Stat6 Regulation of In Vivo IL-4 Responses
This information is current as
of June 15, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2000; 164:2303-2310; ;
doi: 10.4049/jimmunol.164.5.2303
http://www.jimmunol.org/content/164/5/2303
This article cites 74 articles, 44 of which you can access for free at:
http://www.jimmunol.org/content/164/5/2303.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
References
Fred D. Finkelman, Suzanne C. Morris, Tatyana Orekhova,
Masaaki Mori, Debra Donaldson, Steven L. Reiner, Nancy
L. Reilly, Lisa Schopf and Joseph F. Urban, Jr.
Stat6 Regulation of In Vivo IL-4 Responses1
Fred D. Finkelman,2* Suzanne C. Morris,* Tatyana Orekhova,* Masaaki Mori,3*
Debra Donaldson,† Steven L. Reiner,‡ Nancy L. Reilly,‡ Lisa Schopf,§ and Joseph F. Urban, Jr.§
I
nfectious agents can induce rodent and human T cells to secrete polarized sets of cytokines that influence the host’s ability to defend against the pathogen (1, 2). No single set of
cytokines is universally protective for the host: protection against
intracellular parasites is generally promoted by type 1 cytokines,
such as IFN-␥, but inhibited by type 2 cytokines, such as IL-4 (1),
while protection against gastrointestinal nematode parasites is promoted by the type 2 cytokines, IL-4, IL-9, and IL-13, but inhibited
by IFN-␥ (2, 3).
Appreciation of these consequences of T cell cytokine production has promoted investigation of the mechanisms that regulate
the differentiation of naive CD4⫹ T cells into Th1 or Th2 cells.
Considerable progress has been made toward elucidating the
mechanisms that induce type 1 cytokine production. Ligation of
cellular receptors that recognize characteristics shared by multiple
pathogens, called pattern recognition molecules (4 – 6), can stimulate APC to secrete IL-12, IL-18, IFN-␣, and IFN-␤, which promote the development of a type 1 cytokine response and inhibit the
development of a type 2 response (7–10).
Identification of the mechanisms that induce type 2 cytokine
responses, in general, and IL-4 responses, in particular, has been
more difficult. Several in vitro studies and some in vivo studies
indicate that IL-4, itself, stimulates the development of a type 2
*Division of Immunology, University of Cincinnati College of Medicine, Cincinnati,
OH 45267, and Cincinnati Veterans Administration Medical Center, Cincinnati, OH
45220; †Genetics Institute, Cambridge, MA 02140; ‡University of Pennsylvania
School of Medicine, Philadelphia, PA 19104; and §U.S. Department of Agriculture,
Beltsville, MD 20705
Received for publication October 14, 1999. Accepted for publication December
14, 1999.
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.
1
This work was supported in part by the Cincinnati Veterans Administration Medical
Center; U.S. Public Health Service Grants RO1 AI35987, RO1 AI44971, RO1
AI42370; and U.S. Department of Agriculture/Agricultural Research Station/CRIS
Grant 1265-32000-049.
2
Address correspondence and reprint requests to Dr. Fred D. Finkelman, Department
of Internal Medicine, Research Service 151, Cincinnati VAMC, 3200 Vine Street,
Cincinnati, OH 45220. E-mail address: [email protected]
3
Current address: Department of Pediatrics, Yokohama City University School of
Medicine, 3-9 Fuku-ura, Yokohama 236-0004, Japan.
Copyright © 2000 by The American Association of Immunologists
cytokine response and does so by binding to IL-4R␣ and activating
the IL-4R␣-associated signaling molecule, Stat6 (11–18). Observations that CD4⫹ T cells from Stat6-deficient mice fail to differentiate into Th2 cells when stimulated in vitro with Ag or anti-CD3
mAb ⫾ anti-CD28 mAb, even in the absence of IL-12 signaling
(19); that a Stat6 binding site is present on the IL-4 promoter (16);
and that occupancy of this site by Stat6 enhances IL-4 gene transcription (20, 21) are all consistent with the concept that Stat6
signaling is required to induce naive CD4⫹ T cells to differentiate
into polarized Th2 cells.
The view that Stat6 activation by IL-4 is required to induce
naive CD4⫹ T cells to differentiate into Th2 cells requires a source
for the IL-4 that would prime the T cell response. Several sources
for this initial IL-4 have been suggested, including mast cells, basophils, eosinophils, and NK T cells (22–26). Although each of
these cell types can produce IL-4, none has been found to be necessary for the development of an in vivo IL-4 response (27–31).
These observations are compatible with the possibility that the in
vivo IL-4 response by conventional CD4⫹ T cells can be primed
by multiple redundant sources of IL-4, so that deletion of any one
source might have little effect on in vivo IL-4 production.
An in vivo observation, however, challenges the view that Stat6
signaling is required to induce IL-4 production: Stat6-deficient
mice immunized with an affinity-purified goat Ab to mouse IgD
(GaM␦)4 (4) make an IL-4 response that has the same magnitude
and kinetics as the response made by wild-type mice (31). It is
unlikely that the large Stat6-independent IL-4 response in GaM␦immunized mice is made by cells other than CD4⫹ T cells (IL-4
production in GaM␦-immunized wild-type mice is blocked by antiCD4 mAb (32) and only CD4⫹ T cells from these mice express
increased IL-4 mRNA (33)) or that it is coming from classic NK
T cells (the response is ␤2-microglobulin independent and class II
MHC dependent (31)). Furthermore, studies of mice in which the
absence of a functional IL-4R␣ gene eliminated any possibility of
IL-4 priming of T cell IL-4 production demonstrated that T cell
IL-4 responses can be generated in the absence of IL-4R␣ signaling (34).
4
Abbreviations used in this paper: GaM␦, affinity-purified goat anti-mouse IgD Ab;
APF, Ascaris pseudocoelomic fluid; CCCA, Cincinnati cytokine capture assay; CFSE,
carboxyfluorescein diacetate succinimidyl ester.
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Although in vitro development of a Th2 response from naive CD4ⴙ T cells is Stat6 dependent, mice immunized with a goat Ab
to mouse IgD have been reported to produce a normal primary IL-4 response in Stat6-deficient mice. Experiments have now been
performed with mice immunized with more conventional Ags or inoculated with nematode parasites to account for this apparent
discrepancy. The ability of an immunogen to induce a primary in vivo IL-4 response in Stat6-deficient mice was found to vary
directly with its ability to induce a strong type 2 cytokine-biased response in normal mice. Even immunogens, however, that induce
strong primary IL-4 responses in Stat6-deficient mice induce poor memory IL-4 responses in these mice. Consistent with this,
Stat6-deficient CD4ⴙ T cells make relatively normal IL-4 responses when stimulated in vitro for 3 days with anti-CD3 and
anti-CD28, but poor IL-4 responses if they are later restimulated with anti-CD3. Thus, Stat6 signaling enhances primary IL-4
responses that are made as part of a type 0 cytokine response (mixed type 1 and type 2) and is required for normal development
or survival of Th2 memory cells. The Journal of Immunology, 2000, 164: 2303–2310.
2304
The unexpected results of these studies led us to investigate
whether Stat6-independent IL-4 production in GaM␦-immunized
mice remains IL-4R␣ dependent and whether Stat6-independent
IL-4 production can be induced in vivo by immunogens that are
more conventional than GaM␦. In addition, because host protection against gastrointestinal nematode parasites is Stat6 dependent
(2, 3), we investigated the Stat6 dependence of IL-4 responses to
these worms. Results of these studies demonstrate that the in vivo
IL-4 response to GaM␦ is IL-4R␣ independent as well as Stat6
independent and that all Ags tested can induce at least some IL-4
production in Stat6-deficient mice, although Ags that induce the
strongest and most polarized type 2 responses in normal mice appear to depend least on Stat6 signaling to induce maximal IL-4
production. In contrast to primary IL-4 responses, secondary IL-4
responses are Stat6 dependent even when they are induced by
stimuli that evoke large, Stat6-independent IL-4 primary responses. These observations suggest that Stat6 signaling is less
important for the initial production of IL-4 by naive T cells than
for the generation or survival of memory T cells that secrete IL-4.
