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*
Metabolism Branch, National Cancer Institute,
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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produced by Th1 cells is
required for optimal activation of macrophage microbicidal function
(2, 3), whereas the Th2 cytokines IL-4, IL-10, and IL-13
suppress macrophage activation and permit intracellular parasite growth
(4, 5, 6). The factors regulating the development of Th1 vs
Th2 responses during L. major infection in different strains
of mice has been an area of vigorous investigation. Most mouse strains
produce Th1 responses after L. major infection. However,
infected BALB/c mice produce an early burst of IL-4, which appears to
skew the developing T cell response toward the Th2 subset (7). BALB/c
mice have a subset of CD4 T cells bearing specific TCRs that appear to
have been preprogrammed to secrete IL-4 after activation by L.
major Ags (8). Interference with this IL-4 production
in BALB/c mice by anti-IL-4 Abs or by gene disruption converts the
BALB/c anti-L. major response to a healing response in
most, but not all, cases (9, 10, 11). Conversely, disruption
of the genes for IFN- or IL-12 can switch an L.
major-resistant mouse strain to an L.
major–susceptible strain (12, 13). Thus,
manipulation of the Th cell response has been shown to be critical for
the outcome of L. major infection in mice.
Studies of mutant BCL-6-/- mice have shown
BCL-6 to be an important regulator of Th2 cell responses
(14, 15, 16). BCL-6-/- mice
characteristically develop a spontaneous and fatal Th2-type
inflammation of the heart and lungs. Moreover, immunization of
BCL-6-/- mice with a protein Ag in adjuvant
results in the generation of Th2 cells and Th2-type inflammation.
Interestingly, the Th2 inflammatory response proceeds even in
BCL-6-/-IL-4-/- mice
and BCL-6-/-STAT6-/-
double-mutant mice, suggesting that BCL-6 controls Th2 differentiation
by a pathway independent of signaling through the IL-4R
(16). BCL-6 is a transcriptional repressor protein that
binds DNA motifs that closely resemble the IFN- activation site
motifs recognized by the STAT transcription factors (14).
Furthermore, BCL-6 can repress IL-4-induced transcription mediated by
STAT6 (14). Thus, one hypothesis is that BCL-6 may
regulate Th2 differentiation by modulating transcription induced by
cytokine signaling through STAT proteins.
To further characterize the role of BCL-6 in the development of Th2 cells and Th2 responses, we infected BCL-6-/- mice (on a C57BL/6 x 129 intercrossed background) with L. major. We found that lack of functional BCL-6 resulted in a nonhealing L. major response that correlated with the development of Th2 cells. BCL-6-/-IL-4-/- mice were still susceptible to L. major infection, but BCL-6-/-STAT6-/- mice were resistant. These results indicate that susceptibility to L. major infection in this model depends on signaling through STAT6. IL-4 and IL-13 are both products of Th2 cells, and both signal through STAT6 (17, 18, 19). The fact that disruption of IL-4 alone did not make BCL-6-/- mice resistant to L. major suggests that IL-13 plays a major role in mediating nonhealing L. major responses in the absence of BCL-6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BCL-6-/-, BCL-6-/-IL-4-/-, and BCL-6-/-STAT6-/- mice were generated as previously described (14, 16). The IL-4-/- mouse line was described by Kopf et al. (20). The STAT6-/- mouse line was described by Shimoda et al. (21). Animals were housed in an American Association for the Accreditation of Laboratory Animal Care-approved specific pathogen-free facility. Mouse cages, bedding, water, and food were autoclaved before use. Mice were used for infections between 5 and 8 wk of age. Animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD).
Leishmania parasites
L. major strain V1 (World Health Organization designation MHOM/IL/80/Friedlin, provided by Dr. David Sacks) was maintained in culture and passaged as previously described (22). Mice were infected s.c. in the right hind footpad with 106 viable promastigote-stage parasites.
