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Have Opposing Effects, While TGF-ß Positively Regulates Its Own Production
*
Lymphokine Regulation Unit and
Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production could
not be detected from highly purified naive CD4+/lymphocyte
endothelial cell adhesion molecule (LECAM)-1high cells
following primary stimulation for 36 h with plate-bound
anti-CD3, anti-CD28, and IL-2. This population was subsequently
used to study the differentiation of TGF-ß-producing CD4+
T cells. In further studies, naive
CD4+/LECAM-1high cells from TCR transgenic mice
of both the BALB/c and B10.A backgrounds were stimulated with
T-depleted spleen cells (TDS) and specific peptide in the presence of
various cytokines and/or cytokine antagonists for 5 days, restimulated,
and TGF-ß, IL-4, and IFN- production were measured. Priming
conditions favoring high IL-4 production and/or low IFN- production
greatly enhanced TGF-ß production in secondary cultures. Furthermore,
the presence of IL-10 in cultures was associated with an increase in
TGF-ß production following restimulation. The importance of IL-4 and
IFN- in regulating TGF-ß production was confirmed in studies
showing that cells from IFN--/- mice produced more
TGF-ß, while cells from IL-4-/- mice produced less
TGF-ß compared with wild-type controls. Finally, the addition of
exogenous TGF-ß to priming cultures significantly enhanced the
production of TGF-ß upon restimulation, demonstrating that TGF-ß
has a role in self-regulating its own production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While the factors controlling the development of Th1 and Th2 cells
have been well defined (8, 9, 10, 11, 12, 13), relatively little is known concerning
the factors controlling the development of TGF-ß-producing cells. On
the one hand, there are several reports linking IL-4 production to such
development (14, 15, 16), but this has so far consisted of studies of whole
tissues, bulk cell populations, and cell lines, which show that IL-4-
and TGF-ß-producing cells develop under similar conditions. In
addition, the requirement of IL-4 for TGF-ß induction was questioned
by the studies of Powrie et al. (17), who showed that resolution of a
TGF-ß-dependent experimental colitis occurs following adoptive
transfer of CD4+ T cells from IL-4-/- mice.
In addition, it has been reported that TGF-ß production could be
enhanced by neutralization of IL-12 and IFN- (18) in the absence of
an effect on IL-4 production, again indicating that the generation of
TGF-ß-producing cells occurs independently of the Th2 response. These
observations indicate that the factors influencing TGF-ß production
are less well defined than those regulating IFN- and IL-4
production.
In the studies reported here, we have addressed this latter point by
examining the conditions under which naive CD4+ T cells
differentiate into TGF-ß-producing cells in vitro. We found that such
differentiation occurs under priming conditions favoring induction of
Th2 and inhibition of Th1 responses. This finding was corroborated by
the fact that primed CD4+ T cells from
IL-4-/- mice produced less TGF-ß, while cells from
IFN--/- mice produced more TGF-ß, when compared with
cells from wild-type mice. In further studies, we showed that addition
of IL-10 to primary cultures enhanced the amount of TGF-ß and
inhibited the amount of IFN- produced upon restimulation, consistent
with the fact that inhibiting IFN- directly (as noted above) or
indirectly (i.e., via IL-10) leads to increased TGF-ß production.
Finally, we showed that TGF-ß itself was able to strikingly enhance
production of TGF-ß following restimulation. This effect is both
direct, occurring independently of IL-4 and/or IFN-, and indirect,
occurring through the ability of TGF-ß to inhibit IFN- produced in
priming cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR transgenic
(Tg)2 mice were produced
as previously described (11). All mice used in experiments were
heterozygous for the integration of TCR variable region V
11 and
Vß3 chains and maintained by backcrossing to B10.A mice. Homozygous
6- to 12-week-old female OVA 323–339- specific and
I-Ad-restricted DO11.10TCR-ß Tg mice (19) on a BALB/c
genetic background were kindly provided by Dr. Dennis Loh
(Washington University, St. Louis, MO). IL-4-deficient mice derived on
a BALB/c background were obtained from Nancy Noben-Trauth (NIAID,
Bethesda, MD) (20). IFN--deficient mice derived on a C57BL/6
background (21) were obtained from Genentech (San Francisco, CA).
Virus-free B10.A and BALB/c female mice (6–12 wk old) were obtained
from the Division of Cancer Treatment, National Cancer Institute
(Frederick, MD).
Tissue culture medium
RPMI 1640 medium supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), 1 mM sodium pyruvate, L-glutamine (2 mM), and 2-ME (50 µM) was used for all primary stimulations. For all secondary stimulations, cells were cultured in serum-free medium in which Nutridoma SP was substituted for FCS (Boehringer Mannheim, Indianapolis, IN).
Peptide
Peptides with the sequences corresponding to residues 88–104 of pigeon cytochrome c (KAERADLIAYLKQATAK) and to residues 323–339 (ISQAVHAAHAEINEAGR) of OVA were synthesized by the National Institute of Allergy and Infectious (NIAID) Diseases Laboratory of Molecular Structure.
