IL-9 Is a Susceptibility Factor in Leishmania major Infection by Promoting Detrimental Th2/Type 2 Responses
http://www.100md.com
免疫学杂志 2005年第4期
Abstract
IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.
Introduction
Interleukin-9 is a pleiotropic cytokine produced primarily by Th cells that was originally identified as a mouse T cell growth (1) and mast cell differentiation factor. Other targets ascribed to this cytokine include erythroid precursors, B lymphocytes, eosinophils, bronchial epithelial cells, and neuronal precursors (reviewed in Refs.2 and 3). Because of its restricted production by Th2 clones in vitro (4) and its expression in Th2-type responses in vivo (5, 6, 7), IL-9 is considered to be a Th2 cytokine that can be induced via IL-4-dependent (8) and IL-4-independent (9) pathways. Deregulated IL-9 expression in transgenic mice resulted in profound perturbations of multiple hemopoietic cell lineages with diverse phenotypes. Lymphoma genesis, enhanced Ig expression (10), expansion of B1 cell lymphocyte subset (11), mastocytosis, and eosinophil maturation (12), and parasitic worm expulsion (7, 13) were observed. Lung-specific IL-9 expression led to severe airway inflammation, with infiltration of eosinophils and lymphocytes, mast cell hyperplasia, and increased subepithelial collagen deposition (14). Recently generated IL-9-deficient mice showed a severe impairment of goblet cell hyperplasia and mastocytosis in a pulmonary granuloma model. However, pulmonary fibrosis, eosinophil and lymphocyte infiltration were normal, as was the development of pulmonary Th2 responses (15).
Experimental murine leishmaniasis is a paradigm example of the relationship between the genetic factors that control Th cell differentiation and the outcome of the disease. Healer strains, like C57BL/6, develop predominant Th1 responses with high IFN-, low IL-4 production and protective cellular immune responses, whereas nonhealer strains, like BALB/c, develop predominant Th2 responses with high IL-4, low IFN- production resulting in exacerbation of the disease (16). Leishmania-induced Th2 cytokine responses include IL-4, IL-5, IL-9, and IL-13, respectively. Many studies have shown that IL-4 is a main disease-promoting factor in cutaneous leishmaniasis, and neutralizing IL-4 in vivo by mAb converts nonhealers to healers (reviewed in Refs.17 and 18). Infection studies in BALB/c mice deficient for IL-4, IL-13, IL-4R, or STAT6 are able to contain acute infection with impaired Th2 responses, suggesting that both IL-4 and IL-13 contribute to the susceptible phenotype, which seems to depend on the Leishmania strain used (19). The role of IL-13 remains unclear, with disease-promoting functions during acute leishmaniasis and possible protective functions during the later chronic disease, which were suggested from infection studies using IL-13- or IL-4R-deficient mice, respectively. IL-5 plays a minor role in the overall susceptibility in Leishmania major-infected BALB/c (19).
Very little is known on the role of IL-9 in leishmaniasis. The only two publications available investigated IL-9 expression, showing that IL-9 is induced by L. major infection and transiently expressed during the first days postinfection. Also of interest was the observation that from 4 wk onward, IL-9 synthesis was only observed in susceptible BALB/c but not in resistant C57BL/6 or DBA mice (4, 20). Expression correlated with the expansion of Ag-specific Th2 cells and purified CD4+ T cells produced IL-9 during polyclonal or Ag-specific restimulation (4). No further information is available, with a potential role of IL-9 in leishmaniasis still unexplored.
Recently, it was shown that linking murine IL-9 to OVA results in the formation of a highly immunogenic complex that ensures production of high titers of neutralizing anti-IL-9 Abs in mice (21). Because the absence of T cell help is crucial for B cell tolerance, the coupling of an immunogenic foreign Ag to a self Ag provides physically linked T cell help to B cells, therefore overcoming B cell tolerance. Using this method, IL-9-depleted BALB/c and C57BL/6 mice were generated, infected with L. major, and compared with sham-immunized control BALB/c mice to define a possible role of IL-9 in leishmaniasis by loss of function.
Materials and Methods
Mice and parasites
Mice were kept at the animal facility at the Health Science Faculty, University of Cape Town, under specific pathogen-free conditions.
The L. major MHOM/IL/81/FEBNI strain was maintained by continuous passage in BALB/c mice as previously described (22). Anesthetized mice were infected s.c. in one hind footpad with 2 x 106 stationary-phase metacyclic L. major promastigotes in a final volume of 50 μl of HBSS. Parasites were isolated from skin lesions of infected animals. Parasite burden from homogenized organs was determined by 2-fold limiting dilutions in Schneiders medium (Sigma-Aldrich). Stationary-phase cultures were also used to prepare frozen-and-thawed (F/T; 1 x 107/ml) Ag of L. major promastigotes.
Preparation of IL-9-OVA complexes and immunization protocol
IL-9-OVA complexes were obtained by cross-linking mouse IL-9 and OVA (Sigma-Aldrich) with glutaraldehyde, and purified as previously shown (21). Mice were injected s.c. with 100 μl of 1:1 mixture of 10 μg of IL-9 OVA complexes in PBS and CFA. Two s.c. boosts were performed in IFA at wk 2 and 4. Control mice received an equivalent amount of OVA in Freund’s adjuvant only. Anti-IL-9 titers were measured by the inhibitory activity of the sera on the proliferation of TS1 cells that respond to IL-9. Sera were serially diluted in 96-well plates containing culture medium and incubated in the presence of 2.5 U/ml murine (m)3 IL-9 for 1 h. TS1 cells were incubated at 37°C, 8% CO2 for 3 days, and proliferation was measured by hexoseaminidase activity determination (23).
Cell isolation and CD4+ restimulation
CD4+ T cells were purified (>90% by FACS) from lymph nodes by positive selection with magnetic mouse CD4 Dynabeads and mouse CD4 DETACHaBEAD (Dynal; Robbins Scientific) and differentiation performed as described (24). CD4+ T cells (2 x 106cell/ml) were stimulated with anti-CD3 (145-2C-11; BD Pharmingen) or F/T and cytokine concentration from supernatant determined by ELISA 48 h later.
Macrophage activation
Thioglycolate (3%)-elicited peritoneal exudate cells were harvested at day 6 and further cultured in triplicates at a concentration of 2 x 106/ml in 96-well plates (Nunc) for 4 h. Plastic-adherent macrophages were stimulated with LPS (10 ng/ml; Sigma-Aldrich) and IFN- (100 U/ml; BD Pharmingen). After 48 h, the concentration of NO in supernatants was measured by the Griess reaction. Arginase was determined as previously described (25).
ELISA
Cytokine concentrations were determined by sandwich ELISA. Standards and Abs were purchased from BD Pharmingen and detected using alkaline phosphatase-coupled streptavidin (Southern Biotechnology). Detection limits were as follows: IFN-, IL-9, and IL-13, 46 pg/ml; IL-4, 2 pg/ml. Ag-specific Ig ELISA was performed as previously described (22).
Histology
Tissue samples were fixed in neutral buffered formalin, processed, and 5- to 7-μm sections were stained with H&E.
Statistics
Data are given as mean ± SD, and the differences were tested using the unpaired two-tailed Student’s t test or ANOVA using GraphPad Prism software.