Assaying disease progression
Development of the primary L. major lesion was monitored by measuring the increase in thickness of the inoculated footpad and the thickness of the uninfected contralateral footpad with a vernier caliper. Skin ulceration and necrosis of the infected footpad were scored by eye; necrosis was judged by the presence of brown or black foot tissue. Mice were sacrificed at 5 wk postinfection, at which time the footpads were measured, and the spleen and draining lymph node were removed for further analysis. Lymph nodes and spleens were processed to obtain single cell suspensions, and these cells were used for parasite quantitation assays. Serial dilutions of spleen and lymph node cells were prepared in 96-well plates containing complete medium 199 over blood agar. Parasite growth was scored after incubation at 26°C for 7–10 days, and the greatest dilution at which parasites could be grown was used as the number of parasites per 1% of lymph node or spleen cells, which was the input amount of cells. The number of parasites per lymph node was calculated by multiplying the dilution factor by 100. The total number of lymph node parasite counts was than normalized to parasites per 106 lymph node cells to correct for differences in the sizes of the draining lymph nodes.
T cell stimulations and cytokine quantitation
Single-cell suspensions from draining lymph nodes obtained 5 wk
postinfection were plated at 0.8 x 106/well
in flat-bottom 96-well plates. Cells were stimulated with either Con A
(5 µg/ml) or L. major Ag (10 µg/ml) for 48 h, at
which time the supernatants were harvested for cytokine quantitation.
Murine IFN- and murine IL-4 were measured by ELISA using reagents
obtained from PharMingen (San Diego, CA). The sensitivity of the IL-4
ELISA was 20 pg. The sensitivity of the IFN- ELISA was 0.1 ng.
Murine IL-13 was measured by ELISA with reagents obtained from R&D
Systems (Minneapolis, MN), and the sensitivity of the IL-13 ELISA
was 8 pg.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because BCL-6 negatively regulates the development of Th2 cells,
we decided to test whether BCL-6-/- mice would
be susceptible to infection with the parasite L. major. The
genetic background for the BCL-6-/- mice is a
mix of C57BL/6 (B6) and 129Sv (129) strains, and both the
C57BL/6 and 129 mouse strains are reported to be resistant to L.
major infection (1, 13). Therefore,
BCL-6-/- mice, littermate B6-129 wild-type
mice, and BALB/c mice were infected with L. major by s.c.
injection of 106 parasites in one rear footpad.
The infections were allowed to proceed only 5 wk due to the high
frequency of early death of the BCL-6-/- mice.
Nevertheless, at this time, differences between resistant and
susceptible strains of mice usually become apparent. Indeed, at this
time point, the swelling of both the BCL-6-/-
and BALB/c footpads was significantly greater than that of wild-type
B6-129 footpads (Fig. 1
). Moreover, the
footpads of both BCL-6-/- and BALB/c mice had
characteristic skin ulcers and tissue necrosis (Table I). Next, the number of viable parasites
in the lymph node draining the infection site was assayed. The parasite
loads of BCL-6-/- and BALB/c mice were
comparable, and both types of mice had >2 orders of magnitude more
parasites than wild-type B6-129 mice (Fig. 2). T cell responses to L.
major infection were assayed by testing cytokine production after
polyclonal stimulation of the T cells from the draining lymph nodes.
Both BCL-6-/- and BALB/c mice produced
significantly higher levels of the Th2 cytokines IL-4 and IL-13 than
did wild-type B6-129 mice (Fig. 3). The
BCL-6-/- mice actually made significantly
greater IL-4 and IL-13 than the BALB/c mice, indicating a more potent
Th2 cytokine response to L. major infection in the
BCL-6-/- mice. Infected
BCL6-/- mice also produced elevated levels of
the Th2 cytokine IL-5 (data not shown). IFN- production by
BCL-6-/- mice was not as low as that in BALB/c
mice. Thus, the elevated production of Th2 cytokines in both
BCL-6-/- and BALB/c mice correlated with their
nonhealing responses to the parasite infection. These data, taken
together, show that BCL-6 controls both the production of Th2 cytokines
and the susceptibility to L. major infection.
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In BALB/c mice, disruption of IL-4 function by anti-IL-4 Abs
or IL-4 gene targeting can block susceptibility to L. major
infection (9, 10, 11), although there are exceptions to these
findings (23, 24). We therefore studied the role of IL-4
in the susceptibility of BCL-6-/- mice to
L. major infection by mating the
BCL-6-/- mice to mice genetically incapable of
making IL-4 (IL-4-/- mice) (20).