Recombinant lymphokines
Recombinant mouse IL-2 and IL-10 were purchased from Genzyme
(Cambridge, MA). Mouse rIL-4 was obtained by a baculovirus expression
system utilizing a vector into which the IL-4 gene had been inserted by
C. Watson (Laboratory of Immunology, NIAID). One unit of IL-4 is
equivalent to 
0.5 pg. Mouse rIL-12 was a generous gift of Genetics
Institute (Cambridge, MA). Human TGF-ß was purchased from R&D Systems
(Minneapolis, MN).
Antibodies
Purified monoclonal rat anti-mouse IL-4 (11B11) (22) was
prepared by Verax Corporation (Hanover, NH). Rat anti-mouse IFN-
(XMG 1.2) (23), lymphocyte endothelial cell adhesion molecule (LECAM)-1
(MEL14) (24), was purchased from PharMingen (San Diego, CA). Sheep
anti-IL-12 was a generous gift of Genetics Institute. Chicken
anti-TGF-ß was purchased from R&D Systems. Anti-CD28 ascites was
a generous gift of Dr. James Allison (University of California,
Berkeley, CA). Anti-CD3 (2C11) (25) was a generous gift of Dr. Jeffrey
Bluestone (University of Chicago, Chicago, IL).
Preparation of accessory cells
TDSs were prepared from B10.A mice or BALB/c mice as previously described (10) and used as APCs. There were no T cells noted in the TDSs as assessed by FACS.
Preparation of T cells
CD4+ T cells from transgenic mice were prepared
in the following manner. Pooled lymph node cells and spleen cells were
removed from TCR/Tg, IL-4, or IFN--/- mice (4–8 wk of
age) and passed over a negatively selecting CD4 subset enrichment
column (R&D Systems). Cells were then stained with phycoerythrin
(PE)-labeled anti-CD4 and FITC LECAM-1. These cells were subjected
to FACS with a FACStarPlus (Becton Dickinson, Sunnyvale,
CA). Postsort analysis revealed >99.6%
CD4+/LECAM-1high cells.
Primary and secondary stimulation of Tg T cells
For primary stimulation,
CD4+/LECAM-1high and
CD4+/LECAM-1low T cells obtained by FACS were
stimulated on plates coated with anti-CD3 (10 µg/ml;
PB-anti-CD3) plus soluble anti-CD28 (1:500 serial dilution of
ascites) and IL-2 (10 U/ml) for 36 to 48 h in the presence of
additional cytokines or cytokine antagonists. TGF-ß, IL-4, and
IFN- production were assessed in culture supernatants by
cytokine-specific ELISA.
For secondary stimulation, priming cultures were first established by adding 3 to 5 x 105 sorted CD4+/LECAMhigh T cells to individual wells of 24-well plates in a total volume of 1.5 ml of media with irradiated (1000 rad) TDS cells, Ag (peptide or soluble anti-CD3), and various cytokines and anti-cytokines, as described in Results. After 48 h, cells were transferred to 6-well plates and supplemented with 3 ml of fresh medium only for an additional 2 to 3 days to allow for further expansion. CD4+ T cells were then washed three times, and 1 to 2 x 105 cells in a total volume of 200 µl were restimulated on 96-well microtiter plates coated with anti-CD3 (PB-anti-CD3) in the presence of soluble anti-CD28 (1/500 final dilution) and IL-2 (10 U/ml). Thirty-six hours later, supernatants were collected and assayed for lymphokine. All secondary restimulations were done in serum-free medium in which Nutridoma SP was substituted for FCS. It should be noted that, at the time of restimulation, cells were >99% CD4+ T cells.
Measurement of lymphokine production
IFN- was assayed by a specific two-site ELISA with reference
standard curves using known amounts of recombinant IFN-. The lower
limit of detection was 0.3 ng/ml. A cytokine-specific ELISA kit for
IL-4 was purchased from Endogen (Boston, MA). The lower limit of
sensitivity for the IL-4 ELISA was 3 U/ml. TGF-ß1 and IL-10 levels
were determined by ELISA from kits purchased from Genzyme. The lower
limit of detection was 50 to 100 pg/ml for TGF-ß1 and 30 to 60 pg/ml
for IL-10. All measurements of TGF-ß were done after supernatants
were acidified as per the instructions by the manufacturer. All TGF-ß
detected was latent, since there was no TGF-ß detected if the
supernatants were not acidified. In all experiments, serial dilutions
of supernatants were used to measure cytokine content for IL-4,
IFN-, and TGF-ß to ensure that all values were obtained from the
linear portion of the standard curve. In addition, IL-4, IFN-, and
TGF-ß production were all assessed from the same supernatants.
Supernatants were assayed in triplicate for all experiments. The SEM
was <10% in all experiments.
Statistical analysis
Normally distributed continuous variable comparisons were done employing the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our initial studies, we determined the TGF-ß production from naive and memory CD4+ T cells after primary stimulation in vitro in the absence of accessory cells. Accordingly, CD4+/LECAM-1high and CD4+/LECAM-1low T cells representing naive and memory cells, respectively, were obtained by cell sorting from DO11.10 OVA-TCR/Tg mice (BALB/c background) and stimulated on plates coated with anti-CD3 in the presence of soluble anti-CD28 and IL-2. After 36 h, culture supernatants were harvested and assayed for secreted TGF-ß.