Results
Effective production of anti-IL-9 autoantibodies
IL-9-deficient mice were generated by immunization with mIL-9, chemically complexed to OVA. For this purpose, BALB/c and C57BL/6 mice were immunized with three injections of IL-9 cross-linked to OVA. Two weeks after the last injection, the anti-IL-9 response was evaluated by measuring inhibitory activities of the sera in a bioassay by using an IL-9-dependent T cell line TS1. As shown in a representative experiment for BALB/c (Fig. 1a) and C57BL/6 (b), sera were found to strongly inhibit IL-9-induced proliferation, independent of the mouse strain used. Half-maximal inhibition of 2.5 U/ml mIL-9 was obtained at mean serum dilutions of 3 and 7 x 10–4 in BALB/c and C57BL/6 mice, respectively (shown in Fig. 1c). The vaccination was 1) specific for IL-9, because proliferation of TS1 by IL-4 was not influenced from sera, and 2) long lasting, with only slight reductions in the observed inhibition titers after a year of vaccination (data not shown), as reported before (21).
FIGURE 1. Induction of IL-9 specific autoantibodies. a and b, Eight mice per group of BALB/c mice (a) or C57BL/6 mice (b) were immunized with IL-9 complexed to OVA in CFA (IL-9-OVA) or with OVA alone to produce anti-IL-9 autoantibodies. Sera was collected 2 wk after the last immunization and tested in serial dilutions for IL-9 inhibition in an IL-9-dependent TS1 cell proliferation assay. c, The serum titer able to inhibit 50% of TS1 cell proliferation with 2.5 U/ml mIL-9 is shown from individual mice, including the mean (horizontal line). Shown is a representative of three independent experiments.
Anti-IL-9 vaccination increases resistance in L. major-infected BALB/c mice
To determine a possible role of IL-9 in leishmaniasis, IL-9-OVA-immunized or control OVA-immunized BALB/c or C57BL/6 mice were infected with 2 x 106 virulent L. major (MHOM/IL/81/FEBNI) metacyclic promastigotes into one hind footpad. As expected, control BALB/c mice developed massive footpad swelling (Fig. 2a) with ulceration and necrosis (indicated by the asterisks) from wk 3 onwards. Mice had to be killed at wk 8 postinfection due to disease progression, with necrosis and ulceration in all infected control BALB/c mice. In contrast, IL-9-OVA-immunized BALB/c mice stabilized footpad swelling on a moderate level within the first 4 wk postinfection. Parasite burden in the draining popliteal lymph node (Fig. 2b) and in the infected footpad (c) was significantly lower in IL-9-OVA-immunized BALB/c mice compared with infected control BALB/c mice at 8 wk postinfection and confirmed at wk 5 and 9 in independent experiments. Histopathology developed from wk 3 onwards in the control OVA-immunized BALB/c mice with severe bone destruction in the footpad at wk 8 (data not shown). IL-9-OVA-immunized BALB/c mice more than doubled their time span until ulceration and necrosis developed, which started from wk 10 onward with termination of the experiment at wk 17 postinfection with similar disease progression as observed in the control group 9 wk earlier. In contrast, the healer strain C57BL/6 developed a transient and moderate swelling during the first weeks and subsequently developed resistance to L. major infection without an observed effect of IL-9 vaccination. Conclusively, these data showed that anti-IL-9 vaccination was able to delay disease progression during L. major infection in the nonhealer BALB/c mouse strain.
FIGURE 2. L. major infection of anti-IL-9-vaccinated mice. a, Three weeks after the last immunization, BALB/c (, ) and C57BL/6 (, ) mice, either immunized with IL-9-OVA (, ), or sham-immunized with OVA only (, ) were injected in the left hind footpad with 2 x 106 metacyclic promastigotes of L. major (MHOM/IL/81/FEBNI). The course of the infection was monitored weekly by measuring infected and noninfected hind footpads. Values are expressed as means ± SD of eight mice per group of a representative of three independent experiments. The onset of ulceration and necrosis from individual mice is marked with an asterisk. Parasite load from the draining popliteal lymph node (b) or infected footpad (c) of individual mice was determined by 2-fold limiting dilutions. Mean (—) ± SD from four mice per group at wk 8 postinfection; ***, p < 0.001; **, p < 0.01, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Similar results were obtained at wk 5 and 9 (p < 0.01) in two independent experiments.
Anti-IL-9 vaccination leads to a shift to protective Th1/type 1 responses
To investigate possible mechanisms to explain the observed increased resistance, L. major-specific Th polarization was determined during infection. Mitogenic- or Ag-specific restimulation of CD4+ T cells, isolated from the draining lymph node of IL-9-OVA-immunized BALB/c mice, produced significantly higher IFN- but lower IL-4 levels compared with cells from sham-immunized mice (Fig. 3). Reduced IL-4 production was consistent with impaired Th2 effector cytokines, because IL-9 and IL-13 were significantly impaired in IL-9-vaccinated BALB/c mice (Fig. 3). As expected, the healer strain C57BL/6 showed a predominant L. major-specific Th1-polarized response with high IFN- and very low IL-4 production (Fig. 3) without an Ag-specific effect of IL-9 OVA vaccination. These results, first, suggest an influence on Th cell differentiation by IL-9 and, second, confirm that Th2 cells are major IL-9 producers during leishmaniasis. This resulted in slightly increased type 1 Ab responses but significantly impaired Ab-specific type 2 responses in comparison to control BALB/c mice (Fig. 4). IL-9-OVA or sham-immunized C57BL/6 mice showed no significant differences in their Ab responses. Together, these results show evidence for a shift toward Th1/type 1 responses by neutralizing endogenous IL-9 in BALB/c mice.
FIGURE 3. Th cell differentiation. CD4+ T cells from the draining lymph node were isolated and incubated with anti-CD3 (), L. major Ag (), or medium only (). IFN-, IL-4, IL-9, and IL-13 were determined by ELISA, with values expressed as mean ± SD of triplicate cultures from four mice per group at wk 5 (a), wk 8 (b), or at wk 9 (data not shown) postinfection in independent experiments. Values of p compared with sham-immunized BALB/c mice were calculated by paired Student’s t test.
FIGURE 4. L. major-induced Ab responses. At the same time point, sera were analyzed from BALB/c (, ) and C57BL/6 (, ) mice, either immunized with IL-9-OVA (, ), or sham-immunized with OVA only (, ). L. major Ag-specific IgG1, IgG2a, or total IgE titers were determined by ELISA using end-point dilution or a known standard for IgE (mean + SD of five mice per group). *, p < 0.01, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Shown is a representative of three independent experiments.
Increased NO killing effector macrophage activity
Macrophages are the major cellular host for L. major where amastigotes propagate in the phagolysosome. NO is the crucial killing effector molecule against leishmaniasis, produced by IFN--stimulated and iNOS-induced classical macrophages. To determine the influence of IL-9-OVA immunization on L. major-specific killing effector functions, macrophages were isolated from thioglycolate-elicited peritoneal exudate cells of infected mice and restimulated with IFN-/LPS to determine their killing effector function. Macrophages from 5-wk-infected mice showed no differences (data not shown). Macrophages from 8-wk-infected IL-9-OVA-immunized BALB/c mice showed small but significant increased induced NO synthase (iNOS)-catalyzed NO production compared with cells from sham-immunized BALB/c (Fig. 5a). This was verified by showing a striking reduction in urea production in the earlier (Fig. 5b), which is a side product of arginase 1 activity. This differential outcome can be explained by competition between iNOS and arginase 1 for the common substrate L-arginine. IL-9-OVA immunization had no effect on the NO production of C57BL/6 mice.
FIGURE 5. Macrophage NO production. Eight weeks postinfection, thioglycolate-elicited, adherent peritoneal macrophages were restimulated with LPS/IFN-, and the production of NO (a) and urea (b) was determined after 48 h. Values represent the mean ± SD from triplicate cultures. **, p 0.01; *, p 0.05, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Shown is a representative of two independent experiments.
