Download Induction of Th2 type immunity in a mouse system

From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
IMMUNOBIOLOGY
Induction of Th2 type immunity in a mouse system reveals a novel
immunoregulatory role of basophils
Keunhee Oh,1 Tao Shen,1 Graham Le Gros,2 and Booki Min1,3
1Department
3Laboratory
of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, OH; 2Malaghan Institute of Medical Research, Wellington, New Zealand;
of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
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-tocell contact to exert the inhibitory effect
on Th1 differentiation. Stimulation of na-
ive 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.
(Blood. 2007;109:2921-2927)
© 2007 by The American Society of Hematology
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, CD40Ldependent 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-tocell 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.
Submitted July 25, 2006; accepted November 19, 2006. Prepublished
online as Blood First Edition Paper, November 28, 2006; DOI 10.1182/blood2006-07-037739.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
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.
2921
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2922
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
OH et al
Isolation of basophils
Mice were subcutaneously implanted with a miniosmotic pump (Alzet;
Durect, Cupertino, CA) containing 5 ␮g 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 Fc␥R
blocking antibody (2.4G2), cells were then labeled with anti-Fc⑀RI–FITC
(MAR-1; eBioscience, San Diego, CA) and incubated with anti-FITC
microbeads (Miltenyi Biotec, Auburn, CA). Fc⑀RI-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 ␮M 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 ␮g/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 ␮g PCC protein (Sigma). In some experiments, mice were
pretreated with 5 ␮g 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 ␮g/mL) and anti-CD28 (3 ␮g/mL) for 6 hours in the presence
of 2 ␮M 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)–IFN␥ (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
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 Fc⑀RI and c-kit. Fc⑀RI/c-kit double positive cells represent mast cells,
while Fc⑀RI 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 ␮g IL-3. Seven days after pump implantation, cells from bone marrow, liver, spleen, and lymph nodes were harvested and stained for CD49b
and Fc⑀RI. Proportions of CD49b⫹ Fc⑀RI⫹ 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).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
(Fc⑀RI⫹ c-kit⫹), and basophils (Fc⑀RI⫹ 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, Fc⑀RI, 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, Fc⑀RI⫹ 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 Nbinfected 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.
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
BASOPHILS AND TH2 IMMUNITY
2923
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 IFN␥ 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 IFN␥ 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).
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 IFN␥ 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
(Fc⑀RI⫹ c-kit⫹) and basophils (Fc⑀RI⫹ 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 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 ␮M 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 IFN␥–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 ␮g/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.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2924
OH et al
(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 Th1promoting 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 IFN␥ 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 IFN␥
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
Figure 3. IL-4 produced by basophils is primarily responsible for basophilinduced 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
IFN␥ 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 ␮g/mL anti–IL-3 or anti–IL-13 antibodies were added.
All the experiments were repeated more than 3 times with similar results.
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
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).
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 IFN␥
production by basophils
From the coculture experiments with G4 basophils, we noticed that the
proportions of CD4 T cells producing IFN␥ 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
IFN␥-producing cells, while development of IL-4–producing CD4 T
cells was still compromised in the absence of basophil-derived IL-4.
Inhibition of IFN␥ 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 IFN␥ production from the CD4 T cells up
to a comparable level of IFN␥ 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 IFN␥ 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 IFN␥ production, culture in Transwell abolished such
inhibition, restoring IFN␥ production (Figure 4B). These results demonstrate that IL-4–deficient basophils, although incapable of inducing Th2
differentiation, suppress IFN␥ production by a cell-to-cell contact–
dependent mechanism.
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
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
Figure 4. IL-4–independent and contact-dependent suppression of IFN␥ 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 cytokineproducing 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 IFN␥–producing cells measured by
intracellular staining from 3 independent experiments.
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 IFN␥-producing cells. About 18% and about
15% of the V␤3⫹ (5C.C7) CD4 T cells from lymph nodes and
spleen produced IFN␥, respectively. Higher proportions (46%) of
liver CD4 T cells produced IFN␥. No IL-4–producing cells were
found. On the other hand, there was a substantial reduction of the
IFN␥-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).
BASOPHILS AND TH2 IMMUNITY
2925
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 IFN␥-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 IFN␥ production
or, alternatively, interact with DCs, which subsequently alter DC
functions to activate T cells to produce IFN␥.
Naive CD4 T cells primed in vivo within a basophil-enriched
environment displayed substantially diminished IFN␥ 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 IFN␥ production was rather independent of
the level of basophil accumulation, as a comparable level of
reduction of IFN␥ 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.
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.
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 ␮g 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 IFN␥ production of V␤3⫹ CD4 T cells. Experiments were repeated 3 times
with similar results.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2926
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
OH et al
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/EBP␣) 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 Fc⑀RI-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 memorycell 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 Th1mediated immune responses such as autoimmunity.
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.
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: [email protected].
References
1. Seder RA, Paul WE. Acquisition of lymphokineproducing phenotype by CD4⫹ T cells. Annu Rev
Immunol. 1994;12:635-673.