As shown in Table I
,
CD4+/LECAM-1high (naive) T cells in primary
cultures produced low to undetectable amounts of TGF-ß as well as low
to undetectable amounts of IFN- or IL-4. In contrast,
CD4+/LECAM-1low (memory) T cells stimulated in
a similar fashion produced low but detectable amounts of TGF-ß (205
pg/ml) as well as substantial amounts of IL-4 along with low amounts of
IFN-. To determine whether these levels of TGF-ß production could
be influenced by the presence or absence of other cytokines in the
cultures, we also stimulated cells in the presence of added cytokines
and anti-cytokine Abs. As also shown in Table I, we found that the
addition of IL-4, but not anti-IL-4 or anti-IFN-, induced
the production of small but detectable amounts of TGF-ß (215 pg/ml)
in cultures of naive (LECAM-1high) T cells, but none of
these additions appreciably altered TGF-ß production in cultures of
memory (LECAM-1low) T cells.
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, naive T
cells stimulated with OVAp and TDS still do not produce detectable
TGF-ß, whereas memory CD4+ T cells produced both TGF-ß
and IL-4. Overall, these studies show that memory CD4+ T
cells produce low amounts of TGF-ß in association with the production
of large amounts of IL-4 and minimal amounts of IFN-, while naive
CD4+ T cells produce little or no TGF-ß upon primary
stimulation in vitro. Thus, naive
CD4+/LECAM-1high cells constitute a defined
population of cells that is suitable for the study of the
differentiation of cells capable of producing TGF-ß following in
vitro priming.
Priming of naive CD4+/LECAM-1high T
cells (derived from either BALB/c or B10.A mice) for TGF-ß production
is enhanced by the presence of IL-4 and absence of IFN- in the
priming cultures
In light of the above studies, we next determined the ability of
CD4+/LECAM-1high (naive) T cells from
TCR/Tg mice to undergo differentiation into TGF-ß-producing T cells
when primed in vitro under various conditions in secondary cultures. In
these studies, we utilized CD4+ T cells from OVA-TCR/Tg
mice derived on a BALB/c background (as in the studies above) as well
as CD4+ T cells from cytochrome c
(CYTO-C)-TCR/Tg mice derived on a B10.A background, recognizing that
mice of these respective backgrounds produce different levels of IL-4
and IFN- under "neutral" baseline priming conditions (i.e., in
the absence of exogenous cytokines) that could affect the generation of
TGF-ß-producing cells (12). Accordingly, naive
CD4+/LECAM-1high cells from OVA-TCR/Tg mice and
CYTO-C-TCR/Tg mice were stimulated in priming cultures for 5 days with
TDS plus OVAp or CYTO-C peptide, respectively, then extensively washed
and restimulated in secondary cultures with immobilized anti-CD3
plus soluble anti-CD28 and IL-2 for 2 days, after which cell
culture supernatants were harvested and assayed for cytokines. As shown
in Table II, depicting the mean values
obtained from three independent studies, naive CD4+ T cells
from OVA-TCR/Tg mice primed under these baseline conditions produced
much higher levels of TGF-ß following secondary stimulation than
similarly primed T cells from CYTO-C-TCR/Tg mice. In the studies of
OVA-TCR/Tg mice (Table II, Expt.1), the high baseline production of
TGF-ß by naive T cells was enhanced by the addition of IL-4 or
anti-IL-12 plus anti-IFN- to the priming cultures
(p < 0.05), whereas addition of anti-IL-4
diminished TGF-ß production (p < 0.05).
Furthermore, in the studies of naive T cells from CYTO-C-TCR/Tg mice,
the low level of baseline production of TGF-ß was enhanced by the
addition of IL-4, either in the presence (p <
0.05) or absence (p < 0.05) of anti-IL-12
plus anti-IFN- in the priming cultures. Addition of
anti-IL-12 plus anti-IFN- alone to the priming cultures also
led to an increase in TGF-ß production compared with what was
produced under baseline priming conditions. These studies show that the
baseline generation of TGF-ß-producing cells is influenced by the
strain of origin of the naive CD4+ T cells. In addition,
they show that regardless of baseline TGF-ß production, such
generation is positively regulated by priming in the presence of IL-4
and is negatively regulated by priming in the presence of IL-12 and
IFN-. It should be noted that, while it is likely that the ability
of IL-12 to down-regulate TGF-ß is mediated through IFN-, a direct
role for IL-12 cannot be excluded (10).
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-producing
cells. To investigate this possibility, we measured IL-4 and IFN-
production from the same cultures used to assess TGF-ß production to
see whether there was a positive correlation between TGF-ß and IL-4
production and a negative correlation between TGF-ß and IFN-
production. As shown in Table II (Expt. 1), appreciable amounts of IL-4
and IFN- are present in supernatants of secondary cultures of cells
primed under baseline conditions. Furthermore, addition of IL-4 or
anti-IL-12 plus anti-IFN- to priming cultures led to an
increase in both TGF-ß and IL-4 production and markedly reduced
IFN- production. By contrast, addition of anti-IL-4 to the
priming cultures led to a marked reduction in IL-4 production as well
as a decrease in TGF-ß production. Overall, these results show that
increased TGF-ß production occurs under conditions that favor high
IL-4 and low IFN- production in secondary cultures.