IL-9 acts downstream from IL-4R-mediated functions
To determine whether anti-IL-9 vaccination is dependent on IL-4- or IL-13-mediated functions, L. major infection studies in IL-9-OVA-immunized BALB/c IL-4R-deficient mice were performed. These mice are IL-4 and IL-13 unresponsive, because the IL-4R chain is a crucial component of the IL-4 and IL-13 receptor (19). Anti-IL-9 vaccination was similarly effective in IL-4R-deficient mice with a half-maximal inhibition of 2.5 U/ml mIL-9, obtained at mean serum dilution of 5 x 104 (data not shown). Anti-IL-9 or sham-immunized control or BALB/c IL-4R-deficient mice (eight mice per group) were infected with 2 x 106 virulent L. major (MHOM/IL/81/FEBNI) metacyclic promastigotes into one hind footpad, and the swelling of the footpad was monitored. As previously shown using a different L. major strain, IL-4R-deficient BALB/c mice were resistant, with slightly increased footpad swelling compared with C57BL/6 (Fig. 6a). Although IL-9-OVA immunization had the described protective effect in BALB/c mice, shown by delayed footpad swelling, and disease progression (Fig. 6a), as well as reduced parasite burden (b), no effect was observed in IL-9-vaccinated BALB/c IL-4R-deficient mice compared with sham-vaccinated controls. CD4+ T cell IFN-, IL-4, and IL-9 responses after Ag- or CD3-specific restimulation were similar between the IL-9- and sham-immunized IL-4R-deficient mice, with no detectable IL-9 found (Fig. 6c). Together, these data suggest that IL-9 vaccination had no influence on the balance of Th differentiation in this mouse strain. This conclusion was confirmed, because IgG1, -2a, and IgE Ag-specific Ab responses (data not shown) and macrophage NO production were also similar (Fig. 6d). Together, these data suggest that IL-9 acts downstream from IL-4/IL-13-mediated functions.
FIGURE 6. No effect on leishmaniasis in IL-9-OVA-immunized IL-4R-deficient BALB/c mice. Eight IL-9-OVA-immunized IL-4R-deficient BALB/c () or sham-immunized IL-4R-deficient BALB/c (?) were injected in the left hind footpad with 2 x 106 metacyclic promastigotes of L. major (MHOM/IL/81/FEBNI). a, The course of the infection was monitored weekly by measuring infected and noninfected hind footpads. At wk 5 postinfection, three mice per group were sacrificed and further analyzed. b and c, The draining popliteal lymph node was isolated, and parasite burden (b) and anti-CD3 and Leishmania Ag-specific IFN-, IL-4, and IL-9 production (c) was determined after 48 h from the supernatant. Values are expressed as means ± SD. d, Thioglycolate-elicited peritoneal macrophages were LPS/IFN- stimulated, and the NO concentration in the supernatant was measured.
Discussion
IL-9-deficient mice were generated by immunization with mIL-9, which was chemically complexed to OVA. This approach induced high titers of neutralizing anti-IL-9 Abs, as shown before (21). In this study, we present data showing evidence for the first time that IL-9 is a susceptible factor in L. major infection. Experimentally, this was shown by in vivo IL-9 neutralization, which substantially delayed disease progression of nonhealer BALB/c mice. Delayed leishmaniasis was accompanied by reduced parasite burden, footpad swelling, and milder histopathology during acute infection. As a consequence, IL-9-vaccinated BALB/c mice more than doubled their time span upon infection until deleterious leishmaniasis developed. Neutralization of endogenous IL-9 caused impaired Th2/type 2 responses with a shift toward protective Th1/type 1 responses, as determined by cytokine production of Ag-specific restimulated CD4+ T cells, isolated from the draining lymph nodes. This was confirmed ex vivo by reduced Ag-specific type 2 Ab isotype production in the blood of these mice. We concluded from these data that IL-9 is involved in promoting Th responses toward deleterious Th2/type 2 immune responses during leishmaniasis. The theoretical possibility of Th2 cell depletion due to direct anti-IL-9-specific Ab binding to its receptor is unlikely due to the restricted surface expression of the IL-9R among T cell subpopulations (26) and the observed normal Ag-specific IgG1 and IgE responses during immunization with Aspergillus extract or infection with Trichinella in IL-9-vaccinated mice (21). Despite the fact that IL-9 can act as a growth factor for certain T cell clones (1) and IL-9 overexpression in vivo can lead to thymic lymphomas (10), a Th2-promoting effect was an unexpected finding, because no effect on Th differentiation has been found until now. In vitro and in vivo (Nippostrongylus brasiliensis and schistosoma egg) Th differentiation was found to be normal in a genetically engineered IL-9-deficient 129sv mouse strain (15). Together, this may indicate that the observed influence of IL-9 on Th responses is restricted to certain diseases, like L. major infection, and/or certain genetic backgrounds influencing T cell responses. In any case, the present observations provide the first demonstration that IL-9 is an important element in the physiological regulation of the Th1/Th2 balance in vivo.
With respect to the analysis of IL-9 mode of action, we are currently not able to distinguish whether IL-9 has a direct effect on early steps in L. major-induced Th differentiation or alternatively may promote Th2 effector cell expansion. The latter possibility is more attractive, because IL-9 is barely detectable at the onset of an L. major infection and is mainly produced by differentiated Th2 effector cells (see Fig. 3) during acute leishmaniasis. This may also explain why IL-9 vaccination had no measurable effect during L. major infection in C57BL/6 as well as IL-4R-deficient BALB/c mice. Both mouse strains showed dominant Th1 responses during infection with very little IL-9 production after CD4+ T cell restimulation (Figs. 3 and 6). Therefore, it can be envisaged that IL-9 does act downstream from IL-4-mediated Th2 differentiation, and may be an important element to maintain Th2-dominated response to L. major. Clearly, further investigations are needed to fully explore the effects of IL-9 on Th responses in L. major infection.
IL-9 vaccination resulted in improved classical macrophage effector functions. This was determined by increased NO production but reduced urea production, the latter a side product of arginase 1 catalyzation from LPS/IFN--restimulated macrophage. Indeed, NO is a crucial effector molecule able to kill amastigotes within the macrophages (27). NO is catalyzed by the enzyme inducible NO synthase (NOS2), which competes with arginase 1 for the substrate arginine (28). Abrogating iNOS function, either by in vivo blocking or by using iNOS-deficient mice does lead to disease progression in healer strains (29, 30). Arginase 1, in contrast is induced by (Th2-produced) IL-4/IL-13-activated alternative macrophages in the nonhealer strain BALB/c (25) and catalyzes arginine to L-ornithine and urea. L-Ornithine plays a pivotal role in polyamine precursor metabolism in the host as well as in the Leishmania parasite (31). Because arginase I produced by alternatively activated macrophages supports the growth of intracellular Leishmania parasite (32), the observed impairment in IL-9-vaccinated mice together with the increased killing effector function in classically activated macrophages may explain the reduced parasite burden found in vaccinated mice. Also of importance, the observed increased macrophage killing effector function can be explained as a direct consequence of the increased IFN- production due to the found shift toward Th1 cytokine responses in IL-9-vaccinated and L. major-infected BALB/c mice. However, this does not exclude an additional and direct action by IL-9 on macrophages and, vice versa, a possible effect on IL-9-stimulated macrophages on Th cells. Although IL-9R surface expression was never found in freshly isolated mouse macrophages, some macrophage cell lines are expressing low levels of the receptor (26). This may also be the case in vivo during L. major infection. A recent study showing that rIL-9 was able to protect mice from Gram-negative bacterial shock and the correlation of a reduction in early IL-12, IFN-, and TNF, together with an increase in IL-10 (33), may also implicate a possible monocyte/macrophage effect of IL-9.