Mice
Stat6-deficient mice on a mixed C57BL/6-129 background (14) were a gift
of Dr. James Ihle (Memphis, TN); a separately produced line of Stat6deficient mice (13), which had been bred onto a BALB/c background, was
a gift of Dr. Michael Grusby (Cambridge, MA). IL-4R␣-deficient mice
(35), which were produced on a BALB/c background, were bred at National Institutes of Health (Bethesda, MD). W/Wv, ␮MT, and appropriate
control mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). Stat6-deficient mice and appropriate controls were bred in the laboratory animal facility at the Cincinnati Veterans Administration Medical
Center (Cincinnati, OH). All mice were age and sex matched with controls
in any given experiment.
Immunological reagents
GaM␦ and normal goat IgG were produced and purified as described (36).
Hybridomas that secrete a cytotoxic rat IgG anti-mouse CD4 mAb (GK1.5)
(37), a stimulatory anti-CD3⑀ mAb (145-2C11) (38), or a neutralizing antiIFN-␥ mAb (R46-A2) (39) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). Hybridomas that produce neutralizing
or nonneutralizing rat IgG anti-IL-4 mAbs (BVD4-1D11 and BVD624G2.3, respectively) (40) were obtained from the ATCC with the permission of the DNAX Research Institute (Palo Alto, CA) and the assistance of
Dr. Robert Coffman (DNAX). Hybridomas that produce the control rat
IgG2b mAb, J1.2, or a nonneutralizing anti-IFN-␥ mAb (AN18) (41) were
gifts of Dr. John Abrams and Anne O’Garra, respectively (DNAX). All of
these hybridomas were grown as ascites in Pristane-primed athymic nude
mice. All mAbs were purified from ascites by salt precipitation and ionexchange chromatography, as described (42), using isotype-specific antisera
(Miles Labs, Naperville, IL) and gel-double diffusion analysis to identify Abrich fractions. Human IL-2 and mAbs specific for mouse IL-4, IFN-␥, CD28,
and CD3 that were used for in vitro studies were obtained as described (43).
To produce a rabbit Ab to murine IL-13R␣2, rabbits were immunized
with 80% pure CHO cell-derived T7-epitope-tagged murine IL-13R␣2
(44). Antiserum from one rabbit was assayed for IL-13R␣2 binding by
ELISA. The Ab was purified by protein A-Sepharose affinity chromatography and adjusted to a concentration of 1 mg/ml. Specificity was confirmed by FACS and Western blotting.
IL-13R␣2-binding activity was demonstrated with a BIACORE 2000.
Briefly, 1200 resonance units of IL-13R␣2-Fc or irrelevant receptor-Fc
fusion proteins were immobilized on a dextran chip using standard coupling procedures. IL-13, IL-13 ⫹ IL-13R␣2-Fc, or IL-13 ⫹ IL-13R␣2-Fc
⫹ anti-IL-13R␣2 Ab was injected and binding of IL-13 to IL-13R␣1 was
quantified as an increase in resonance units. Addition of 10 ␮g/ml of rabbit
IgG anti-IL-13R␣2 Ab restored IL-13 binding to IL-13R␣1 in the presence
of IL-13R␣2 (increase of 120 resonance units).
OVA (grade V; Sigma, St. Louis, MO) was dissolved in PBS and centrifuged at 14,000 ⫻ g for 3 min before use to remove large aggregates.
Alkaline phosphatase conjugated to streptavidin was purchased from Jackson ImmunoResearch (West Grove, PA). Purified, mouse rIL-4 was a generous gift of the Schering-Plough Research Institute (Kenilworth, NJ). Purified, mouse rIFN-␥ was a gift of Genzyme (Cambridge, MA). Some
mAbs were biotin conjugated, using biotin N-hydroxy-succinimide (Calbiochem-Behring, La Jolla, CA), as described (32), at a 1:10 (w/w) biotin:
protein ratio.
Ascaris pseudocoelomic fluid (APF), a strong allergen, was prepared as
follows: Adult male and female worms were freshly recovered from the
intestines of pigs at a local abattoir. The worms were kept at room temperature in a PBS solution in transit to the laboratory, where they were
washed in PBS several times. The pseudocoelom was cut with a scissors
and the fluid drained into a collection flask. The fluid was clarified by
centrifugation at 10,000 rpm for 20 min at 4°C and the supernatant stored
frozen at ⫺20°C until used.
Nematode parasites
Nippostrongylus brasiliensis larvae (L3), Heligmosomoides polygyrus larvae (L3), and Trichinella spiralis muscle stage larvae were prepared as
described (45– 47). Mice were inoculated by s.c. injection with 500
N. brasiliensis L3 or by oral gavage with 200 H. polygyrus L3 or 50 T.
spiralis muscle stage larvae. In experiments that examined responses to a
second infection, primary infections were cured by treating mice by oral gavage with 1 mg of pyrantal pamoate (Strongid T; Pfizer, New York, NY) (47).
Cincinnati cytokine capture assay
The Cincinnati cytokine capture assay (CCCA) was used to monitor in vivo
production of IL-4 and IFN-␥. This assay allows cytokines to accumulate
in serum by capturing them in vivo with neutralizing IgG mAbs that inhibit
their excretion, utilization, and catabolism (48). This increases the ability
to detect IL-4 in serum by ⬎1000-fold and is specific (no IL-4 response is
detected in IL-4-deficient mice). To capture secreted IL-4, mice were injected i.v. with 10 ␮g of biotin-BVD4-1D11. Mice were bled 2– 4 h later,
and serum levels of IL-4-biotin-anti-IL-4 complexes were determined by
ELISA, using microtiter plates coated with BVD6-24G2.3. This mAb recognizes an IL-4 epitope that is distinct from that recognized by the neutralizing anti-IL-4 mAb, BVD4-1D11, that was injected into mice. A
CCCA for IFN-␥ was performed similarly by injecting mice with 50 ␮g of
biotin-R46-A2 and coating microtiter plates with AN18.
Serum IL-13 assay
Mouse IL-13 ELISA kits were purchased from R&D Systems (Minneapolis, MN) and were performed according to the manufacturer’s instructions, with the exception that rabbit IgG anti-IL-13R␣2 Ab (100 ␮g/ml)
was added during the primary incubation period to a 1/10 or 1/100 dilution
of serum samples and incubated overnight at 4°C, to dissociate IL-13 from
serum sIL-13R␣2. Duplicate serum samples were examined from each animal. OD readings were converted to pg/ml using a standard curve and the
appropriate dilution factor. Rabbit IgG anti-mouse IL-13R␣2 had no effect
on the assay curve for purified mouse IL-13. Addition of mouse serum to
an IL-13 standard decreased assay sensitivity.
To determine whether addition of rabbit anti-mouse IL-13R␣2 Ab
would restore assay sensitivity, we compared the effect of adding rabbit
IgG anti-mouse IL-13R␣2 (100 ␮g/ml) or an irrelevant rabbit IgG Ab during the primary incubation period to the IL-13 standard diluted in 50%
serum. ELISAs were performed as described and demonstrated 100% recovery of IL-13 standard curve sensitivity, up to an IL-13 dose of 250
ng/ml.