Finally, as shown in Table I, TGF-ß production by
CD4+/LECAM-1low (memory) T cells stimulated
with anti-CD3 plus CD28 plus IL-2 was not appreciably affected by
addition of cytokines or cytokine antagonists to the cultures. This
result suggests that TGF-ß production from previously activated cells
may be relatively unaffected by changes in the cytokine environment. To
address this issue more directly,
CD4+/LECAM-1high T cells were stimulated under
baseline priming conditions and then restimulated in secondary cultures
in the presence or absence of cytokine antagonists, after which TGF-ß
production was measured. Cells stimulated under baseline conditions as
described above and restimulated with anti-CD3 plus CD28 plus IL-2
produced 801 pg/ml of TGF-ß. TGF-ß production following secondary
stimulation in the presence of anti-IFN- or anti-IL-4 was
857 pg/ml and 771 pg/ml, respectively. Thus, it is apparent that, once
cells become committed to making TGF-ß in priming cultures, they are
relatively resistant to further regulation by IL-4 or IFN-, at least
over the 36-h time period of the restimulation culture.
IL-10 enhances priming of CD4+ T cells for TGF-ß production
Based on the observation that IL-10 is a potent inhibitor of
IFN- and IL-12 production, it was of interest to determine what
effect IL-10 would have on the induction of TGF-ß-producing cells. As
shown in Table IIIA, in data
from two independent experiments, the presence of IL-10 in primary
cultures led to a twofold increase in TGF-ß production in secondary
cultures (p < 0.02). This was associated with
a decrease in IFN-, suggesting that the presence of IL-10 may exert
a positive but indirect influence on the induction of TGF-ß-producing
cells through its inhibition of IFN-. It should be noted that, while
IL-4 production was also increased by the presence of IL-10, this did
not appear to contribute to the increase in TGF-ß production, since
the addition of anti-IL-4 did not substantially diminish production
of TGF-ß.
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B), addition of IL-10 to priming cultures
in the presence or absence of anti-IL-4 led to a threefold increase
in production of TGF-ß following restimulation, consistent with what
was seen above. In addition, the presence of IL-10 also led to a
threefold increase in IL-10 production that was abrogated by addition
of anti-IL-4, suggesting that IL-4 had a role in the induction of
IL-10-producing cells. This was supported by the finding that addition
of IL-4 to priming cultures led to a striking increase in the
production of IL-10 following restimulation. These results show that
optimal priming for IL-10 and TGF-ß are somewhat different. While the
IL-10 effect of TGF-ß priming is independent of IL-4, the IL-10
effect of IL-10 priming appears dependent on IL-4. The ability of IL-4 to enhance both TGF-ß and IL-10 raises the question of whether IL-4 is enhancing TGF-ß indirectly through IL-10. To address this possibility, naive CD4+/LECAM-1high T cells from IL-10-deficient mice and control wild-type mice were primed under neutral conditions or in the presence of IL-4, and TGF-ß production was assessed following restimulation. CD4+ T cells from IL-10-/- mice stimulated under neutral conditions produced 245 pg/ml of TGF-ß compared with 600 pg/ml from control mice, demonstrating that IL-10-/- mice make relatively less TGF-ß than do control mice. Furthermore, addition of IL-4 to cultures of IL-10-/- mice increased TGF-ß production from 245 pg/ml to 635 pg/ml, providing strong evidence that IL-4 can induce TGF-ß in the complete absence of IL-10.
CD4+ T cells from IFN--/- mice
have an increased capacity to produce TGF-ß
Since IFN- appears to inhibit TGF-ß priming, we next wished
to determine whether CD4+ T cells from
IFN--/- mice were biased toward increased TGF-ß
production. In these studies, CD4+/LECAM-1high
(naive) T cells from IFN--/- and
IFN-+/+ mice were stimulated in priming cultures under
baseline conditions in the presence or absence of cytokines and/or
cytokine antagonists and were then evaluated for their production of
TGF-ß, IL-4, and IFN- following restimulation under baseline
conditions in secondary culture. As shown in Table IV, in results combined from three
independent experiments, TGF-ß production by cells from
IFN--/- mice primed under baseline conditions was
modestly increased over that of similarly primed cells from
IFN-+/+ mice. Furthermore, the addition of IL-4 to
priming cultures of cells from IFN--/- mice resulted
in a modest increase in both TGF-ß and IL-4 production. Finally,
addition of anti-IFN- plus anti-IL-12 to priming cultures
containing naive cells from IFN-+/+ mice resulted in an
increase in TGF-ß production similar to the amount detected with
IFN--/- mice primed under neutral conditions. These
data are thus entirely consistent with the effects of IFN- and IL-4
on priming for TGF-ß obtained in normal mice. In addition, they
emphasize the fact that naive CD4+ T cells from
IFN--/- mice can be positively primed by IL-4 to
produce increased amounts of TGF-ß, providing evidence that IL-4 has
a direct positive effect (i.e., IFN- independent) on the
differentiation of TGF-ß-producing cells.