In conclusion, we uncovered the role of IL-9 as a susceptibility factor in L. major infection by promoting detrimental Th2/type 2 response. Indeed, IL-9-vaccinated mice are excellent models to study the role of IL-9 in human diseases, because the system is time inducible, an advantage to classical gene-targeted mice. Our results further extend the efforts made to generate autoantibodies capable of regulating biological processes with IL-9.
Acknowledgments
We thank E. Smith, R. Peterson, M. Simpson, and the animal facility staff for excellent technical assistance. Drs. C. Renauld and M. Kopf are thanked for critically reading the manuscript.
Footnotes
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 Medical Research Council and National Research Foundation of South Africa and the Belgian Federal Service for Scientific, Technical and Cultural Affairs, the Actions de Recherche Concertées, Communauté Fran?aise de Belgique. F.B. is holder of a Wellcome Trust Research Senior Fellowship for Medical Science in South Africa (Grant 056708/Z/99).
2 Address correspondence and reprint requests to Dr. Frank Brombacher, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, S27.1, Observatory 7925, Anzioroad, South Africa. E-mail address: fbrombac@uctgsh1.uct.ac.za
3 Abbreviations used in this paper: m, murine; iNOS, induced NO synthase.
Received for publication June 24, 2004. Accepted for publication November 19, 2004.
References
Uyttenhove, C., R. J. Simpson, J. Van Snick. 1988. Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity. Proc. Natl. Acad. Sci. USA 85:6934.
Renauld, J. C., F. Houssiau, J. Louahed, A. Vink, J. Van Snick, C. Uyttenhove. 1993. Interleukin-9. Adv. Immunol. 54:79.
Demoulin, J. B., J. C. Renauld. 1998. Interleukin 9 and its receptor: an overview of structure and function. Int. Rev. Immunol. 16:345.
Gessner, A., H. Blum, M. Rollinghoff. 1993. Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology 189:419
Grencis, R. K., L. Hultner, K. J. Else. 1991. Host protective immunity to Trichinella spiralis in mice: activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74:329.
Svetic, A., K. B. Madden, X. D. Zhou, P. Lu, I. M. Katona, F. D. Finkelman, J. F. Urban, Jr, 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
Faulkner, H., J. C. Renauld, S. J. Van, R. K. Grencis. 1998. Interleukin-9 enhances resistance to the intestinal nematode Trichuris muris. Infect. Immun. 66:3832.
Kopf, M., G. G. Le, M. Bachmann, M. C. Lamers, H. Bluethmann, G. Koehler. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362:245.
Monteyne, P., J. C. Renauld, B. J. Van, D. W. Dunne, F. Brombacher, J. P. Coutelier. 1997. IL-4-independent regulation of in vivo IL-9 expression. J. Immunol. 159:2616.
Renauld, J. C., N. van der Lugt, A. Vink, M. van Roon, C. Godfraind, G. Warnier, H. Merz, A. Feller, A. Berns, J. Van Snick. 1994. Thymic lymphomas in interleukin 9 transgenic mice. Oncogene 9:1327.
Vink, A., G. Warnier, F. Brombacher, J. C. Renauld. 1999. Interleukin 9-induced in vivo expansion of the B-1 lymphocyte population. J. Exp. Med. 189:1413.
Louahed, J., Y. Zhou, W. L. Maloy, P. U. Rani, C. Weiss, Y. Tomer, A. Vink, J. Renauld, J. Van Snick, N. C. Nicolaides, et al 2001. Interleukin 9 promotes influx and local maturation of eosinophils. Blood 97:1035.
Faulkner, H., N. Humphreys, J. C. Renauld, J. Van Snick, R. Grencis. 1997. Interleukin-9 is involved in host protective immunity to intestinal nematode infection. Eur. J. Immunol. 27:2536.
Temann, U. A., P. Ray, R. A. Flavell. 2002. Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology. J. Clin. Invest. 109:29.
Townsend, J. M., G. P. Fallon, J. D. Matthews, P. Smith, E. H. Jolin, N. A. McKenzie. 2000. IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity 13:573.
Heinzel, F. P., M. D. Sadick, B. J. Holaday, R. L. Coffman, R. M. Locksley. 1989. Reciprocal expression of interferon- or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59.
Louis, J., H. Himmelrich, L. C. Parra, C. F. Tacchini, P. Launois. 1998. Regulation of protective immunity against Leishmania major in mice. Curr. Opin. Immunol. 10:459
Fowell, D. J., R. M. Locksley. 1999. Leishmania major infection of inbred mice: unmasking genetic determinants of infectious diseases. BioEssays 21:510
Brombacher, F.. 2000. The role of interleukin-13 in infectious diseases and allergy. BioEssays 22:646.
Nashed, B. F., Y. Maekawa, M. Takashima, T. Zhang, K. Ishii, T. Dainichi, H. Ishikawa, T. Sakai, H. Hisaeda, K. Himeno. 2000. Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major. Microbes Infect. 2:1435
Richard, M., R. K. Grencis, N. E. Humphreys, J. C. Renauld, J. Van Snick. 2000. Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris-infected mice. Proc. Natl. Acad. Sci. USA 97:767.
Mohrs, M., B. Ledermann, G. Kohler, A. Dorfmuller, A. Gessner, F. Brombacher. 1999. Differences between IL-4- and IL-4 receptor -deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J. Immunol. 162:7302.
Landegren, U.. 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase: applications to detection of lymphokines and cell surface antigens. J. Immunol. Methods 67:379.
Mohrs, M., C. Holscher, F. Brombacher. 2000. Interleukin-4 receptor -deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infect. Immun. 68:1773.
Herbert, D. R., C. Holscher, M. Mohrs, B. Arendse, A. Schwegmann, M. Radwanska, M. Leeto, R. Kirsch, P. Hall, H. Mossmann, et al 2004. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20:623.
Druez, C., P. Coulie, C. Uyttenhove, J. Van Snick. 1990. Functional and biochemical characterization of mouse P40/IL-9 receptors. J. Immunol. 145:2494.
Lemesre, J. L., D. Sereno, S. Daulouede, B. Veyret, N. Brajon, P. Vincendeau. 1997. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigote and axenically grown amastigote forms. Exp. Parasitol. 86:58.
Hesse, M., M. Modolell, A. C. La Flamme, M. Schito, J. M. Fuentes, A. W. Cheever, E. J. Pearce, T. A. Wynn. 2001. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J. Immunol. 167:6533.
Stenger, S., H. Thuring, M. Rollinghoff, C. Bogdan. 1994. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J. Exp. Med. 180:783.
Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu, W. Muller, S. Moncada, F. Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375:408.
Roberts, S. C., M. J. Tancer, M. R. Polinsky, K. M. Gibson, O. Heby, B. Ullman. 2004. Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania: characterization of gene deletion mutants. J. Biol. Chem. 279:23668.
Iniesta, V., L. C. Gomez-Nieto, I. Molano, A. Mohedano, J. Carcelen, C. Miron, C. Alonso, I. Corraliza. 2002. Arginase I induction in macrophages, triggered by Th2-type cytokines, supports the growth of intracellular Leishmania parasites. Parasite Immunol. 24:113.
Grohmann, U., J. Van Snick, F. Campanile, S. Silla, A. Giampietri, C. Vacca, J. C. Renauld, M. C. Fioretti, P. Puccetti. 2000. IL-9 protects mice from Gram-negative bacterial shock: suppression of TNF-, IL-12, and IFN-, and induction of IL-10. J. Immunol. 164:4197.[(Berenice Arendse, Jacques)
IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.