In vitro stimulation of cytokine production and identification of
IL-4- and IFN-␥-producing cells
Peripheral lymph node and spleen cells were depleted of CD8⫹ T cells,
labeled with CFSE, and cultured with 2 ␮g/ml each of soluble anti-CD3
and CD28 mAbs plus 2.5 U of human IL-2, as described (43). After 3 days,
cells were stimulated for 4 h with PMA and ionomycin, stained with tricolor-labeled anti-CD4 mAb, permeabilized, stained with PE-labeled antiIL-4, and analyzed by flow cytometry for CFSE and PE staining of tricolor
positive cells (43). A second aliquot of CD8⫹ T cell-depleted cells was
cultured for 6 days with soluble anti-CD28 and anti-CD3 mAbs plus IL-2,
washed, recultured for 24 h with plate-bound anti-CD3 mAb, stimulated
for 4 h with PMA and ionomycin, permeabilized, and stained with FITC
anti-IFN-␥ and PE IL-4, then analyzed by flow cytometry for FITC and PE
staining (43).
Results
IL-4R␣-deficient mice make a strong IL-4 response to GaM␦
Our observation that Stat6-deficient mice make a normal IL-4 response to GaM␦ left open the possibility that IL-4 can prime CD4⫹
T cells to secrete IL-4 through a Stat6-independent mechanism. To
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Materials and Methods
Stat6 DEPENDENCE OF THE IL-4 RESPONSE
The Journal of Immunology
2305
FIGURE 1. GaM␦ induces IL-4R␣-independent IL-4 production.
BALB/c mice that were heterozygous or homozygous for a defective IL4R␣ allele (5/group) were injected i.v. with 1 mg of GaM␦ and were
monitored for IL-4 production by CCCA before and 3 and 5 days after
GaM␦ injection. Geometric means and SEs are shown in this figure and in
all subsequent figures.
The in vivo IL-4 response to a standard protein Ag is more
Stat6 dependent than the response to a potent allergen
To determine whether the in vivo IL-4 response to a conventional
Ag is as Stat6 independent as the GaM␦-induced IL-4 response, we
used the CCCA to measure in vivo IL-4 production in wild-type
and Stat6-deficient mice immunized with chicken OVA, an Ag that
induces a type 0 cytokine response in mice (49). To avoid the use
of adjuvant, which might modify cytokine responses, mice were
injected i.p. once per day with 1 mg of OVA, a dose that we have
previously found to induce a large IgG1 anti-OVA Ab response
(our unpublished data). IL-4 production was detectable in OVAimmunized wild-type mice by day 3, peaked on day 6, and was still
considerable on day 10 (Fig. 2, upper panel). In contrast, Stat6deficient mice immunized and tested at the same time by the same
protocol developed an IL-4 response that was only detectable at
FIGURE 3. Stat6 dependence of IL-4 production differs in mice infected with N. brasiliensis or T. spiralis. Wild-type and Stat6-deficient
mice on a mixed C57BL/6-129 background (5/group) were inoculated s.c.
with 500 N. brasiliensis L3 or orally with 50 T. spiralis muscle stage
larvae. Mice were monitored for IL-4 production by CCCA before inoculation and 3, 5, 7, 10, and 13 days after inoculation with N. brasiliensis or
2, 4, 6, and 8 days after inoculation with T. spiralis.
day 6 and was only 37% as large as the wild-type response at that
time point. When immunized daily i.p. with 50 ␮l of APF, a potent
allergen (50, 51), Stat6-deficient mice again made a smaller IL-4
response than wild-type mice; however, IL-4 responses were detectable in both strains at all time points, and peak values in the
Stat6-deficient mice were closer (69%) to those in the wild-type
mice (Fig. 2, lower panel).
Stat6-deficient mice make normal IL-4 responses to an initial
N. brasiliensis infection, but a reduced IL-4 response to an
initial T. spiralis infection
FIGURE 2. The in vivo IL-4 response to APF is less Stat6 dependent
than the response to OVA. Wild-type and Stat6-deficient mice on a mixed
C57BL/6-129 background (5/group) were immunized daily i.p. with 1 mg
of OVA in 0.2 ml of saline or with 50 ␮l of APF, diluted to 0.2 ml with
0.15 M NaCl. IL-4 production was monitored by CCCA before and 3, 6,
and 10 days after the initial immunization.
Results of the previous experiment suggested that Stat6 signaling
might be more important for the induction of an IL-4 response by
a conventional Ag (OVA) than by Ags that induce a strongly biased type 2 cytokine response (GaM␦ and APF) (33, 50, 51). To
determine whether an analogous result would be observed if mice
were inoculated with a gastrointestinal nematode parasite that induces a relatively pure type 2 response (N. brasiliensis) (3, 52) or
with a gastrointestinal nematode parasite that induces a more
mixed cytokine response (T. spiralis) (53), primary IL-4 responses
were assayed by CCCA at several time points in wild-type and
Stat6-deficient mice following inoculation with either of these parasites. Identical IL-4 responses developed in wild-type and Stat6deficient mice inoculated with N. brasiliensis; however, IL-4 responses in T. spiralis-infected mice were only ⬃10% as large as
those in wild-type mice (Fig. 3). No differences were observed in
the survival of either N. brasiliensis or T. spiralis during the initial
6 days of infection, by which time IL-4 responses had reached
peak or near-peak levels in wild-type mice, although wild-type
mice cleared infections during the subsequent 4 to 8 days, while
Stat6-deficient mice developed chronic infections (3 and data not
shown).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
test this possibility, IL-4 secretion was measured following GaM␦
injection in BALB/c mice that were heterozygous or fully deficient
for a functional IL-4R␣ allele (Fig. 1). IL-4 levels, as detected by
the CCCA, were considerably higher in IL-4R␣-deficient mice
than in IL-4R␣ heterozygotes. This may reflect absent IL-4 utilization in IL-4R␣-deficient mice, rather than increased IL-4 production (GaM␦ induces similar IL-4 mRNA responses in wild-type
and IL-4R␣-deficient mice (N. Noben-Trauth, unpublished data)),
but clearly demonstrates that no IL-4 signal is required for GaM␦
induction of a strong IL-4 response.
2306
Stat6 DEPENDENCE OF THE IL-4 RESPONSE
type mice (data not shown). Thus, even though Stat6 signaling
does not contribute to primary IL-4 or IL-13 responses to H. polygyrus, it appears to be required for the generation or survival of
cells that make a normal memory type 2 cytokine response when
mice are reinfected with this parasite. Studies of IL-4 production in
wild-type and Stat6-deficient mice infected with N. brasiliensis
and studies of IL-4 gene expression and secretion in BALB/c wildtype and Stat6-deficient mice infected with Leishmania major also
demonstrated normal IL-4 expression in Stat6-deficient mice during a primary infection, but delayed or decreased IL-4 expression
in these mice during a recall response (data not shown).