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production from total/unsorted CD4+ T cells from
IFN--/- and IFN-+/+ mice following in
vitro priming and restimulation, since this cell population contains a
minor population of LECAMlow (memory) CD4+ T
cells that reflect in vivo priming and could thus influence the in
vitro priming of the majority population of LECAM-1high
(naive) CD4+ T cells. It should be noted that, in these
experiments, comparison between total/unsorted CD4+ T cells
from IFN--/- and IFN-+/+ (wild-type)
mice are valid, since these populations contained similar numbers of
naive and memory cells (see Table IV legend). As shown in Table IV,
total/unsorted CD4+ T cells from IFN--/-
mice primed under baseline conditions produced threefold more TGF-ß
(p < 0.002) and two- to fourfold more IL-4
compared with similarly primed cells from the IFN-+/+
mice. Furthermore, addition of anti-IL-4 to the priming cultures
led to decreases in both TGF-ß and IL-4 production. The fact that
neutralization of IL-4 in priming cultures using total/unsorted
CD4+ T cells from IFN--/- mice diminishes
both IL-4 and TGF-ß provides additional evidence that IL-4 has a
direct positive (IFN--independent) effect on the generation of
TGF-ß-producing T cells. IL-4 is not essential for TGF-ß priming
The data presented so far suggest an important role for IL-4 in
the generation of cells producing TGF-ß, either directly or via an
influence on IFN- production. To further address the role of IL-4 in
TGF-ß production, we performed studies similar to those described
above with IFN--/- mice with both
CD4+/LECAM-1high (naive) T cells and
total/unsorted CD4+ T cells derived from
IL-4-/- and IL-4+/+ mice (both on a BALB/c
background). As shown in Table V, in
results combined from three independent experiments, naive T cells from
IL-4-/- mice primed under baseline conditions and then
evaluated for cytokine production after secondary stimulation produced
two- to threefold less TGF-ß than similarly primed cells from
IL-4+/+ mice (p < 0.02). This
increased production of TGF-ß from cells of IL-4+/+ mice
occurred concomitantly with high production of IL-4 but little IFN-
in the secondary culture. Furthermore, addition of IL-4 to the priming
cultures led to a two- to threefold increase in TGF-ß by cells of
IL-4-/- mice, while addition of anti-IFN- plus
anti-IL-12 to the priming cultures was associated with only a
modest increase in TGF-ß production by cells from these
mice.
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(Expts.2 and 3), total/unsorted T cells from
IL-4-/- mice primed under baseline conditions and then
evaluated for cytokine production after secondary stimulation produced
three- to fourfold less TGF-ß than cells from IL-4+/+
mice primed in a similar manner. This observation, taken in conjunction
with the observation that the addition of anti-IL-4 to priming
cultures of cells from IL-4+/+ mice greatly diminished
TGF-ß production in secondary cultures (Expt.3), is consistent with
the view that IL-4—while not essential—may be important for optimal
generation of TGF-ß-producing cells. Finally, addition of
anti-IFN- plus anti-IL-12 to the priming cultures of
total/unsorted cells from IL-4-/- mice led to a 50%
increase in the production of TGF-ß in both experiments, consistent
with IFN- having a negative influence on TGF-ß induction. These
data thus provide further support for the idea that IFN- has a
direct negative (IL-4-independent) effect in the generation of
TGF-ß-producing cells. While the above results clearly show that cells producing TGF-ß can differentiate in the absence of IL-4, they do not rule out the possibility that other Th2-type cytokines such as IL-13 operate in the absence of IL-4 to influence TGF-ß induction. To address this possibility, studies of cells from IL-4-/- mice were conducted in which IL-13 or a neutralizing Ab to IL-13 was added to the priming cultures. CD4+/LECAM-1high T cells from IL-4-/- mice primed under baseline conditions and restimulated produced 386 pg/ml of TGF-ß. Addition of IL-13 or anti-IL-13 to priming cultures induced 340 pg/ml and 396 pg/ml of TGF-ß, respectively, following restimulation. Based on these data, it is unlikely that IL-13 regulates TGF-ß production in these mice.