Introduction
Interleukin-9 is a pleiotropic cytokine produced primarily by Th cells that was originally identified as a mouse T cell growth (1) and mast cell differentiation factor. Other targets ascribed to this cytokine include erythroid precursors, B lymphocytes, eosinophils, bronchial epithelial cells, and neuronal precursors (reviewed in Refs.2 and 3). Because of its restricted production by Th2 clones in vitro (4) and its expression in Th2-type responses in vivo (5, 6, 7), IL-9 is considered to be a Th2 cytokine that can be induced via IL-4-dependent (8) and IL-4-independent (9) pathways. Deregulated IL-9 expression in transgenic mice resulted in profound perturbations of multiple hemopoietic cell lineages with diverse phenotypes. Lymphoma genesis, enhanced Ig expression (10), expansion of B1 cell lymphocyte subset (11), mastocytosis, and eosinophil maturation (12), and parasitic worm expulsion (7, 13) were observed. Lung-specific IL-9 expression led to severe airway inflammation, with infiltration of eosinophils and lymphocytes, mast cell hyperplasia, and increased subepithelial collagen deposition (14). Recently generated IL-9-deficient mice showed a severe impairment of goblet cell hyperplasia and mastocytosis in a pulmonary granuloma model. However, pulmonary fibrosis, eosinophil and lymphocyte infiltration were normal, as was the development of pulmonary Th2 responses (15).
Experimental murine leishmaniasis is a paradigm example of the relationship between the genetic factors that control Th cell differentiation and the outcome of the disease. Healer strains, like C57BL/6, develop predominant Th1 responses with high IFN-, low IL-4 production and protective cellular immune responses, whereas nonhealer strains, like BALB/c, develop predominant Th2 responses with high IL-4, low IFN- production resulting in exacerbation of the disease (16). Leishmania-induced Th2 cytokine responses include IL-4, IL-5, IL-9, and IL-13, respectively. Many studies have shown that IL-4 is a main disease-promoting factor in cutaneous leishmaniasis, and neutralizing IL-4 in vivo by mAb converts nonhealers to healers (reviewed in Refs.17 and 18). Infection studies in BALB/c mice deficient for IL-4, IL-13, IL-4R, or STAT6 are able to contain acute infection with impaired Th2 responses, suggesting that both IL-4 and IL-13 contribute to the susceptible phenotype, which seems to depend on the Leishmania strain used (19). The role of IL-13 remains unclear, with disease-promoting functions during acute leishmaniasis and possible protective functions during the later chronic disease, which were suggested from infection studies using IL-13- or IL-4R-deficient mice, respectively. IL-5 plays a minor role in the overall susceptibility in Leishmania major-infected BALB/c (19).
Very little is known on the role of IL-9 in leishmaniasis. The only two publications available investigated IL-9 expression, showing that IL-9 is induced by L. major infection and transiently expressed during the first days postinfection. Also of interest was the observation that from 4 wk onward, IL-9 synthesis was only observed in susceptible BALB/c but not in resistant C57BL/6 or DBA mice (4, 20). Expression correlated with the expansion of Ag-specific Th2 cells and purified CD4+ T cells produced IL-9 during polyclonal or Ag-specific restimulation (4). No further information is available, with a potential role of IL-9 in leishmaniasis still unexplored.
Recently, it was shown that linking murine IL-9 to OVA results in the formation of a highly immunogenic complex that ensures production of high titers of neutralizing anti-IL-9 Abs in mice (21). Because the absence of T cell help is crucial for B cell tolerance, the coupling of an immunogenic foreign Ag to a self Ag provides physically linked T cell help to B cells, therefore overcoming B cell tolerance. Using this method, IL-9-depleted BALB/c and C57BL/6 mice were generated, infected with L. major, and compared with sham-immunized control BALB/c mice to define a possible role of IL-9 in leishmaniasis by loss of function.
Materials and Methods
Mice and parasites
Mice were kept at the animal facility at the Health Science Faculty, University of Cape Town, under specific pathogen-free conditions.
The L. major MHOM/IL/81/FEBNI strain was maintained by continuous passage in BALB/c mice as previously described (22). Anesthetized mice were infected s.c. in one hind footpad with 2 x 106 stationary-phase metacyclic L. major promastigotes in a final volume of 50 μl of HBSS. Parasites were isolated from skin lesions of infected animals. Parasite burden from homogenized organs was determined by 2-fold limiting dilutions in Schneiders medium (Sigma-Aldrich). Stationary-phase cultures were also used to prepare frozen-and-thawed (F/T; 1 x 107/ml) Ag of L. major promastigotes.
Preparation of IL-9-OVA complexes and immunization protocol
IL-9-OVA complexes were obtained by cross-linking mouse IL-9 and OVA (Sigma-Aldrich) with glutaraldehyde, and purified as previously shown (21). Mice were injected s.c. with 100 μl of 1:1 mixture of 10 μg of IL-9 OVA complexes in PBS and CFA. Two s.c. boosts were performed in IFA at wk 2 and 4. Control mice received an equivalent amount of OVA in Freund’s adjuvant only. Anti-IL-9 titers were measured by the inhibitory activity of the sera on the proliferation of TS1 cells that respond to IL-9. Sera were serially diluted in 96-well plates containing culture medium and incubated in the presence of 2.5 U/ml murine (m)3 IL-9 for 1 h. TS1 cells were incubated at 37°C, 8% CO2 for 3 days, and proliferation was measured by hexoseaminidase activity determination (23).
Cell isolation and CD4+ restimulation
CD4+ T cells were purified (>90% by FACS) from lymph nodes by positive selection with magnetic mouse CD4 Dynabeads and mouse CD4 DETACHaBEAD (Dynal; Robbins Scientific) and differentiation performed as described (24). CD4+ T cells (2 x 106cell/ml) were stimulated with anti-CD3 (145-2C-11; BD Pharmingen) or F/T and cytokine concentration from supernatant determined by ELISA 48 h later.
Macrophage activation
Thioglycolate (3%)-elicited peritoneal exudate cells were harvested at day 6 and further cultured in triplicates at a concentration of 2 x 106/ml in 96-well plates (Nunc) for 4 h. Plastic-adherent macrophages were stimulated with LPS (10 ng/ml; Sigma-Aldrich) and IFN- (100 U/ml; BD Pharmingen). After 48 h, the concentration of NO in supernatants was measured by the Griess reaction. Arginase was determined as previously described (25).
ELISA
Cytokine concentrations were determined by sandwich ELISA. Standards and Abs were purchased from BD Pharmingen and detected using alkaline phosphatase-coupled streptavidin (Southern Biotechnology). Detection limits were as follows: IFN-, IL-9, and IL-13, 46 pg/ml; IL-4, 2 pg/ml. Ag-specific Ig ELISA was performed as previously described (22).
Histology
Tissue samples were fixed in neutral buffered formalin, processed, and 5- to 7-μm sections were stained with H&E.
Statistics
Data are given as mean ± SD, and the differences were tested using the unpaired two-tailed Student’s t test or ANOVA using GraphPad Prism software.