BCL-6-/-IL-4-/-
double-mutant mice and IL-4-/- littermates were
infected with L. major and analyzed 5 wk after infection.
BCL-6-/-IL-4-/- mice
developed footpad swelling and parasite counts comparable in magnitude
to those observed in BCL-6-/- mice and
significantly greater than those observed in either
IL-4-/- or wild-type mice (Figs. 1
and 2). In
fact, the footpad pathology in
BCL-6-/-IL-4-/- mice
was worse than that in BCL-6-/- mice in terms
of both skin ulceration and tissue necrosis (Table I). Another
indication of L. major disease progression is the
dissemination of parasites to the spleen, typically observed in
L. major-susceptible mice. The spleens of wild-type,
BCL-6-/-, IL-4-/-, and
BCL-6-/-IL-4-/- mice were therefore assayed
for the presence of parasites (Table II).
Although the spleens of only one of 10 wild-type mice and one of five
IL-4-/- mice were positive for parasites, every
spleen examined from BCL-6-/- and
BCL-6-/-IL-4-/- mice
contained parasites. Thus, in terms of footpad pathology and parasite
load, the disruption of IL-4 gene expression did not affect the
progression of L. major infection in
BCL-6-/- mice. Because
IL-4-/- mice do not produce IL-4, we assessed
the generation of a Th2 response by measuring IL-5 and IL-13 secretion
by T cells from the draining lymph nodes of infected mice. Although T
cells from infected
BCL-6-/-IL-4-/- mice
produced only modestly elevated (2x) levels of IL-5 compared with T
cells from infected IL-4-/- mice (data not
shown), the
BCL-6-/-IL-4-/- T cells
produced 10-fold more IL-13 than the IL-4-/- T
cells (Fig. 3). T cells from infected
BCL-6-/-IL-4-/- mice
produced 3-fold less IFN- than T cells from infected
IL-4-/- mice (Fig. 3). Thus, as judged by the
elevated secretion of IL-13 and reduced secretion of IFN-,
BCL-6-/-IL-4-/- mice
developed a Th2-like response during L. major infection,
although the levels of IL-13 produced were lower than those observed in
infected BCL-6-/- mice (Fig. 3).
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Because STAT6 is a critical transducer of signaling by IL-4
and IL-13, we tested whether disruption of STAT6 would affect the
susceptibility of BCL-6-/- mice to L.
major infection.
BCL-6-/-STAT6-/- mice
and STAT6-/- littermates were infected
with L. major and analyzed 5 wk after infection.
STAT6-/- mice displayed parasite counts in the
range of wild-type mice (Fig. 2).