2. Noben-Trauth N, Hu-Li J, Paul WE. IL-4 secreted
from individual naive CD4⫹ T cells acts in an autocrine manner to induce Th2 differentiation. Eur
J Immunol. 2002;32:1428-1433.
3. Gregory GD, Brown MA. Mast cells in allergy and
autoimmunity: implications for adaptive immunity.
Methods Mol Biol. 2006;315:35-50.
4. Voehringer D, Reese TA, Huang X, Shinkai K,
Locksley RM. Type 2 immunity is controlled by
IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system. J Exp
Med. 2006;203:1435-1446.
5. Min B, Prout M, Hu-Li J, et al. Basophils produce
IL-4 and accumulate in tissues after infection with
a Th2-inducing parasite. J Exp Med. 2004;200:
507-517.
6. Godfrey DI, Kronenberg M. Going both ways: immune regulation via CD1d-dependent NKT cells.
J Clin Invest. 2004;114:1379-1388.
7. Luccioli S, Brody DT, Hasan S, Keane-Myers A,
Prussin C, Metcalfe DD. IgE(⫹), Kit(-), I-A/I-E(-)
myeloid cells are the initial source of Il-4 after antigen challenge in a mouse model of allergic pulmonary inflammation. J Allergy Clin Immunol.
2002;110:117-124.
8. Mukai K, Matsuoka K, Taya C, et al. Basophils
play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. Immunity. 2005;
23:191-202.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 APRIL 2007 䡠 VOLUME 109, NUMBER 7
9. Falcone FH, Dahinden CA, Gibbs BF, et al. Human basophils release interleukin-4 after stimulation with Schistosoma mansoni egg antigen. Eur
J Immunol. 1996;26:1147-1155.
10. Gauchat JF, Henchoz S, Mazzei G, et al. Induction of human IgE synthesis in B cells by mast
cells and basophils. Nature. 1993;365:340-343.
11. Yanagihara Y, Kajiwara K, Basaki Y, et al. Cultured basophils but not cultured mast cells induce
human IgE synthesis in B cells after immunologic
stimulation. Clin Exp Immunol. 1998;111:136-143.
12. Gessner A, Mohrs K, Mohrs M. Mast cells, basophils, and eosinophils acquire constitutive IL-4
and IL-13 transcripts during lineage differentiation
that are sufficient for rapid cytokine production.
J Immunol. 2005;174:1063-1072.
13. Mitre E, Taylor RT, Kubofcik J, Nutman TB. Parasite antigen-driven basophils are a major source
of IL-4 in human filarial infections. J Immunol.
2004;172:2439-2445.
14. Hu-Li J, Pannetier C, Guo L, et al. Regulation of
BASOPHILS AND TH2 IMMUNITY
expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene. Immunity. 2001;14:1-11.
15. Yoshimoto T, Tsutsui H, Tominaga K, et al. IL-18,
although antiallergic when administered with IL12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci U S A. 1999;96:
13962-13966.
16. Dvorak AM. Ultrastructural analysis is necessary and
sufficient for identification of basophils and mast
cells. Chem Immunol Allergy. 2005;85:10-67.
17. Kullberg MC, Berzofsky JA, Jankovic DL, et al. T
cell-derived IL-3 induces the production of IL-4 by
non-B, non-T cells to amplify the Th2-cytokine
response to a non-parasite antigen in Schistosoma mansoni-infected mice. J Immunol. 1996;
156:1482-1489.
18. Lantz CS, Boesiger J, Song CH, et al. Role for
interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature. 1998;
392:90-93.
19. Hida S, Tadachi M, Saito T, Taki S. Negative control of basophil expansion by IRF-2 critical for the
2927
regulation of Th1/Th2 balance. Blood. 2005;106:
2011-2017.
20. Arinobu Y, Iwasaki H, Gurish MF, et al. Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis. Proc Natl
Acad Sci U S A. 2005;102:18105-18110.
21. Gibbs BF, Haas H, Falcone FH, et al. Purified human peripheral blood basophils release interleukin-13 and preformed interleukin-4 following immunological activation. Eur J Immunol. 1996;26:
2493-2498.
22. Haas H, Falcone FH, Schramm G, et al. Dietary
lectins can induce in vitro release of IL-4 and
IL-13 from human basophils. Eur J Immunol.
1999;29:918-927.
23. Patella V, Florio G, Petraroli A, Marone G. HIV-1
gp120 induces IL-4 and IL-13 release from human
Fc epsilon RI⫹ cells through interaction with the VH3
region of IgE. J Immunol. 2000;164:589-595.
24. Mitre E, Nutman TB. Basophils, basophilia and
helminth infections. Chem Immunol Allergy. 2006;
90:141-156.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2007 109: 2921-2927
doi:10.1182/blood-2006-07-037739 originally published
online November 28, 2006
Induction of Th2 type immunity in a mouse system reveals a novel
immunoregulatory role of basophils
Keunhee Oh, Tao Shen, Graham Le Gros and Booki Min
Updated information and services can be found at:
http://www.bloodjournal.org/content/109/7/2921.full.html
Articles on similar topics can be found in the following Blood collections
Immunobiology (5489 articles)
Phagocytes (969 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.