The memory IL-4 response produced by H. polygyrus-infected
wild-type mice is CD4⫹ T cell dependent and mast cell and Ab
independent
Stat6 signaling is required to generate a normal memory type 2
cytokine response in mice infected with a gastrointestinal
nematode parasite
Because many of the in vitro studies that demonstrated a Stat6
requirement for IL-4 production restimulated cultured T cells to
induce an IL-4 response, it was possible that the Stat6 requirement
reflected Stat6 dependence of generation of memory T cells that
rapidly produce IL-4 upon restimulation, rather than Stat6 dependence of a primary IL-4 response. To examine this possibility, we
compared the Stat6 dependence of IL-4 production during primary
and second inoculations of mice with H. polygyrus, a gastrointestinal nematode parasite that normally stimulates a type 2 cytokine
response (54). To determine whether results were specific for IL-4
or generalizable to other type 2 cytokines, we also evaluated IL-13
responses in the same experiments. As was the case with mice
given a primary N. brasiliensis infection, wild-type and Stat6-deficient mice made similar IL-4 responses to an initial H. polygyrus
infection (Fig. 4). Primary H. polygyrus-induced IL-13 responses
were also Stat6 independent. In wild-type mice, the IL-4 response
to a second H. polygyrus infection (i.e., the memory IL-4 response)
was equal in magnitude, but considerably more rapid than the IL-4
response to a primary H. polygyrus infection, while secondary
IL-13 responses were both larger and more rapid than primary
IL-13 responses in these mice. In contrast, IL-4 and IL-13 responses in Stat6-deficient mice to a second H. polygyrus infection
were more like normal primary than normal memory responses:
the IL-4 response was large, but developed more slowly than the
memory IL-4 response in wild-type mice, and the IL-13 response
was considerably smaller than the memory IL-13 response in wildtype mice. The failure of Stat6-deficient mice to make normal IL-4
and IL-13 responses to a second inoculation with H. polygyrus was
not a result of a less severe infection in Stat6-deficient than in
wild-type mice; in fact, the magnitude of the second infection was
considerably more severe in the Stat6-deficient than in the wild-
Stat6 promotes Th2 cell priming, but not the initial T cell IL-4
response, in vitro
To determine whether the normal primary IL-4 response and defective memory IL-4 response that we observed in vivo in Stat6deficient mice could also be demonstrated in vitro, we evaluated
the Stat6 dependence of IL-4 production by CD8⫹ T cell-depleted
lymph node and spleen cells following primary stimulation with
IL-2 plus soluble anti-CD3 and anti-CD28 mAbs, and secondary
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Stat6 signaling contributes to a 2°, but not a 1° type 2 cytokine response to H. polygyrus. Wild-type and Stat6-deficient mice on a
mixed C57BL/6-129 background (10/group) were inoculated orally with
200 infective, third stage H. polygyrus larvae. After 14 days, mice were
treated with pyrantal pamoate to terminate infection. Mice were reinoculated orally with 200 infective, third stage H. polygyrus larvae 14 days
later. IL-4 levels were monitored by CCCA in five mice of each strain
before and 1, 3, 5, 7, 9, 11, and 15 days after a first inoculation and, in five
different mice of each strain, immediately before and 1, 3, 5, 7, 9, 11, and
15 days after a second inoculation. Serum IL-13 levels were determined at
the same time points, as described in Materials and Methods.
These observations suggested that Stat6 signaling is required for
the changes in cell physiology that allow memory T cells to rapidly
produce IL-4 upon restimulation. An alternative possibility, however, is that the rapid IL-4 response that accompanies a second
H. polygyrus infection results from the release of preformed IL-4
from non-T cells. Mast cells or basophils, for example, which can
produce and store IL-4 (24), might be stimulated to release this
cytokine by cross-linking H. polygyrus-specific IgE bound to their
high affinity IgE receptors. This response might be defective in
Stat6-deficient mice, which produce little or no IgE (13–15).
For this reason, experiments were performed to differentiate T
cell from mast cell or basophil-mediated IL-4 production during
the first day of a second H. polygyrus infection. First, the kinetics
of IL-4 production following a second H. polygyrus infection was
determined. Relatively little IL-4 was secreted 4 – 6 h after the
challenge inoculation, while large quantities were released 8 –10 h
after inoculation and still larger quantities 12–14 h after inoculation (Fig. 5A). This result is more suggestive of new synthesis and
secretion of IL-4 in response to worm inoculation, as opposed to
rapid release of preformed IL-4 stores. Second, IL-4 responses
were compared during first and second H. polygyrus infections in
wild-type and W/Wv mice, which have ⬍1% of the normal number
of mast cells (55). W/Wv mice made normal IL-4 responses to
primary and second infections with H. polygyrus (Fig. 5B). Because some basophils are present in W/Wv mice (56) and these
cells can secrete IL-4 in response to cross-linking of cell membrane Fc⑀RI or Fc␥RIII (24), we also examined IL-4 responses to
primary and second H. polygyrus infections in ␮MT mice that
cannot secrete IL-4 through this mechanism because they lack B
cells and Ig (57). ␮MT mice made normal IL-4 responses to both
primary and second infections with H. polygyrus (Fig. 5C). To
confirm that the rapid IL-4 response to a second H. polygyrus
inoculation is CD4⫹ T cell derived, we tested the ability of a
cytotoxic anti-CD4 mAb (GK1.5), administered 1 day before a
second H. polygyrus inoculation, to inhibit the rapid IL-4 response.
In contrast to mast cell or Ig deficiencies, this treatment reduced
the early IL-4 response to a second H. polygyrus infection to an
undetectable level (Fig. 5D). Thus, the rapid, Stat6-dependent,
memory IL-4 response to H. polygyrus appears to be derived from
CD4⫹ T cells, rather than mast cells or basophils.
The Journal of Immunology
stimulation with plate-bound anti-CD3 mAb. CD8⫹ T cell-depleted lymph node and spleen cells were labeled with CFSE to
allow determination of the number of cell divisions that had occurred during culture, then cultured with anti-CD3 and anti-CD28
mAbs, stained for surface CD4 and cytoplasmic IL-4 after 3 days
of culture, and analyzed by flow cytometry for number of cell
divisions and IL-4 content of CD4⫹ cells. Similar and substantial
numbers of IL-4-containing CD4⫹ T cells were first observed
when cells from both wild-type and Stat6-deficient mice had divided three times (Fig. 6). In contrast, the number of anti-CD3/
CD28-primed CD4⫹ T cells that expressed IL-4, but not IFN-␥,
following restimulation with anti-CD3 mAb, was more than
20-fold greater for cells from wild-type than from Stat6deficient mice.
The rapid in vivo IL-4 response to anti-CD3 mAb is Stat6
independent
Our observations that Stat6-deficient mice are not primed by infection with a gastrointestinal nematode parasite to rapidly pro-
FIGURE 6. IL-4 expression, but not Th2 development, occurs in vitro
in the absence of Stat6-mediated signaling. Lymph node and spleen cells
from unimmunized wild-type and Stat6-deficient mice were depleted of
CD8⫹ cells, labeled with CFSE, and stimulated with soluble anti-CD3 and
anti-CD28 mAbs in the presence of IL-2. Cells were stained for surface
CD4 and cytoplasmic IL-4 and analyzed by flow cytometry on day 3 for
CFSE and intracellular IL-4 expression by CD4⫹ cells. CD8-depleted
lymph node cells that were not labeled with CFSE were cultured as above,
then washed and restimulated on day 6 with plate-bound anti-CD3 mAb,
and analyzed the following day by flow cytometry for cytoplasmic IL-4
and IFN-␥ expression by CD4⫹ cells.