BCL-6-/-STAT6-/- mice,
in marked contrast to BCL-6-/- and
BCL-6-/-IL-4-/- mice,
displayed parasite counts that were not significantly greater than
those observed in littermate STAT6-/- mice
(Fig. 2). Consistent with the lower parasite counts, the footpad
pathology of BCL-6-/-STAT6-/- mice
was also less severe than that of
BCL-6-/- mice and
BCL-6-/-IL-4-/- mice (Fig. 1 and Table I). Most strikingly, none of the
BCL-6-/-STAT6-/- mice
displayed dissemination of parasites to the spleen. Taken together,
these results show that the loss of STAT6 greatly decreases the
susceptibility BCL-6-/- mice to infection with
L. major.
T cells from infected
BCL-6-/-STAT6-/- mice
produced more IL-13 than those from STAT6-/-
littermate controls, but the IL-13 response was less vigorous than that
in BCL-6-/- or
BCL-6-/-IL-4-/- mice
(Fig. 3). The
BCL-6-/-STAT6-/- T
cells did not secrete detectable IL-4 and did secrete somewhat more
IFN- than STAT6-/- controls. Thus, the
strong Th2-like response observed in infected
BCL-6-/- mice was attenuated by the absence of
STAT6, although T cells from
BCL-6-/-STAT6-/- mice
still produced elevated levels of IL-13.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BCL-6-/- mice display two prominent immune defects: an abnormal development of Th2 cells and an inability of B cells to generate a germinal center response (14). The B cell defect in these mice is unlikely to play a major role in their response to L. major infection; a recent study showed that the presence or the absence of B cells does not affect the course of L. major infection in either C57BL/6 or BALB/c mice (25). Moreover, both BCL-6-/- and BCL-6-/-STAT6-/- mice have similar B cell defects (A. L. Dent, unpublished observations), but differ in their resistance L. major infection. On the other hand, the uncontrolled Th2 cell differentiation in BCL-6-/- mice could well contribute to their susceptibility to L. major infection. Previous studies of BALB/c mice (with a wild-type BCL-6 gene) have suggested that IL-4 is required for the generation of a Th2-dominated immune response to L. major (9, 10, 11). Unlike other models of Th2 differentiation, however, the production of Th2 cytokines in BCL-6-/- mice is independent of functional IL-4 and STAT6 genes (16). Indeed, we observed that BCL-6-/-STAT6-/- mice and BCL-6-/-IL-4-/- mice developed Th2-type heart and lung inflammation at the same rate as BCL-6-/- mice. This novel pathway to Th2 development in BCL-6-/- mice presumably explains why BCL-6-/-IL-4-/- mice were able to develop a nonhealing, Th2-like response to L. major. The fact that BCL-6-/-STAT6-/- mice develop Th2-type inflammatory disease at the same rate as BCL-6-/- mice contrasts with the finding that these two strains differ in their susceptibility to L. major infection. Thus, the inflammatory phenotype characteristic of BCL-6-/- mice is genetically separable from the L. major susceptibility phenotype. This is noteworthy because it demonstrates that the L. major susceptibility of BCL-6-/- mice is not merely secondary to a general immunodeficiency associated with their inflammatory disease.
In this study we found that
BCL-6-/-IL-4-/- mice
developed severe L. major infections, while
BCL-6-/-STAT6-/- mice
were resistant to L. major infection. IL-4 and IL-13 are
homologous proteins and are the only two cytokines that have been shown
to physiologically activate the STAT6 transcription factor
(17, 18, 19). Because the absence of IL-4 did not alter the
susceptibility of BCL-6-/- mice to L.
major, the present results suggest that IL-13 signaling through
STAT6 contributes to the pathological, nonhealing L. major
response in BCL-6-/- mice. A role for IL-13 in
L. major infection is supported by Noben-Trauth et al., who
observed significant differences in susceptibility of
IL-4-/- BALB/c mice vs
IL-4R-/- BALB/c mice when infected with the
L. major substrain IR173 (23). Moreover, other
recent studies have found that IL-13 is an important factor for
controlling intestinal parasite infection (26, 27, 28). In
studies with IL-4-/- mice,
IL-4R
-/- mice, and
STAT6-/- mice, IL-13 was a more potent factor
than IL-4 in the expulsion of N. brasiliensis parasites from
the intestine (27, 28). In addition, a recent study using
IL-13-deficient mice has shown IL-13 to be critical for the management
of infection with the intestinal worm Trichuris muris
(26). Thus, the accumulated evidence suggests that IL-4
and IL-13 have both unique and overlapping functions in immune
responses to parasites.
It is not clear at present what cell type would be critically affected by IL-13 during L. major infection. Naive T cells have IL-4R but not IL-13 receptors, and consequently, IL-13 cannot drive Th2 differentiation in vitro (29). However, a recent report demonstrated that T cells from IL-13-/- mice are deficient in Th2 generation in vitro (30). This defect did not reflect a role for IL-13 during the in vitro culture, because addition of exogenous IL-13 did not reverse the defect in Th2 differentiation of IL-13-/- T cells. Rather, it appears that IL-13 exerted an effect on T cells in vivo that impaired their ability to respond to IL-4 and differentiate to Th2 cells both in vivo and in vitro. Thus, it is possible that IL-13 might augment Th2 responses in vivo and thereby influence the magnitude of the Th2 response of BCL-6-/- mice to during L. major infection. This possibility is compatible with the observation that T cells from BCL-6-/-STAT6-/- mice produced lower levels of Th2 cytokines at 5 wk postinfection than did T cells from BCL-6-/- mice and BCL-6-/-IL-4-/- mice at 5 wk postinfection. Although BCL-6-/-STAT6-/- T cells can produce high levels of Th2 cytokines 2 wk after immunization with a protein Ag plus adjuvant (16), STAT6-mediated IL-13 signaling may be required to maintain Th2 responses in BCL-6-/-STAT6-/- mice at longer time points.