Results
Effective production of anti-IL-9 autoantibodies
IL-9-deficient mice were generated by immunization with mIL-9, chemically complexed to OVA. For this purpose, BALB/c and C57BL/6 mice were immunized with three injections of IL-9 cross-linked to OVA. Two weeks after the last injection, the anti-IL-9 response was evaluated by measuring inhibitory activities of the sera in a bioassay by using an IL-9-dependent T cell line TS1. As shown in a representative experiment for BALB/c (Fig. 1a) and C57BL/6 (b), sera were found to strongly inhibit IL-9-induced proliferation, independent of the mouse strain used. Half-maximal inhibition of 2.5 U/ml mIL-9 was obtained at mean serum dilutions of 3 and 7 x 10–4 in BALB/c and C57BL/6 mice, respectively (shown in Fig. 1c). The vaccination was 1) specific for IL-9, because proliferation of TS1 by IL-4 was not influenced from sera, and 2) long lasting, with only slight reductions in the observed inhibition titers after a year of vaccination (data not shown), as reported before (21).
FIGURE 1. Induction of IL-9 specific autoantibodies. a and b, Eight mice per group of BALB/c mice (a) or C57BL/6 mice (b) were immunized with IL-9 complexed to OVA in CFA (IL-9-OVA) or with OVA alone to produce anti-IL-9 autoantibodies. Sera was collected 2 wk after the last immunization and tested in serial dilutions for IL-9 inhibition in an IL-9-dependent TS1 cell proliferation assay. c, The serum titer able to inhibit 50% of TS1 cell proliferation with 2.5 U/ml mIL-9 is shown from individual mice, including the mean (horizontal line). Shown is a representative of three independent experiments.
Anti-IL-9 vaccination increases resistance in L. major-infected BALB/c mice
To determine a possible role of IL-9 in leishmaniasis, IL-9-OVA-immunized or control OVA-immunized BALB/c or C57BL/6 mice were infected with 2 x 106 virulent L. major (MHOM/IL/81/FEBNI) metacyclic promastigotes into one hind footpad. As expected, control BALB/c mice developed massive footpad swelling (Fig. 2a) with ulceration and necrosis (indicated by the asterisks) from wk 3 onwards. Mice had to be killed at wk 8 postinfection due to disease progression, with necrosis and ulceration in all infected control BALB/c mice. In contrast, IL-9-OVA-immunized BALB/c mice stabilized footpad swelling on a moderate level within the first 4 wk postinfection. Parasite burden in the draining popliteal lymph node (Fig. 2b) and in the infected footpad (c) was significantly lower in IL-9-OVA-immunized BALB/c mice compared with infected control BALB/c mice at 8 wk postinfection and confirmed at wk 5 and 9 in independent experiments. Histopathology developed from wk 3 onwards in the control OVA-immunized BALB/c mice with severe bone destruction in the footpad at wk 8 (data not shown). IL-9-OVA-immunized BALB/c mice more than doubled their time span until ulceration and necrosis developed, which started from wk 10 onward with termination of the experiment at wk 17 postinfection with similar disease progression as observed in the control group 9 wk earlier. In contrast, the healer strain C57BL/6 developed a transient and moderate swelling during the first weeks and subsequently developed resistance to L. major infection without an observed effect of IL-9 vaccination. Conclusively, these data showed that anti-IL-9 vaccination was able to delay disease progression during L. major infection in the nonhealer BALB/c mouse strain.
FIGURE 2. L. major infection of anti-IL-9-vaccinated mice. a, Three weeks after the last immunization, BALB/c (, ) and C57BL/6 (, ) mice, either immunized with IL-9-OVA (, ), or sham-immunized with OVA only (, ) were injected in the left hind footpad with 2 x 106 metacyclic promastigotes of L. major (MHOM/IL/81/FEBNI). The course of the infection was monitored weekly by measuring infected and noninfected hind footpads. Values are expressed as means ± SD of eight mice per group of a representative of three independent experiments. The onset of ulceration and necrosis from individual mice is marked with an asterisk. Parasite load from the draining popliteal lymph node (b) or infected footpad (c) of individual mice was determined by 2-fold limiting dilutions. Mean (—) ± SD from four mice per group at wk 8 postinfection; ***, p < 0.001; **, p < 0.01, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Similar results were obtained at wk 5 and 9 (p < 0.01) in two independent experiments.
Anti-IL-9 vaccination leads to a shift to protective Th1/type 1 responses
To investigate possible mechanisms to explain the observed increased resistance, L. major-specific Th polarization was determined during infection. Mitogenic- or Ag-specific restimulation of CD4+ T cells, isolated from the draining lymph node of IL-9-OVA-immunized BALB/c mice, produced significantly higher IFN- but lower IL-4 levels compared with cells from sham-immunized mice (Fig. 3). Reduced IL-4 production was consistent with impaired Th2 effector cytokines, because IL-9 and IL-13 were significantly impaired in IL-9-vaccinated BALB/c mice (Fig. 3). As expected, the healer strain C57BL/6 showed a predominant L. major-specific Th1-polarized response with high IFN- and very low IL-4 production (Fig. 3) without an Ag-specific effect of IL-9 OVA vaccination. These results, first, suggest an influence on Th cell differentiation by IL-9 and, second, confirm that Th2 cells are major IL-9 producers during leishmaniasis. This resulted in slightly increased type 1 Ab responses but significantly impaired Ab-specific type 2 responses in comparison to control BALB/c mice (Fig. 4). IL-9-OVA or sham-immunized C57BL/6 mice showed no significant differences in their Ab responses. Together, these results show evidence for a shift toward Th1/type 1 responses by neutralizing endogenous IL-9 in BALB/c mice.
FIGURE 3. Th cell differentiation. CD4+ T cells from the draining lymph node were isolated and incubated with anti-CD3 (), L. major Ag (), or medium only (). IFN-, IL-4, IL-9, and IL-13 were determined by ELISA, with values expressed as mean ± SD of triplicate cultures from four mice per group at wk 5 (a), wk 8 (b), or at wk 9 (data not shown) postinfection in independent experiments. Values of p compared with sham-immunized BALB/c mice were calculated by paired Student’s t test.
FIGURE 4. L. major-induced Ab responses. At the same time point, sera were analyzed from BALB/c (, ) and C57BL/6 (, ) mice, either immunized with IL-9-OVA (, ), or sham-immunized with OVA only (, ). L. major Ag-specific IgG1, IgG2a, or total IgE titers were determined by ELISA using end-point dilution or a known standard for IgE (mean + SD of five mice per group). *, p < 0.01, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Shown is a representative of three independent experiments.
Increased NO killing effector macrophage activity
Macrophages are the major cellular host for L. major where amastigotes propagate in the phagolysosome. NO is the crucial killing effector molecule against leishmaniasis, produced by IFN--stimulated and iNOS-induced classical macrophages. To determine the influence of IL-9-OVA immunization on L. major-specific killing effector functions, macrophages were isolated from thioglycolate-elicited peritoneal exudate cells of infected mice and restimulated with IFN-/LPS to determine their killing effector function. Macrophages from 5-wk-infected mice showed no differences (data not shown). Macrophages from 8-wk-infected IL-9-OVA-immunized BALB/c mice showed small but significant increased induced NO synthase (iNOS)-catalyzed NO production compared with cells from sham-immunized BALB/c (Fig. 5a). This was verified by showing a striking reduction in urea production in the earlier (Fig. 5b), which is a side product of arginase 1 activity. This differential outcome can be explained by competition between iNOS and arginase 1 for the common substrate L-arginine. IL-9-OVA immunization had no effect on the NO production of C57BL/6 mice.
FIGURE 5. Macrophage NO production. Eight weeks postinfection, thioglycolate-elicited, adherent peritoneal macrophages were restimulated with LPS/IFN-, and the production of NO (a) and urea (b) was determined after 48 h. Values represent the mean ± SD from triplicate cultures. **, p 0.01; *, p 0.05, compared with sham-immunized BALB/c mice, calculated by paired Student’s t test. Shown is a representative of two independent experiments.