The presence of TGF-ß in priming cultures has a positive effect on the generation of TGF-ß-producing cells
Recognizing that cytokines, e.g., IL-4, play an important role in
their own regulation, we next studied the effect that TGF-ß itself
had on the induction of TGF-ß-producing cells. As shown in Table VI, stimulation of naive
CD4+ T cells in priming cultures in the presence of
TGF-ß (using T cells derived from either OVA (Expt. 1) or
CYTO-C-TCR/Tg (Expts. 2–4) mice) led to enhanced TGF-ß production in
secondary cultures. These data are consistent with two potential
mechanisms of such self induction: 1) a direct effect of TGF-ß on T
cells; and 2) an indirect effect mediated by the ability of TGF-ß to
inhibit IFN- production. Evidence in support of an indirect effect
through inhibition of IFN- is shown in experiment No. 1 using
CD4+ T cells from OVA-TCR/Tg, in which the presence of
TGF-ß in priming cultures and enhancement of TGF-ß production in
secondary cultures was associated with complete inhibition of IFN-
production in secondary cultures but no increase in the production of
IL-4. In addition, in experiment No. 4, partial reversal of the
enhancement of TGF-ß priming by the presence of TGF-ß in the
primary cultures was seen by the addition of IL-12 to the priming
cultures, which was associated with increased IFN- production.
Evidence in support of a direct effect of TGF-ß on TGF-ß induction
is shown in experiments No. 2 and 4 using CD4+ T cells from
CYTO-C-TCR/Tg mice. In these studies, addition of TGF-ß to priming
cultures resulted in a two- to fivefold increase in TGF-ß production
in the absence of detectable IFN- production. Thus, TGF-ß is able
to positively regulate its own induction.
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production when added to priming cultures in the
presence of IL-4 or IL-12. The addition of TGF-ß to priming cultures
in which IL-4 was also added (experiment #3) abrogated the increase in
IL-4 production normally seen with priming in the presence of IL-4
alone but maintained priming for TGF-ß production. Furthermore, the
addition of TGF-ß to priming cultures in which IL-12 was also added
abrogated most of the increase in IFN- production following
restimulation caused by IL-12. Thus, the presence of exogenous TGF-ß
in priming cultures appears to have a negative regulatory influence on
IL-12 and IL-4 priming for IFN- and IL-4, respectively, but still
augments the amount of TGF-ß produced in secondary cultures. Finally,
it should be noted that in all of the studies reported here, priming
cultures were done in complete media containing 10% FCS that itself
contained approximately 100 pg/ml of TGF-ß by ELISA (data not shown).
Since addition of anti-TGF-ß did not alter production of any of
the cytokines tested (Table VI), it is unlikely that the levels of
endogenous TGF-ß present in the cultures influences priming for
TGF-ß. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-4 production, respectively).
Using this system, we first showed that
CD4+/LECAM-1high T cells (naive) did not
produce detectable TGF-ß or IL-4 36 h after primary stimulation
under baseline conditions. This indicated that naive T cells were a
suitable starting point for evaluating factors regulating the
differentiation of TGF-ß-producing T cells. By contrast,
CD4+/LECAM-1low (memory) T cells produced
measurable, albeit low, amounts of TGF-ß under baseline conditions;
the level of TGF-ß produced by these cells was not appreciably
influenced by the presence or absence of IL-4 or IFN-, cytokines
shown to influence TGF-ß production of naive T cells (see below).
Thus, CD4+ T cells expressing a marker consistent with
previous activation appear to be relatively resistant to further
regulation following short-term in vitro stimulation. It should be
noted, however, that such nascent memory cells may later regain some
ability to be regulated, since LECAM-1low (memory) cells
isolated from mouse lymphoid cells and then primed for several days and
restimulated produced variable amounts of TGF-ß, depending on the
conditions present in the priming culture (our unpublished
observations).
In further studies, we showed that two somewhat interrelated priming
conditions favor the differentiation of naive CD4+ T cells
into cells producing TGF-ß: one was the presence of IL-4 and the
second was the presence of the Th1 cytokine antagonists, anti-IL-12
plus anti-IFN- Abs, in the priming culture. This was seen most
dramatically in cultures of naive TCR/Tg CD4+ T cells
derived from a strain of mice (B10.A) that produces relatively low
amounts of TGF-ß under baseline conditions but was also seen with
similarly stimulated cells derived from a strain of mice (BALB/c) that
produces relatively high amounts of TGF-ß under baseline conditions.
Moreover, this result was supported by the fact that the emergence of
TGF-ß-producing cells in secondary cultures correlated with the
induction of cells producing low amounts of IFN- and high amounts
of IL-4.
Additional support for the importance of low IFN- and high IL-4
during priming for induction of TGF-ß-producing cells was obtained
from studies of IFN--/- and IL-4-/-
mice. The most informative finding in these studies was that
total/unsorted CD4+ T cells (containing both
LECAM-1high (naive) cells and LECAM-1low
(memory) cells) of IFN--/- mice produced far higher
levels of TGF-ß after priming under baseline conditions than did
similar cells of IFN-+/+ controls; this correlated with
the lack of IFN- in cultures containing cells from the
IFN--/- mice. Conversely, total/unsorted
CD4+ T cells of IL-4-/- mice produced far
lower levels of TGF-ß after priming under baseline conditions than
did similar cells of IL-4+/+ control mice. This correlated
with the high levels of IFN- and lack of IL-4 in the cultures
containing cells from the IL-4-/- mice. Finally, the
presence of IL-10 in priming cultures leading to increased TGF-ß
production in secondary cultures was associated with decreased IFN-.