duce IL-4 upon reexposure to that parasite and that CD4⫹ lymph
node cells from Stat6-deficient mice are not induced by culture
with anti-CD3 and anti-CD28 mAbs to generate a rapid IL-4 response upon in vitro restimulation with anti-CD3 mAb raised the
possibility that Stat6 signaling is universally required to promote
the differentiation, survival, or growth of T cells that can rapidly be
induced to produce IL-4. To investigate this possibility, we compared 4-h in vivo IL-4 responses in wild-type and Stat6-deficient
mice to injection of anti-CD3 mAb. Most of the in vivo IL-4 response to this mAb is derived from T cells that express NK cell
markers and are restricted by MHC class I-like CD1 molecules,
rather than by conventional MHC class I- or MHC class II-restricted T cells (31, reviewed in Ref. 58). Stat6-deficient mice were
found to make large, rapid IL-4 and IFN-␥ responses to anti-CD3
mAb that are indistinguishable in magnitude from those made by
wild-type mice of the same background strain (Fig. 7). Thus, Stat6independent pathways exist for the generation, expansion, and survival of T cells that can rapidly produce IL-4, and NK T cells are
FIGURE 7. CD3 stimulation induces similar in vivo IL-4 and IFN-␥
responses in wild-type and Stat6-deficient mice. Wild-type and Stat6-deficient mice (5/group), on a mixed C57BL/6, 129 background (5/group),
were injected i.v. with 10 ␮g of biotin-BVD4-1D11 (anti-IL-4) and 50 ␮g
of biotin-R46-A2 (anti-IFN-␥) or with these mAbs plus 10 ␮g of 145-2C11
(anti-CD3⑀ mAb). Mice were bled 4 h later, and serum levels of captured
IL-4 and IFN-␥ were determined by ELISA.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. The rapid IL-4 response made by mice given a second infection with H. polygyrus is CD4⫹ T cell derived. A, BALB/c mice (5/
group) were inoculated orally with 200 infective, third stage H. polygyrus
larvae. After 14 days, mice were treated with pyrantal pamoate to terminate
infection. Mice were reinoculated orally with 200 infective, third stage
H. polygyrus larvae 13 days later. IL-4 production was monitored by
CCCA 2 days before and 4, 8, and 12 h after the second worm inoculation.
B, Mast cell-deficient (W/Wv) mice or mice with a normal phenotype on
the same background (10/group) were inoculated orally with 200 infective,
third stage H. polygyrus larvae. After 14 days, mice were treated with
pyrantal pamoate to terminate infection. Mice were reinoculated orally
with 200 infective, third stage H. polygyrus larvae 14 days later. IL-4
production was monitored in five mice of each type by CCCA before and
1 day after the initial inoculation with H. polygyrus and, in additional
groups of five mice of each type, immediately before and 1 day after the
second inoculation with H. polygyrus. C, B cell-deficient (␮MT) mice or
normal mice on the same background (C57BL/6) (10/group) were treated
and tested as in B. D, Stat6-deficient and wild-type mice on a BALB/c
background (10/group) were inoculated orally with 200 infective, third
stage H. polygyrus larvae. After 14 days, mice were treated with pyrantal
pamoate to terminate infection. Fourteen days after that, mice were injected
i.v. with 1 mg of either anti-CD4 mAb (GK1.5) or an isotype-matched
control mAb (J1.2). One day later, all mice were reinoculated orally with
200 infective, third stage H. polygyrus larvae. IL-4 production was monitored in five mice of each group by CCCA 1 day after the second inoculation with H. polygyrus.
2307
2308
not a likely source of the Stat6-dependent, rapid, secondary IL-4
responses made in vivo by worm-inoculated mice and in vitro by
CD4⫹ anti-CD3/anti-CD28 mAb-primed lymph node cells.
Discussion
is not apparent why some of these parasites are such strong inducers of Stat6-independent type 2 cytokine responses. The ability of
parasite-derived allergens, such as APF, to induce a strong IL-4
response in the absence of Stat6 signaling, even if administered i.p.
rather than orally, makes it unlikely that mucosal Ag processing
and presentation is critical for Stat6-independent elicitation of IL-4
production. Instead, worm-derived allergens, and probably the living parasites, may directly provide signals to T cells that substitute
for Stat6 signaling or induce APCs to express molecules that have
the same effect on T cells.
The ability of Stat6-deficient mice to produce in vivo IL-4 responses is consistent with suggestions that initial production of
IL-4 by T cells may be a stochastic event that is promoted by rapid
T cell proliferation (43, 77), rather than an event programmed by
Stat6 signaling. Our observations clearly exclude Stat6 signaling
as a requirement for the initial T cell IL-4 response, but leave open
the possibility that another, as yet unidentified, factor is required to
induce naive T cells to secrete IL-4.
The inability of Stat6-deficient mice to generate normal memory
IL-4 responses following a second infection with H. polygyrus or
N. brasiliensis, even though these parasites induce Stat6-deficient
mice to make normal primary IL-4 responses, is consistent with a
previous observation that T cells from N. brasiliensis-infected IL4R␣-deficient mice make a markedly diminished IL-4 response
when restimulated in vitro with anti-CD3 mAb (34). Taken together, these observations suggest that IL-4R␣-associated Stat6
signaling may have a nonredundant role in the generation of a
normal memory Th2 cell population. This difference in the Stat6
requirements for the in vivo generation of primary type 2 cytokine
responses vs a secondary Th2 response is also consistent with in
vitro observations that anti-IL-4 or anti-IL-4R␣ mAb does not inhibit IL-4 production by naive CD4⫹ T cells stimulated for the first
time with Ag plus CD28 ligands or anti-TCR ␤ and anti-CD28
mAbs, but prevents these cells from rapidly producing IL-4 and
IL-5 upon restimulation (12, 71, 72) and that restimulation of T
cells from Stat6-deficient mice with anti-CD3 or anti-CD3 and
anti-CD28 mAbs fails to elicit IL-4 production (13–15).
It is possible, but unlikely, that the Stat6 requirement for development of normal secondary IL-4 and IL-13 responses results from
absence of a positive feedback loop in which IL-4 stimulation of
Stat6 stimulates further IL-4 production. As seen in Figs. 3 and 4,
the primary IL-4 responses to H. polygyrus and N. brasiliensis,
which last several days, persist as long in Stat6-deficient mice as
in wild-type mice. These responses are sufficiently long that they
should provide time for a positive feedback loop to influence response magnitude. During the time that IL-4 is being actively produced (primary infection), there is no difference in the magnitude
of the IL-4 response between the wild-type and Stat6-deficient
mice. It is only when H. polygyrus infections are terminated by
drug treatment, with cessation of IL-4 production, and mice are
later reinoculated with H. polygyrus that the IL-4 response is defective. Because the difference between wild-type and Stat6-deficient mice appears when there is no detectable IL-4 production
(the period between primary and secondary immunizations) rather
than when IL-4 is being actively produced (the course of the primary immunization), it seems likely that defective generation of T
cells that rapidly produce IL-4 upon restimulation with Ag, a failure of these cells to survive in the absence of continuing antigenic
or IL-4 stimulation, or absent Stat6-mediated clonal expansion that
is selective for memory cell precursors, rather than absent Stat6mediated clonal expansion of IL-4-secreting cells, accounts for the
defective rapid IL-4 response to a second H. polygyrus infection in
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Observations reported in this study demonstrate that in vivo antigenic stimulation can induce a primary Stat6-independent, IL4R␣-independent IL-4 response that is normal in its magnitude and
kinetics, and demonstrate that the Stat6 signaling requirement for
in vivo generation of a primary IL-4 response is variable, but
never, in our experience, absolute. We cannot totally rule out the
possibility that the primary IL-4 responses that develop in immunized Stat6-deficient mice are made by cells other than CD4⫹ T
cells. This possibility seems unlikely, however, because previous
studies with mice immunized with GaM␦ or inoculated with
N. brasiliensis or H. polygyrus indicated that CD4⫹ T cells account for nearly all of the splenic and mesenteric lymph node IL-4
mRNA response during a primary immunization (Ref. 54 and
K. B. Madden, unpublished data). It is also possible that special T
cell populations, such as NK T cells, are responsible for the Stat6independent IL-4 production that we have observed. It is unlikely
that classical NK T cells, which are CD1 restricted (58), account
for this IL-4 production, because previous studies have demonstrated normal IgE and IL-4 production in ␤2-microglobulin-deficient mice immunized with OVA and other soluble proteins (59 –
61), GaM␦ (31), or N. brasiliensis (62), even though ␤2microglobulin-deficient mice do not express CD1 (58). This does
not eliminate the possibility that primary IL-4 responses in Stat6deficient mice are made by nonclassical NK T cells, which are not
CD1 restricted (63, 64). However, the slow development of in vivo
primary IL-4 responses to soluble Ags or worm infection, as opposed to the rapid in vivo IL-4 responses to anti-CD3 mAb treatment (which stimulates IL-4 production in both classical and nonclassical NK T cells (31)) makes it less likely that even
nonclassical NK T cells are responsible for primary IL-4 responses
in Stat6-deficient mice.