IL-9 acts downstream from IL-4R-mediated functions
To determine whether anti-IL-9 vaccination is dependent on IL-4- or IL-13-mediated functions, L. major infection studies in IL-9-OVA-immunized BALB/c IL-4R-deficient mice were performed. These mice are IL-4 and IL-13 unresponsive, because the IL-4R chain is a crucial component of the IL-4 and IL-13 receptor (19). Anti-IL-9 vaccination was similarly effective in IL-4R-deficient mice with a half-maximal inhibition of 2.5 U/ml mIL-9, obtained at mean serum dilution of 5 x 104 (data not shown). Anti-IL-9 or sham-immunized control or BALB/c IL-4R-deficient mice (eight mice per group) were infected with 2 x 106 virulent L. major (MHOM/IL/81/FEBNI) metacyclic promastigotes into one hind footpad, and the swelling of the footpad was monitored. As previously shown using a different L. major strain, IL-4R-deficient BALB/c mice were resistant, with slightly increased footpad swelling compared with C57BL/6 (Fig. 6a). Although IL-9-OVA immunization had the described protective effect in BALB/c mice, shown by delayed footpad swelling, and disease progression (Fig. 6a), as well as reduced parasite burden (b), no effect was observed in IL-9-vaccinated BALB/c IL-4R-deficient mice compared with sham-vaccinated controls. CD4+ T cell IFN-, IL-4, and IL-9 responses after Ag- or CD3-specific restimulation were similar between the IL-9- and sham-immunized IL-4R-deficient mice, with no detectable IL-9 found (Fig. 6c). Together, these data suggest that IL-9 vaccination had no influence on the balance of Th differentiation in this mouse strain. This conclusion was confirmed, because IgG1, -2a, and IgE Ag-specific Ab responses (data not shown) and macrophage NO production were also similar (Fig. 6d). Together, these data suggest that IL-9 acts downstream from IL-4/IL-13-mediated functions.
FIGURE 6. No effect on leishmaniasis in IL-9-OVA-immunized IL-4R-deficient BALB/c mice. Eight IL-9-OVA-immunized IL-4R-deficient BALB/c () or sham-immunized IL-4R-deficient BALB/c (?) were injected in the left hind footpad with 2 x 106 metacyclic promastigotes of L. major (MHOM/IL/81/FEBNI). a, The course of the infection was monitored weekly by measuring infected and noninfected hind footpads. At wk 5 postinfection, three mice per group were sacrificed and further analyzed. b and c, The draining popliteal lymph node was isolated, and parasite burden (b) and anti-CD3 and Leishmania Ag-specific IFN-, IL-4, and IL-9 production (c) was determined after 48 h from the supernatant. Values are expressed as means ± SD. d, Thioglycolate-elicited peritoneal macrophages were LPS/IFN- stimulated, and the NO concentration in the supernatant was measured.
Discussion
IL-9-deficient mice were generated by immunization with mIL-9, which was chemically complexed to OVA. This approach induced high titers of neutralizing anti-IL-9 Abs, as shown before (21). In this study, we present data showing evidence for the first time that IL-9 is a susceptible factor in L. major infection. Experimentally, this was shown by in vivo IL-9 neutralization, which substantially delayed disease progression of nonhealer BALB/c mice. Delayed leishmaniasis was accompanied by reduced parasite burden, footpad swelling, and milder histopathology during acute infection. As a consequence, IL-9-vaccinated BALB/c mice more than doubled their time span upon infection until deleterious leishmaniasis developed. Neutralization of endogenous IL-9 caused impaired Th2/type 2 responses with a shift toward protective Th1/type 1 responses, as determined by cytokine production of Ag-specific restimulated CD4+ T cells, isolated from the draining lymph nodes. This was confirmed ex vivo by reduced Ag-specific type 2 Ab isotype production in the blood of these mice. We concluded from these data that IL-9 is involved in promoting Th responses toward deleterious Th2/type 2 immune responses during leishmaniasis. The theoretical possibility of Th2 cell depletion due to direct anti-IL-9-specific Ab binding to its receptor is unlikely due to the restricted surface expression of the IL-9R among T cell subpopulations (26) and the observed normal Ag-specific IgG1 and IgE responses during immunization with Aspergillus extract or infection with Trichinella in IL-9-vaccinated mice (21). Despite the fact that IL-9 can act as a growth factor for certain T cell clones (1) and IL-9 overexpression in vivo can lead to thymic lymphomas (10), a Th2-promoting effect was an unexpected finding, because no effect on Th differentiation has been found until now. In vitro and in vivo (Nippostrongylus brasiliensis and schistosoma egg) Th differentiation was found to be normal in a genetically engineered IL-9-deficient 129sv mouse strain (15). Together, this may indicate that the observed influence of IL-9 on Th responses is restricted to certain diseases, like L. major infection, and/or certain genetic backgrounds influencing T cell responses. In any case, the present observations provide the first demonstration that IL-9 is an important element in the physiological regulation of the Th1/Th2 balance in vivo.
With respect to the analysis of IL-9 mode of action, we are currently not able to distinguish whether IL-9 has a direct effect on early steps in L. major-induced Th differentiation or alternatively may promote Th2 effector cell expansion. The latter possibility is more attractive, because IL-9 is barely detectable at the onset of an L. major infection and is mainly produced by differentiated Th2 effector cells (see Fig. 3) during acute leishmaniasis. This may also explain why IL-9 vaccination had no measurable effect during L. major infection in C57BL/6 as well as IL-4R-deficient BALB/c mice. Both mouse strains showed dominant Th1 responses during infection with very little IL-9 production after CD4+ T cell restimulation (Figs. 3 and 6). Therefore, it can be envisaged that IL-9 does act downstream from IL-4-mediated Th2 differentiation, and may be an important element to maintain Th2-dominated response to L. major. Clearly, further investigations are needed to fully explore the effects of IL-9 on Th responses in L. major infection.
IL-9 vaccination resulted in improved classical macrophage effector functions. This was determined by increased NO production but reduced urea production, the latter a side product of arginase 1 catalyzation from LPS/IFN--restimulated macrophage. Indeed, NO is a crucial effector molecule able to kill amastigotes within the macrophages (27). NO is catalyzed by the enzyme inducible NO synthase (NOS2), which competes with arginase 1 for the substrate arginine (28). Abrogating iNOS function, either by in vivo blocking or by using iNOS-deficient mice does lead to disease progression in healer strains (29, 30). Arginase 1, in contrast is induced by (Th2-produced) IL-4/IL-13-activated alternative macrophages in the nonhealer strain BALB/c (25) and catalyzes arginine to L-ornithine and urea. L-Ornithine plays a pivotal role in polyamine precursor metabolism in the host as well as in the Leishmania parasite (31). Because arginase I produced by alternatively activated macrophages supports the growth of intracellular Leishmania parasite (32), the observed impairment in IL-9-vaccinated mice together with the increased killing effector function in classically activated macrophages may explain the reduced parasite burden found in vaccinated mice. Also of importance, the observed increased macrophage killing effector function can be explained as a direct consequence of the increased IFN- production due to the found shift toward Th1 cytokine responses in IL-9-vaccinated and L. major-infected BALB/c mice. However, this does not exclude an additional and direct action by IL-9 on macrophages and, vice versa, a possible effect on IL-9-stimulated macrophages on Th cells. Although IL-9R surface expression was never found in freshly isolated mouse macrophages, some macrophage cell lines are expressing low levels of the receptor (26). This may also be the case in vivo during L. major infection. A recent study showing that rIL-9 was able to protect mice from Gram-negative bacterial shock and the correlation of a reduction in early IL-12, IFN-, and TNF, together with an increase in IL-10 (33), may also implicate a possible monocyte/macrophage effect of IL-9.