This ability of IL-10 to regulate TGF-ß may help explain why
IL-10-/- mice have a propensity to develop inflammatory
colitis (27). Taken together, these studies provide strong support for
the view that the generation of cells producing high amounts of TGF-ß
occurs under conditions that are similar to those required for the
induction of Th2 cells, namely low amounts of IL-12 and IFN- and
high amounts of IL-4.
Based on the conclusions above, one major mechanism by which IL-4 and
IL-12/IFN- reciprocally regulate the differentiation of
TGF-ß-producing cells is via the capacity of these cytokines to
cross-regulate one another. This, however, should not obscure the fact
that the data obtained show that both IL-4 and IFN- have direct
(opposing) effects on TGF-ß production. The evidence for a direct
effect of IL-4 is supported by preliminary studies that show that
highly purified naive CD4+ T cells primed with
PB-anti-CD3 in the absence of APCs (and thus in the absence of
cells capable of producing IL-12) differentiated into cells producing
TGF-ß, provided that IL-4 was added to the culture (data not shown).
In addition, as shown here in experiments with cells from
IFN--/- mice, the presence of anti-IL-4 in the
priming cultures diminished the production of TGF-ß in secondary
cultures, and the presence of IL-4 in the priming cultures augmented
the production of TGF-ß in the secondary cultures, demonstrating that
IL-4 has a direct effect on TGF-ß production. With regard to the
mechanisms by which IL-4 can directly influence TGF-ß production, two
possibilities should be considered. One is that IL-4 has a specific
effect on the differentiation of nascent TGF-ß-producing cells, and
another is that IL-4 enhances TGF-ß production in cells already
committed to producing TGF-ß. Some evidence for the latter
possibility is inherent in the fact that the TGF-ß promoter contains
a potential STAT 6 binding site, and STAT 6 has been specifically
linked to IL-4 signaling functions. There is also evidence to show that
IFN- is an independent regulator of TGF-ß production. This
includes the fact that the generation of TGF-ß-producing cells occurs
in the complete absence of IL-4 (i.e., IL-4-/- mice) and
that addition of anti-IFN- plus anti-IL-12 to priming
cultures of total/unsorted cells from IL-4-/- mice caused
an increase in TGF-ß production in secondary cultures. In addition,
cells from IFN--/- mice produced more TGF-ß than did
cells from IFN-+/+ mice, even if the latter’s
production of IL-4 was blocked by the presence of anti-IL-4 in the
priming culture. In considering all these data, we would conclude that
the regulation of the differentiation of TGF-ß-producing cells with
regard to IL-4 and IFN- consists of at least three components: 1) a
direct positive effect of IL-4 that is important for optimal generation
of TGF-ß-producing cells; 2) an interactive effect of IFN- and
IL-4 whereby IL-4 down-regulates IFN- production and responsiveness
to IL-12 or IL-4 enhances IL-10 production, leading to increased
production of TGF-ß; and 3 a direct negative effect of IFN-
that ensures that, in low IFN-/IL-12 environments, TGF-ß can be
produced regardless of the presence of IL-4.
A final issue of interest is the role of TGF-ß itself in the
regulation of Th1/Th2 responses, as well as it role in its own
induction. With regard to TGF-ß regulation of Th1/Th2 responses,
previous in vitro and in vivo studies have yielded contrasting results.
Several reports have shown that TGF-ß enhanced the generation of Th1
cells in vitro (28, 29); however, recent work by Schmitt et al. (30)
showed that TGF-ß can promote or inhibit Th1 development depending on
the mouse strain used and the amount of IL-2 present in the priming
culture (31). In vivo studies are also discordant as to the role of
TGF-ß in regulating Th1/Th2 responses. In experimental models of
toxoplasmosis or leishmanial infection, TGF-ß administration clearly
inhibited the generation of a Th1 response (1, 2). By contrast, TGF-ß
administration to mice infected with Candida albicans
delayed progression of disease concomitant with lower levels of IL-4
but not of IFN- (32). In the present studies, we also examined the
role of both endogenously produced and exogenously added TGF-ß in the
priming of naive CD4+ T cells for IL-4 and IFN-
production as well as for TGF-ß production. We found that addition of
TGF-ß to priming cultures containing IL-12 markedly reduced the
induction of IFN- production; it should be noted, however, that this
inhibition was not Th1 specific, since the addition of TGF-ß also
suppressed the generation of IL-4-producing cells. In addition, in
Table IIIB, the presence of TGF-ß in priming cultures
markedly enhanced induction of TGF-ß-producing cells but did not
appreciably alter production of IL-10 following restimulation (data not
shown). Thus, the addition of TGF-ß to priming cultures has a direct
positive effect on the generation of TGF-ß-producing cells as well as
an indirect effect mediated by the ability of TGF-ß to inhibit
IFN- production. This ability of TGF-ß to positively regulate its
own production is consistent with several previous reports showing that
TGF-ß1 enhanced mRNA expression from a variety of transformed cell
lines (33, 34, 35); however, while these data provided evidence for
autoregulation of TGF-ß expression by TGF-ß1, they were not done
using normal primary CD4+ T cells. In addition, further
studies demonstrated that mRNA expression of TGF-ß and secretion of
protein may not be correlative (36). Thus, our studies provide the
first evidence that TGF-ß may be the central mechanism controlling
TGF-ß production from naive CD4+ T cells. Furthermore, as
alluded to above, the presence of high amounts of IL-4 and low amounts
of IFN- may set up the conditions for initial TGF-ß production.