In mice immunized with protein Ags or inoculated with gastrointestinal nematode parasites, the Stat6 dependence of the primary
IL-4 response was greatest for immunogens that induce a mixed,
type 0 cytokine response and least for immunogens that induce a
strong, heavily biased type 2 cytokine response. This observation
suggests that Stat6 signaling may promote IL-4 production during
a primary response indirectly, by inhibiting the IL-4-suppressive
effects of cytokines such as IL-12 and IL-18 (10), or that Stat6
enhances other stimuli that promote IL-4 production, such as CD4
or CD28 signaling (12, 65–72), when those signals are relatively
weak, but not when those signals are strong. The simplest possibility is that strong T cell costimulation, in the absence of molecules such as IL-12, IL-18, and IFN-␣/␤/␥, which inhibit type 2
cytokine production, is all that is needed to induce an optimal type
2 cytokine response. This view is consistent with previous in vitro
and in vivo observations that induction of primary mouse and human T cell IL-4 responses by Ag or anti-CD3 mAb requires CD4
and CD28 costimulation (12, 27, 65, 67, 70, 72–74) and in vitro
observations that primary in vitro IL-4 responses are not always
inhibited by anti-IL-4 or anti-IL-4R mAbs (12, 71, 72). This view
is also consistent with the remarkable ability of GaM␦ to induce
Stat6-independent IL-4 production in vivo; in addition to inducing
most B cells to process and present goat IgG, this Ab most likely
optimizes CD4 and CD28 costimulation by directly increasing B
cell MHC class II and CD86 expression (75, 76). Because little is
known about the cells and costimuli that are involved in the presentation of Ags derived by gastrointestinal nematode parasites, it
Stat6 DEPENDENCE OF THE IL-4 RESPONSE
The Journal of Immunology
Acknowledgments
We thank Colleen Byrd for technical assistance, Michael Grusby and
James Ihle for their generous gifts of Stat6-deficient mice, Nancy NobenTrauth for her generous gift of IL-4R␣-deficient mice and for allowing us
to mention her unpublished data, DNAX Research Institute for the gift of
mAbs, and the Schering-Plough Research Institute for mouse IL-4.
References
1. Locksley, R. M., and S. L. Reiner. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.
2. Finkelman, F. D., T. Shea-Donohue, J. Goldhill, C. A. Sullivan, S. C. Morris,
K. B. Madden, W. C. Gause, and J. F. Urban, Jr. 1997. Cytokine regulation of
host defense against parasitic gastrointestinal nematodes. Annu. Rev. Immunol.
15:505.
3. Urban, J. F., Jr., N. Noben-Trauth, D. D. Donaldson, K. B. Madden, S. C. Morris,
M. Collins, and F. D. Finkelman. 1998. IL-13, IL-4R␣, and Stat6 are required for
the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8:255.
4. Medzhitov, R., and C. A. Janeway, Jr. 1997. Innate immunity: the virtues of a
nonclonal system of recognition. Cell 91:295.
5. Janeway, C. A., Jr. 1998. Presidential address to the American Association of
Immunologists: the road less traveled by: the role of innate immunity in the
adaptive immune system. J. Immunol. 161:539.
6. Fraser, I. P., H. Koziel, and R. A. Ezekowitz. 1998. The serum mannose-binding
protein and the macrophage mannose receptor are pattern recognition molecules
that link innate and adaptive immunity. Semin. Immunol. 10:363.
7. Finkelman, F. D., A. Svetic, I. Gresser, C. Snapper, J. Holmes, P. P. Trotta,
I. M. Katona, and W. C. Gause. 1991. Regulation by interferon-␣ of immunoglobulin isotype selection and lymphokine production in mice. J. Exp. Med. 174:
1179.
8. Trinchieri, G. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive
immunity. Annu. Rev. Immunol. 13:251.
9. Krieg, A. M., L. Love-Homan, A. K. Yi, and J. T. Harty. 1998. CpG DNA
induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428.
10. Okamura, H., S. Kashiwamura, H. Tsutsui, T. Yoshimoto, and K. Nakanishi.
1998. Regulation of interferon-␥ production by IL-12 and IL-18. Curr. Opin.
Immunol. 10:259.
11. Le Gros, G., S. Z. Ben-Sasson, R. Seder, F. D. Finkelman, and W. E. Paul. 1990.
Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and
IL-4 are required for in vitro generation of IL-4-producing cells. J. Exp. Med.
172:921.
12. Croft, M., and S. L. Swain. 1995. Recently activated naive CD4 T cells can help
resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation
to secretion of T helper 2-type cytokines. J. Immunol. 154:4269.
13. Kaplan, M. H., U. Schindler, S. Smiley, and M. Grusby. 1996. Stat6 is required
for mediating responses to IL-4 and for development of Th2 cells. Immunity
4:313.
14. Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson,
R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al. 1996. Lack
of IL-4-induced Th2 response and IgE class switching in mice with disrupted
Stat6 gene. Nature 380:630.
15. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura,
K. Nakanishi, N. Yoshida, T. Kishimoto, and S. Akira. 1996. Essential role of
Stat6 in IL-4 signaling. Nature 380:627.
16. Kubo, M., J. Ransom, D. Webb, Y. Hashimoto, T. Tata, and T. Nakayama. 1997.
T-cell subset-specific expression of the IL-4 gene is regulated by a silencer element and STAT6. EMBO J. 16:4007.
17. Murphy, K. 1998. T lymphocyte differentiation in the periphery. Curr. Opin.
Immunol. 10:226.
18. O’Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275.
19. Kaplan, M. H., and M. J. Grusby. 1998. Regulation of T helper cell differentiation
by STAT molecules. J. Leukocyte Biol. 64:2.
20. Kotanides, H., and N. C. Reich. 1996. Interleukin-4-induced STAT6 recognizes
and activates a target site in the promoter of the interleukin-4 receptor gene.
J. Biol. Chem. 271:25555.
21. Szabo, S. J., L. H. Glimcher, and I. C. Ho. 1997. Genes that regulate interleukinexpression in T cells. Curr. Opin. Immunol. 9:776.
22. Ben-Sasson, S. Z., G. Le Gros, D. H. Conrad, F. D. Finkelman, and W. E. Paul.
l990. Cross-linking Fc receptors stimulate splenic non-B, non-T cells to secrete
IL-4 and other lymphokines. Proc. Natl. Acad. Sci. USA 87:1421.
23. Conrad, D. H., S. Z. Ben-Sasson, G. Le Gros, F. D. Finkelman, and W. E. Paul.
1990. Infection with Nippostrongylus brasiliensis or injection of anti-IgD antibodies markedly enhances Fc-receptor-mediated interleukin 4 production by
non-B, non-T cells. J. Exp. Med. 171:1497.
24. Seder, R. A., W. E. Paul, A. M. Dvorak, S. J. Sharkis, A. Kagey-Sobotka, Y. Niv,
F. D. Finkelman, S. A. Barbieri, S. J. Galli, and M. Plaut. 1991. Mouse splenic
and bone marrow cell populations that express high affinity Fc⑀ receptors and
produce IL-4 are highly enriched in basophils. Proc. Natl. Acad. Sci. USA 88:
2835.
25. Yoshimoto, T., and W. E. Paul. 1994. CD4⫹ NK1.1⫹ T cells promptly produced
IL-4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179:1285.