Alternatively, IL-13 may regulate the response of non-T cells during L. major infection. IL-13 may play a critical role in L. major infection due to its ability to inhibit macrophage activation (6, 31). Activated macrophages are important for the elimination of the L. major parasite, and IL-13 may modulate the L. major response by inhibiting macrophage function during the infection. Recently, it was demonstrated that IL-13 can inhibit the production of nitric oxide by activated wild-type macrophages but not activated STAT6-/- macrophages (32). Thus, the healing response of BCL-6-/-STAT6-/- mice to L. major infection might be due to the lack of an inhibitory action by IL-13 on STAT6-deficient macrophages, thereby allowing these macrophages to effectively control the growth of the parasite. BCL-6-/-STAT6-/- mice may therefore have lower Th2 responses in response to L. major infection because the macrophages have controlled the infection, which would result in less T cell stimulation and differentiation.
STAT6 has also been shown to be an important factor in the murine immune response to L. mexicana (33). In contrast to infection of mice with L. major, L. mexicana forms chronic infections in most strains of mice, including C57BL/6 and 129/Sv. Although not as well characterized as the murine response to L. major, the nonhealing response of mice in response to L. mexicana infection correlates with the induction of a Th2-type response. Disruption of IL-4 signaling by mutation of the STAT6 gene therefore prevents the formation of a nonhealing response to L. mexicana infection, resulting in the formation of a healing Th1 type of response (33). Although the study by Stamm et al. (33) reveals a critical role for STAT6 in regulating the murine immune response to Leishmania infection, no mention was made of the role of IL-13.
The response of BCL-6-/- mice to L. major infection again highlights the critical role of BCL-6 in regulating T cell differentiation to Th2 cells in vivo. The genetic absence of BCL-6 was sufficient to convert a Th1 immune response to L. major to a response in which Th2 cytokines predominated. Indeed, T cells from infected BCL-6-/- mice produced higher levels of Th2 cytokines than did T cells from infected BALB/c mice. The mechanisms by which BCL-6 regulates Th cell differentiation are not known at present. BCL-6 is not constituitively expressed by T cells, and BCL-6 is not expressed after mitogenic activation of T cells in vitro (A. L. Dent, unpublished observations). However, BCL-6 is highly expressed in vivo in germinal center T cells as well as in a subset of CD30-positive T cells scattered throughout the T cells zones of secondary lymphoid organs (34, 35). The in vivo activation signals that lead to this high level of BCL-6 expression in T cells are not known. Conceivably, though, the expression of BCL-6 in this T cell subset may be necessary to block a pathway to Th2 differentiation in vivo (reviewed in Ref. 36). This pathway is distinct from the previously described Th2 differentiation pathways in that it can proceed in the absence of IL-4 and STAT6. Future work will address whether BCL-6 induction is required in all T cells to prevent Th2 differentiation or whether this novel, BCL-6-dependent, regulatory pathway is engaged in only a subset of T cells responding to certain antigenic challenges.
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Footnotes |
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Received for publication March 5, 1999. Accepted for publication June 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is the activating lymphokine. J. Clin. Invest. 72:1506.
as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670.8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
-independent mechanism. J. Exp. Med. 171:115.-deficient mice infected with Leishmania major. J. Exp. Med. 179:1367. c chain and activate STAT6, STAT3 and STAT5 proteins in normal human B cells. FEBS Lett. 393:53.[Medline]
, and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8:255.[Medline]
-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses. Curr. Biol. 8:669.[Medline]
mRNA translational activation in lipopolysaccharide-induced mouse macrophages. J. Biol. Chem. 272:14394.This article has been cited by other articles:
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H.-C. Chang, S. Zhang, and M. H. Kaplan Neonatal Tolerance in the Absence of Stat4- and Stat6- Dependent Th Cell Differentiation J. Immunol., October 15, 2002; 169(8): 4124 - 4128. [Abstract] [Full Text] [PDF]
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N. Noben-Trauth, J. Hu-Li, and W. E. Paul Conventional, Naive CD4+ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation J. Immunol., October 1, 2000; 165(7): 3620 - 3625. [Abstract] [Full Text] [PDF]
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