In conclusion, we uncovered the role of IL-9 as a susceptibility factor in L. major infection by promoting detrimental Th2/type 2 response. Indeed, IL-9-vaccinated mice are excellent models to study the role of IL-9 in human diseases, because the system is time inducible, an advantage to classical gene-targeted mice. Our results further extend the efforts made to generate autoantibodies capable of regulating biological processes with IL-9.
Acknowledgments
We thank E. Smith, R. Peterson, M. Simpson, and the animal facility staff for excellent technical assistance. Drs. C. Renauld and M. Kopf are thanked for critically reading the manuscript.
Footnotes
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 Medical Research Council and National Research Foundation of South Africa and the Belgian Federal Service for Scientific, Technical and Cultural Affairs, the Actions de Recherche Concertées, Communauté Fran?aise de Belgique. F.B. is holder of a Wellcome Trust Research Senior Fellowship for Medical Science in South Africa (Grant 056708/Z/99).
2 Address correspondence and reprint requests to Dr. Frank Brombacher, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, S27.1, Observatory 7925, Anzioroad, South Africa. E-mail address: fbrombac@uctgsh1.uct.ac.za
3 Abbreviations used in this paper: m, murine; iNOS, induced NO synthase.
Received for publication June 24, 2004. Accepted for publication November 19, 2004.
References
Uyttenhove, C., R. J. Simpson, J. Van Snick. 1988. Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity. Proc. Natl. Acad. Sci. USA 85:6934.
Renauld, J. C., F. Houssiau, J. Louahed, A. Vink, J. Van Snick, C. Uyttenhove. 1993. Interleukin-9. Adv. Immunol. 54:79.
Demoulin, J. B., J. C. Renauld. 1998. Interleukin 9 and its receptor: an overview of structure and function. Int. Rev. Immunol. 16:345.
Gessner, A., H. Blum, M. Rollinghoff. 1993. Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology 189:419
Grencis, R. K., L. Hultner, K. J. Else. 1991. Host protective immunity to Trichinella spiralis in mice: activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74:329.
Svetic, A., K. B. Madden, X. D. Zhou, P. Lu, I. M. Katona, F. D. Finkelman, J. F. Urban, Jr, 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
Faulkner, H., J. C. Renauld, S. J. Van, R. K. Grencis. 1998. Interleukin-9 enhances resistance to the intestinal nematode Trichuris muris. Infect. Immun. 66:3832.
Kopf, M., G. G. Le, M. Bachmann, M. C. Lamers, H. Bluethmann, G. Koehler. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362:245.
Monteyne, P., J. C. Renauld, B. J. Van, D. W. Dunne, F. Brombacher, J. P. Coutelier. 1997. IL-4-independent regulation of in vivo IL-9 expression. J. Immunol. 159:2616.
Renauld, J. C., N. van der Lugt, A. Vink, M. van Roon, C. Godfraind, G. Warnier, H. Merz, A. Feller, A. Berns, J. Van Snick. 1994. Thymic lymphomas in interleukin 9 transgenic mice. Oncogene 9:1327.
Vink, A., G. Warnier, F. Brombacher, J. C. Renauld. 1999. Interleukin 9-induced in vivo expansion of the B-1 lymphocyte population. J. Exp. Med. 189:1413.
Louahed, J., Y. Zhou, W. L. Maloy, P. U. Rani, C. Weiss, Y. Tomer, A. Vink, J. Renauld, J. Van Snick, N. C. Nicolaides, et al 2001. Interleukin 9 promotes influx and local maturation of eosinophils. Blood 97:1035.
Faulkner, H., N. Humphreys, J. C. Renauld, J. Van Snick, R. Grencis. 1997. Interleukin-9 is involved in host protective immunity to intestinal nematode infection. Eur. J. Immunol. 27:2536.
Temann, U. A., P. Ray, R. A. Flavell. 2002. Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology. J. Clin. Invest. 109:29.
Townsend, J. M., G. P. Fallon, J. D. Matthews, P. Smith, E. H. Jolin, N. A. McKenzie. 2000. IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity 13:573.
Heinzel, F. P., M. D. Sadick, B. J. Holaday, R. L. Coffman, R. M. Locksley. 1989. Reciprocal expression of interferon- or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59.
Louis, J., H. Himmelrich, L. C. Parra, C. F. Tacchini, P. Launois. 1998. Regulation of protective immunity against Leishmania major in mice. Curr. Opin. Immunol. 10:459
Fowell, D. J., R. M. Locksley. 1999. Leishmania major infection of inbred mice: unmasking genetic determinants of infectious diseases. BioEssays 21:510
Brombacher, F.. 2000. The role of interleukin-13 in infectious diseases and allergy. BioEssays 22:646.
Nashed, B. F., Y. Maekawa, M. Takashima, T. Zhang, K. Ishii, T. Dainichi, H. Ishikawa, T. Sakai, H. Hisaeda, K. Himeno. 2000. Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major. Microbes Infect. 2:1435
Richard, M., R. K. Grencis, N. E. Humphreys, J. C. Renauld, J. Van Snick. 2000. Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris-infected mice. Proc. Natl. Acad. Sci. USA 97:767.
Mohrs, M., B. Ledermann, G. Kohler, A. Dorfmuller, A. Gessner, F. Brombacher. 1999. Differences between IL-4- and IL-4 receptor -deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J. Immunol. 162:7302.
Landegren, U.. 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase: applications to detection of lymphokines and cell surface antigens. J. Immunol. Methods 67:379.
Mohrs, M., C. Holscher, F. Brombacher. 2000. Interleukin-4 receptor -deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infect. Immun. 68:1773.
Herbert, D. R., C. Holscher, M. Mohrs, B. Arendse, A. Schwegmann, M. Radwanska, M. Leeto, R. Kirsch, P. Hall, H. Mossmann, et al 2004. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20:623.
Druez, C., P. Coulie, C. Uyttenhove, J. Van Snick. 1990. Functional and biochemical characterization of mouse P40/IL-9 receptors. J. Immunol. 145:2494.
Lemesre, J. L., D. Sereno, S. Daulouede, B. Veyret, N. Brajon, P. Vincendeau. 1997. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigote and axenically grown amastigote forms. Exp. Parasitol. 86:58.
Hesse, M., M. Modolell, A. C. La Flamme, M. Schito, J. M. Fuentes, A. W. Cheever, E. J. Pearce, T. A. Wynn. 2001. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J. Immunol. 167:6533.
Stenger, S., H. Thuring, M. Rollinghoff, C. Bogdan. 1994. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J. Exp. Med. 180:783.
Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu, W. Muller, S. Moncada, F. Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375:408.
Roberts, S. C., M. J. Tancer, M. R. Polinsky, K. M. Gibson, O. Heby, B. Ullman. 2004. Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania: characterization of gene deletion mutants. J. Biol. Chem. 279:23668.
Iniesta, V., L. C. Gomez-Nieto, I. Molano, A. Mohedano, J. Carcelen, C. Miron, C. Alonso, I. Corraliza. 2002. Arginase I induction in macrophages, triggered by Th2-type cytokines, supports the growth of intracellular Leishmania parasites. Parasite Immunol. 24:113.
Grohmann, U., J. Van Snick, F. Campanile, S. Silla, A. Giampietri, C. Vacca, J. C. Renauld, M. C. Fioretti, P. Puccetti. 2000. IL-9 protects mice from Gram-negative bacterial shock: suppression of TNF-, IL-12, and IFN-, and induction of IL-10. J. Immunol. 164:4197.[(Berenice Arendse, Jacques)