-3 produced by activated T cells plays a major role in splenic non-B non-T cells following Schistosoma infection.17  In addition to activated T cells, basophils themselves produce a substantial level of IL-3 (K.O. and B.M., unpublished results, April 2006). Considering the fact that basophil production is highly associated with Th2 type immunity, it is likely that an additional factor(s) is involved in the basophil production. Recently, it was shown that mice deficient in IRF-2 spontaneously develop Th2 type immune responses that are predominantly mediated by increased production of basophils and their IL-4 production.19  Furthermore, transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) plays an important role in differentiation of precursor cells into basophil precursors.20  More studies are needed to clarify these issues.

Given that IL-3 not only promotes basophil generation but also stimulates basophils to produce cytokines and histamines,15  basophils isolated from IL-3–treated mice may have already activated phenotypes. Notably, however, basophils found in the bone marrow spontaneously express IL-4 mRNA,12  and basophils from unmanipulated G4 knock-in mice spontaneously express GFP and intracellular IL-4 following stimulation.5  Moreover, we employed a positive isolation method to purify basophils using FcϵRI-specific antibody. The positive isolation method may crosslink an IgE receptor that induces basophil activation and cytokine production. One might argue that the degranulated appearance of basophils may account for this possibility. However, granules are less numerous in mouse basophils, and mouse basophils lack secondary and tertiary granules.16  Moreover, when basophils were cultured in vitro in media and a spontaneous IL-4 production was determined by ELISA, we found that the spontaneous IL-4 production was relatively negligible compared with that from in vitro–activated basophils (K.O. and B.M., unpublished results, August 2006). Therefore, although it is possible that in vivo IL-3 treatment that is necessary for the generation of basophils may induce some level of basophil activation, we believe it does not change the conclusion of the current paper. In support of this, we have previously shown that activation of T cells further enhances basophil IL-4 production.5 

It is rather immature to conclude whether basophils are the bona fide innate Th2 type cells that subsequently induce adaptive Th2 type immunity. Kinetics of basophil accumulation following Nb infection turned out to be similar to that of Th2 type CD4 T cells.5  It was recently reported that production of IL-4 and IL-13 by innate type cells, including basophils, was rather dispensable for the induction of IL-4–producing CD4 T-cell responses.4  Therefore, it is possible that in vivo functions of basophils may be not to induce but to amplify Th2 immune responses and possibly to stabilize the generation of Th2-type memory-cell responses. Yet, we cannot exclude a possibility that a small number of activated basophils prior to enhanced generation may be sufficient to influence the subsequent T-cell differentiation pathway by IL-4–dependent and IL-4–independent mechanisms. Indeed, basophils can produce IL-4 within 4 hours after stimulation.21  Moreover, the rapid release of IL-4 by basophils could be easily achieved independently of a developed adaptive immunity, as there are several types of IgE-independent basophil stimulation that lead to IL-4 production.22,23  Alternatively, a contact-dependent mechanism may be more critical in vivo.

In conclusion, our data provide evidence indicating that activation of T cells in an environment of an enhanced level of basophils appears to promote the subsequent development of Th2 type effector immune responses. It is known that basophilia preferentially occurs during parasitic infection, further supporting our observations.24  Future studies should be directed to understanding underlying mechanisms that control basophil generation as well as basophil activation in certain circumstances. The potential of basophils not only to promote Th2 type immunity but also to suppress Th1 type immunity has a particularly important clinical implication; namely, that basophils may provide a novel therapeutic strategy for intervening in the modulation of ongoing Th1-mediated immune responses such as autoimmunity.

Authorship

Contribution: K.O. and B.M. designed research; K.O., T.S., and B.M. performed research; K.O., G.L.G., and B.M. analyzed data; and K.O. and B.M. wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Booki Min, Department of Immunology/NB30, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH; e-mail: minb@ccf.org.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported by start-up funds from the Cleveland Clinic Foundation (B.M.).

We thank Shadi Swaidani for Wright-Giemsa staining experiments and Dr William E. Paul for insightful discussion.

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