26. Sabin, E. A., M. A. Kopf, and E. J. Pearce. 1996. Schistosoma mansoni egginduced early IL-4 production is dependent upon IL-5 and eosinophils. J. Exp.
Med. 184:1871.
27. Brown, D. R., D. J. Fowell, D. B. Corry, R. A. Wynn, N. H. Moskowitz,
A. W. Cheever, R. M. Locksley, and S. L. Reiner. 1996. ␤2-Microglobulindependent NK1.1⫹ T cells are not essential for T helper cell 2 immune responses.
J. Exp. Med. 184:1295.
28. Guery, J. C., F. Galbiati, S. Smiroldo, and L. Adorini. 1996. Selective development of T helper 2 cells induced by continuous administration of low dose soluble
proteins to normal and ␤2-microglobulin-deficient BALB/c mice. J. Exp. Med.
183:484.
29. Von der Weid, T., A. M. Beebe, D. C. Roopenian, and R. L. Coffman. 1996. Early
production of IL-4 and induction of Th2 responses in the lymph node originate
from an MHC class I-independent CD4⫹NK1.1⫺ T cell population. J. Immunol.
157:4421.
30. Smiley, S. T., M. H. Kaplan, and M. J. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science
275:977.
31. Morris, S. C., R. L. Coffman, and F. D. Finkelman. 1998. In vivo IL-4 responses
to anti-IgD antibody are MHC class II-dependent, ␤2-microglobulin-independent,
and Stat6-independent. J. Immunol. 160:3299.
32. Finkelman, F. D., J. Ohara, D. K. Goroff, J. Smith, N. Villacreses, J. J. Mond, and
W. E. Paul. l986. Production of BSF-1 during an in vivo, T-dependent immune
response. J. Immunol. l37:2878.
33. Svetić, A., F. D. Finkelman, Y. C. Jian, C. W. Dieffenbach, D. E. Scott,
K. F. McCarthy, A. D. Steinberg, and W. C. Gause. 1991. Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD
antibody. J. Immunol. 147:2391.
34. Noben-Trauth, N., L. D. Shultz, F. Brombacher, J. F. Urban, H. Gu, and
W. E. Paul. 1997. An interleukin-4 (IL-4)-independent pathway for CD4⫹ T cell
IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. Natl. Acad. Sci.
USA 94:10838.
35. Noben-Trauth, N., G. Kohler, K. Burki, and B. Ledermann. 1996. Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line. Transgenic Res.
5:487.
36. Finkelman, F. D., S. W. Kessler, J. F. Mushinski, and M. Potter. l98l. IgD secreting murine plasmacytomas: identification and characterization of two IgD
myeloma proteins. J. Immunol. l26:680.
37. Wilde, D. B., P. Marrack, J. Kappler, D. P. Dialynas, and F. W. Fitch. 1983.
Evidence implicating L3T4 in class II MHC antigen reactivity: monoclonal antibody GK 1.5 (anti-L3T4) blocks class II MHC antigen-specific proliferation,
release of lymphokines, and binding by cloned murine helper T lymphocyte lines.
J. Immunol. 131:2178.
38. Leo, O., M. Foo, D. H. Sachs, L. E. Samelson, and J. A. Bluestone. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc.
Natl. Acad. Sci. USA 84:1374.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Stat6-deficient mice. The possibility that Stat6 signaling is required to prevent memory cell death is consistent with considerable evidence that IL-4 has antiapoptotic effects, but less consistent
with evidence that these antiapoptotic effects are Stat6 independent
(78, 79). Recent observations that permanent commitment to type
2 cytokine secretion is accompanied by increased DNase sensitivity and demethylation of the IL-4 locus (80) leave open the possibility that these changes are not required to induce a primary IL-4
response. If so, the IL-4 locus may temporarily open in response to
an acute Stat6-independent stimulus, but close once that stimulus
terminates, preventing a rapid IL-4 response to restimulation. In
contrast, Stat6 signaling may permanently open the IL-4 locus,
allowing the generation of memory cells that rapidly produce IL-4
upon restimulation.
In sum, our data, taken together with previous observations (12,
43, 65, 66, 69, 71, 72), suggest the following scheme for the regulation of IL-4 production: Initial IL-4 production during a primary response may require CD4 and CD28 costimulation, but does
not depend on IL-4 itself, although, in some circumstances, it is
promoted by IL-4 activation of Stat6. Thus, no initial production of
IL-4 by non-T cells or specialized T cells should be necessary to
induce conventional CD4⫹ T cells to produce IL-4. IL-4-induced
Stat6 signaling during a primary response may enhance IL-4 production later on in that response, particularly if costimulation is
limiting. Stat6 signaling is not universally required for the generation of a rapid T cell IL-4 response, but makes an important
contribution to the differentiation, survival, and/or clonal expansion of conventional memory T cells that rapidly produce IL-4
upon restimulation.
2309
2310
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
soluble proteins to normal and ␤2-microglobulin-deficient BALB/c mice. J. Exp.
Med. 183:485.
Zhang, Y., K. H. Rogers, and D. B. Lewis. 1996. ␤2-microglobulin-dependent T
cells are dispensable for allergen-induced T helper 2 responses. J. Exp. Med.
184:1507.
Daser, A., B. Gerstenr, R. Hansen, S. Bulfone-Paus, and H. Renz. 1998. Impaired
NK1.1⫹ T cells do not prevent the development of an IgE-dependent allergic
phenotype. Clin. Exp. Allergy 28:950.
Brown, D. R., D. J. Fowell, D. B. Corry, T. A. Wynn, N. H. Moskowitz,
A. W. Cheever, R. M. Locksley, and S. L. Reiner. 1996. ␤2-Microglobulindependent NK1.1⫹ T cells are not essential for T helper cell 2 immune responses.
J. Exp. Med. 184:1295.
Chiu, Y. H., J. Jayawardena, A. Weiss, D. Lee, S. H. Park, A. Dautry-Varsat, and
A. Bendelac. 1999. Distinct subsets of CD1d-restricted T cells recognize selfantigens loaded in different cellular compartments. J. Exp. Med. 189:103.
Behar, S. M., T. A. Podrebarac, C. J. Roy, C. R. Wang, and M. B. Brenner. 1999.
Diverse TCRs recognize murine CD1. J. Immunol. 162:161.
Lu, P., X. di Zhou, S.-J. Chen, M. Moorman, S. C. Morris, F. D. Finkelman,
P. Linsley, J. F. Urban, and W. C. Gause. 1994. CTLA-4 ligands are required to
induce an in vivo IL-4 response to a gastrointestinal nematode parasite. J. Exp.
Med. 180:693.
Lu, P., X. D. Zhou, S. J. Chen., M. Moorman, A. Schoneveld, S. Morris,
F. D. Finkelman, P. Linsley, E. Claassen, and W. C. Gause. 1995. Requirement
of CTLA-4 counter receptors for IL-4 but not IL-10 elevations during a primary
systemic in vivo immune response. J. Immunol. 154:1078.
Corry, D. B., S. L. Reiner, P. S. Linsley, and R. M. Locksley. 1994. Differential
effects of blockade of CD28 –B7 on the development of Th1 or Th2 effector cells
in experimental leishmaniasis. J. Immunol. 153:4142.
Kalinski, P., C. M. U. Hilkens, E. A. Wierenga, T. C. T. M. van der Pouw-Kraan,
R. A. W. van Lier, J. D. Bos, M. L. Kapsenberg, and F. G. M. Snijdewint. 1995.
Functional maturation of human naive T helper cells in the absence of accessory
cells: generation of IL-4-producting T helper cells does not require exogenous
IL-4. J. Immunol. 154:3753.
King, C. L., R. J. Stupi, N. Craighead, C. H. June, and G. Thyphronitis. 1995.