Then TGF-ß, via its production, establishes a positive feedback loop
that can further enhance the magnitude of its production.
One possible caveat to the above conclusions is that the increases in
TGF-ß production under the various priming conditions studied were on
the order of two- to fivefold, which are substantially lower than the
concomitant changes in IL-4 and IFN- production. In addition, the
absolute amount of TGF-ß produced by T cells is lower compared with
the amounts generated of the other cytokines. It should be pointed out,
however, that in an in vivo model of inflammatory colitis, two- to
fourfold differences in TGF-ß production had a clear effect on
suppressing colitis following oral administration of Ag (7).
Furthermore, abrogation of responses of this magnitude by
administration of anti-TGF-ß led to reestablishment of disease.
Finally, while CD4+ T cells may not necessarily be the
major source of TGF-ß in vivo, it is clear from studies of Powrie et
al. that transfer of purified CD4+ T cells is sufficient to
inhibit colitis in a TGF-ß-dependent manner (17). On the basis of
these considerations, it seems likely that the observations on TGF-ß
priming reported here are physiologically relevant. One possibility not
addressed in these studies is that IL-4 and IFN- also regulate
TGF-ß production by non-T cells. This could occur, since, as shown in
these studies, IL-4 and IFN- regulate production of TGF-ß, and
TGF-ß has its own independent effects on TGF-ß production even in
non-T cells (33, 34, 35). Thus, even if a non-T cell lacks receptors for
IL-4 or IFN-, these cytokines could effect TGF-ß production
indirectly by TGF-ß itself.
To conclude, the studies reported here, defining the optimal conditions
for the generation of TGF-ß-producing CD4+ T cells, may
have important implications for the treatment of autoimmune diseases,
especially those diseases mediated by excessive Th1 responses. Thus,
since the inhibition of IL-12/IFN- production can lead to enhanced
TGF-ß and IL-4 production, short-term administration of
anti-IL-12 (or another IL-12 inhibitor) at the time of specific
antigenic challenge may be an effective way to direct a specific T cell
response away from Th1 differentiation without at the same time causing
long-term nonspecific immunosuppression that would impair the
generation of a necessary Th1 response to an intracellular pathogen. A
response directed in this way could be particularly useful in the
amelioration of autoimmune disease, since it has already been shown
that, in experimental models of inflammatory bowel disease (37) or
experimental autoimmune encephalomyelitis (38), administration of
anti-IL-12 at the time of disease induction or even after the
disease is established leads to abrogation of disease progression or
even to resolution of disease. While it is believed that the chief
mechanism of such resolution is the inhibition of the underlying Th1
response, it is important to emphasize that the same anti-IL-12
treatment also leads to the generation of TGF-ß-producing cells, and
thus it is possible that the disease is also being affected by the
secretion of a suppressor cytokine. Finally, the demonstration that the
outcome of oral Ag administration may be manipulated by
coadministration of a cytokine antagonist (anti-IL-12) (18),
coupled with the possibility derived from the studies reported here as
well as recently conducted studies by Weiner and his colleagues
(unpublished observations) demonstrating that oral tolerance and
TGF-ß production are enhanced by coadministration of IL-4, suggests
that oral tolerance may indeed be manipulated to provide more effective
therapy for autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: Tg, transgenic; TDS, T-depleted spleen cell; LECAM, lymphocyte endothelial cell adhesion molecule; OVAp, OVA-peptide; CYTO-C, cytochrome c.
Received for publication December 4, 1997. Accepted for publication February 6, 1998.
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C.-Y. Wu, H. Maeda, C. Contursi, K. Ozato, and R. A. Seder Differential Requirement of IFN Consensus Sequence Binding Protein for the Production of IL-12 and Induction of Th1-Type Cells in Response to IFN-{gamma} J. Immunol., January 15, 1999; 162(2): 807 - 812. [Abstract] [Full Text] [PDF]
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M. E. Wilson, B. M. Young, B. L. Davidson, K. A. Mente, and S. E. McGowan The Importance of TGF-{beta} in Murine Visceral Leishmaniasis J. Immunol., December 1, 1998; 161(11): 6148 - 6155. [Abstract] [Full Text] [PDF]
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W. Chen, W. Jin, and S. M. Wahl Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor {beta} (TGF-{beta}) Production by Murine CD4+ T Cells J. Exp. Med., November 16, 1998; 188(10): 1849 - 1857. [Abstract] [Full Text] [PDF]
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M.F. Neurath, B. Weigmann, S. Finotto, J. Glickman, E. Nieuwenhuis, H. Iijima, A. Mizoguchi, E. Mizoguchi, J. Mudter, P.R. Galle, et al. The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease J. Exp. Med., May 6, 2002; 195(9): 1129 - 1143. [Abstract] [Full Text] [PDF]
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