CD28 activation promotes Th2 subset differentiation by human CD4⫹ cells. Eur.
J. Immunol. 25:587.
Webb, L. M., and M. Feldmann. 1995. Critical role of CD28/B7 costimulation in
the development of human Th2 cytokine-producing cells. Blood 86:3479.
Bix, M., Z-.E. Wang, B. Thiel, N. J. Schork, and R. M. Locksley. 1998. Genetic
regulation of commitment to interleukin 4 production by a CD4⫹ T cell-intrinsic
mechanism. J. Exp. Med. 188:2289.
Yang, L. P., C. E. Demeure, D.-G. Byun, N. Vezzio, and G. Delespesse. 1995.
Maturation of neonatal human CD4 T cells. III. Role of B7 co-stimulation at
priming. Int. Immunol. 7:1987.
Fowell, D. J., J. Magram, C. W. Turck, N. Killeen, and R. M. Locksley. 1997.
Impaired Th2 subset development in the absence of CD4. Immunity 6:559.
Leitenberg, D., Y. Boutin, S. Constant, and K. Bottomly. 1998. CD4 regulation
of TCR signaling and T cell differentiation following stimulation with peptides of
different affinities for the TCR. J. Immunol. 161:1194.
Mond, J. J., E. Seghal, J. Kung, and F. D. Finkelman. l98l. Increased expression
of I-region-associated antigen (Ia) on B cells after cross-linking of surface immunoglobulin. J. Immunol. l27:88l.
Brink, R., C. C. Goodnow, and A. Basten, 1995. IgD expression on B cells is
more efficient than IgM but both receptors are functionally equivalent in upregulation of CD80/CD86 co-stimulatory molecules. Eur. J. Immunol. 25:1980.
Bix, M., and R. M. Locksley. 1998. Independent and epigenetic regulation of the
interleukin-4 alleles in CD4⫹ T cells. Science 281:1352.
Vella, A., T. K. Teague, J. Ihle, J. Kapppler, and P. Marrack. 1997. Interleukin
4 (IL-4) or IL-7 prevents the death of resting T cells: Stat6 is probably not
required for the effect of IL-4. J. Exp. Med. 186:325.
Zamorano, J., and A. D. Keegan. 1998. Regulation of apoptosis by tyrosinecontaining domains of IL-4R␣: Y497 and Y713, but not the STAT6-docking
tyrosines, signal protection from apoptosis. J. Immunol. 161:859.
Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates
cytokine gene expression during T cell differentiation. Immunity 9:765.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
39. Spitalny, G., and E. Havell. 1984. Monoclonal antibody to murine ␥ interferon
inhibits lymphokine-induced antiviral and macrophage tumoricidal activities.
J. Exp. Med. 159:1560.
40. Abrams, J. S., M. G. Roncarolo, H. Yssel, U. Anderson, G. J. Gleich, and
J. Silver. 1992. Strategies of anti-cytokine monoclonal antibody development.
Immunol. Rev. 127:5.
41. Slade, S. J., and J. Langhorne. 1989. Production of interferon-␥ during infection
of mice with Plasmodium chabaudi chabaudi. Immunobiology 179:353.
42. Goroff, D. K., A. Stall, J. J. Mond, and F. D. Finkelman. l986. In vitro and in vivo
B lymphocyte activating properties of monoclonal anti-␦ antibodies. I. Determinants of B lymphocyte activating properties. J. Immunol. l35:2382.
43. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M .A. Mahowald,
J. R. Sider, T. F. Gajewski, C. R. Wang, and S. L. Reiner. 1998. Helper T cell
differentiation is controlled by the cell cycle. Immunity 9:229.
44. Donaldson, D. D., M. J. Whitters, L. J. Fitz, T. Y. Neben, H. Finnerty,
S. L. Henderson, R. M. O’Hara, Jr., D. R. Beier, K. J. Turner, C. R. Wood, and
M. Collins. 1998. The murine IL-13 receptor ␣2: molecular cloning, characterization, and comparison with murine IL-13 receptor ␣1. J. Immunol. 161:2317.
45. Katona, I. M., J. F. Urban, I. Scher, C. Kanellopoulos, and F. D. Finkelman. 1983.
Induction of an IgE immune response in mice infected with Nippostrongylus
brasiliensis: characterization of lymphoid cells with intracytoplasmic or surface
IgE. J. Immunol. l30:350.
46. Alizadeh, H., J. F. Urban, Jr., I. M. Katona, and F. D. Finkelman. 1986. Cells
containing IgE in the intestinal mucosa of mice infected with the nematode parasite Trichinella spiralis are predominantly of a mast cell lineage. J. Immunol.
l37:2555.
47. Urban, J. F., Jr., I. M. Katona, W. E. Paul, and F. D. Finkelman. 1991. Interleukin
4 is important in protective immunity to a gastrointestinal nematode infection in
mice. Proc. Natl. Acad. Sci. USA 88:5513.
48. Finkelman, F. D., and S. C. Morris. 1999. Development of an assay to measure
in vivo cytokine production in the mouse. Int. Immunol. 11:1811.
49. HayGlass, K. T., M. Wang, R. S. Gieni, C. Ellison, and J. Gartner. 1996. In vivo
direction of CD4 T cells to Th1 and Th2-like patterns of cytokine synthesis. Adv.
Exp. Med. Biol. 409:309.
50. McWilliam, A. S., G. A. Stewart, and K. J. Turner. 1987. An immunochemical
investigation of the allergens from Ascaris suum perienteric fluid: cross-reactivity, molecular weight distribution, and correlation with phosphorylcholine-containing compounds. Int. Arch. Allergy Appl. Immunol. 82:125.
51. McGibbon, A. M., J. F. Christie, M. W. Kennedy, and T. D. Lee. 1990. Identification of the major Ascaris allergen and its purification to homogeneity by
high-performance liquid chromatography. Mol. Biochem. Parasitol. 39:163.
52. Finkelman, F. D., K. B. Madden, A. W. Cheever, I. M. Katona, S. C. Morris,
M. K. Gately, B. R. Hubbard, W. C. Gause, and J. F. Urban, Jr. 1994. Effects of
interleukin-12 on immune responses and host protection in mice infected with
intestinal nematode parasites. J. Exp. Med. 179:1563.
53. Ramaswamy, K., D. Negrao-Correa, and R. Bell. 1996. Local intestinal immune
responses to infections with Trichinella spiralis: real-time, continuous assay of
cytokines in the intestinal (afferent) and efferent thoracic duct lymph of rats.
J. Immunol. 156:4328.
54. Svetić, A., K. B. Madden, X. di Zhou, P. Lu, I. M. Katona, F. D. Finkelman,
J. F. Urban, Jr., and W. C. Gause. 1993. A primary intestinal helminthic infection
rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3.
J. Immunol. 150:3434.
55. Kitamura, Y., S. Go, and K. Hatanaka. 1978. Decrease of mast cells in W/Wv
mice and their increase by bone marrow transplantation. Blood 52:447.
56. Steeves, E. B., and J. R. Allen. 1990. Basophils in skin reactions of mast celldeficient mice infested with Dermacentor variabilis. Int. J. Parasitol. 20:655.
57. Kitamura, D., J. Roes, R. Kuhn, and K. Rajewsky. 1991. A B cell-deficient mouse
by targeted disruption of the membrane exon of the immunoglobulin ␮ chain
gene. Nature 350:423.
58. Bendelac, A., M. N. Rivera, S.-H. Park, and J. H. Roarck. 1997. Mouse CD1specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.
59. Guery, J. C., F. Galbiati, S. Smiroldo, and L. Adorini. 1996. Selective development of T helper (Th)2 cells induced by continuous administration of low dose
Stat6 DEPENDENCE OF THE IL-4 RESPONSE