caused by Taenia crassiceps. Received: 29 May 1998 / Accepted: 3 July 1998. Abstract Resistance and susceptibility to different par- asitic diseases have been ...
Parasitol Res (1999) 85: 135 ± 141
Ó Springer-Verlag 1999
ORIGINAL PAPER
L.I. Terrazas á M. Cruz á M. RodrõÂ guez-Sosa R. Bojalil á F. GarcõÂ a-Tamayo á C. Larralde
Th1-type cytokines improve resistance to murine cysticercosis caused by Taenia crassiceps
Received: 29 May 1998 / Accepted: 3 July 1998
Abstract Resistance and susceptibility to dierent parasitic diseases have been associated with the predominance of Th1- or Th2-type immune responses. In experimental murine cysticercosis a Th1 response seems to be involved in resistance, whereas Th2 activity is associated with heavy parasite intensities. To test this notion the roles of Th1- and Th2-type cytokines in infected mice were studied after treatment with anticytokine monoclonal antibodies or with recombinant murine cytokines during early stages of infection. Mice receiving anti-interleukin 10 (IL-10) carried lower parasite intensities than did control mice and developed a strong Th1type response, whereas mice receiving anti-interferon gamma (IFN-c) showed a dramatic increase in susceptibility. Treatment with recombinant cytokines con®rmed these results; mice receiving IFN-c and IL-2 showed low parasite numbers, whereas IL-10 induced a signi®cant increase in parasite loads. Thus, the Th1-type immune response plays a fundamental role in protection against Taenia crassiceps cysticercosis, whereas Th2, at least through IL-10, favors parasite establishment.
Introduction In response to pathogens, naive CD4+ T-cells dierentiate into eector Th1 and/or Th2 cells. Th1 cells pro-
L.I. Terrazas (&) á M. Cruz á F. GarcõÂ a-Tamayo Department of Biology, Facultad de QuõÂ mica, Universidad Nacional AutoÂnoma de MeÂxico, Ciudad Universitaria, MeÂxico D.F. 04510, MeÂxico Tel.: (525) 622-30-98; Fax: (525) 616-20-10 M. RodrõÂ guez-Sosa á R. Bojalil Department of Immunology, Instituto Nacional de CardiologõÂ a Ignacio ChaÂvez, Department of Health Attention, Universidad AutoÂnoma Metropolitana-Xochimilco, MeÂxico, D.F. 14080, Mexico C. Larralde Department of Immunology, Instituto de Investigaciones BiomeÂdicas, U.N.A.M. MeÂxico, D.F 04510, Mexico
duce interleukin 2 (IL-2), interferon gamma (IFN-c), and tumor necrosis factor beta (TNF-b) and are involved in cell-mediated immune reactions. Th2 cells secrete mainly IL-4, IL-5, IL-6, IL-10, and IL-13 and mediate B-cell activation, antibody production, and the regulation of Th1 responses (Mosmann and Coman 1989). In many instances a Th1-type immune response helps to eliminate intracellular microorganisms, whereas a Th2-type response specializes in the control of extracellular pathogens (Cox and Liew 1992; Reiner and Locksley 1993; Stevenson and Tam 1993). Development of an inappropriate immune response can be ineective and even pathogenic to the host (Romagnani 1997). Experimental murine Taenia crassiceps cysticercosis has been used as a laboratory model in which to study the participation of immunologic, genetic, and genderassociated factors of resistance and susceptibility. Notwithstanding the limitations of secondary experimental infection with respect to natural infection with eggs from tapeworms, this model has been useful for the demonstration of important biological factors involved in the outcome of infection by T. crassiceps such as major histocompatibillity complex (MHC), associated dierences in susceptibility, which are principally mediated through the expression of the nonclassic class I MHC Qa-2 antigen (Fragoso et al. 1998) and are related to the hormonal environment where 17-b-estradiol is permissive, whereas androgens appear restrictive of parasite growth (Terrazas et al. 1994; Larralde et al. 1995). The mechanisms involved in parasite-restrictive immunity to murine T. crassiceps cysticercosis are currently known to be associated with T-cell responses. Initial characterization has revealed that neonatal thymectomy of mice greatly increases the susceptibility to T. crassiceps infection and T-cell replacement restores it to normal levels (Bojalil et al.1993), whereas the bulk of antiparasite antibodies do not clearly relate to protection and might even enhance parasite growth (HermaÂnek and Prokopic 1989; Kunz et al. 1991). Recent evidence indicates that experimentally infected mice develop an early Th1-type immune response concomitantly with limited parasite
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growth. Later the immune response is progressively polarized toward a Th2-type response at times when a signi®cant increase in the number of parasites is noted. At this late stage of infection there is a reduced cellular immune response to T. crassiceps soluble antigens accompanied by an elevated production of IL-10, IL-6, and IL-4 as well as IgG1 and IgG2b anti-cysticercal antibodies (Terrazas et al. 1998). These ®ndings have been taken to indicate that the Th1-type response is associated with resistance and that the Th2-type response either is irrelevant or favors parasite growth. In the same direction it has recently been reported that the granulomas surrounding dying T. crassiceps cysticerci present higher levels of IFN-c and IL-2 than of IL-4 and IL-10 (Robinson et al. 1997). The precise contribution of dierent T-helper (Th) or T-cytotoxic-cell subsets and the role of speci®c cytokines produced by these cells in anticysticercosis immunity has not yet been explored, and the idea of a Th1-to-Th2 shift being of signi®cance for the outcome of infection needs to be further tested. For exploration of the role played by several cytokines in the development of T. crassiceps infection, experiments were performed to assess modi®cations in the burdens of T. crassiceps induced by early supplementation with recombinant murine (rm) cytokines of the Th1 or Th2 type. Modi®cations in parasite burdens were also explored using an initial and transient treatment with monoclonal antibodies against the same cytokines.
Chemical Co., St. Louis, Mo. USA) was used as the IgG control antibody. Other groups of mice (®ve per group) were treated i.p. at the moment of infection and daily during the 1st week of infection with recombinant murine cytokines (a) IFN-c + IL-2 (50 ng and 5 U/ mouse, respectively), (b) IL-10 (50 ng/mouse), or (c) IL-4 (50 ng/ mouse) in PBS-BSA 3%. All cytokines were purchased from Pharmingen (San Francisco, Calif. USA). Control mice in this experiment received only PBS-BSA 3%. Infections Experimental infections were achieved by i.p. injection of each mouse with ten small (ca. 2-mm diameter) nonbudding cysticerci of T. crassiceps suspended in 0.3 ml PBS obtained as described above. Resulting individual intensities of infection (number of parasites in each mouse) were measured at 4 weeks after infection, when mice were killed and their peritoneal cavities were opened, and the numbers of cysticerci found inside were counted. Cell preparations and culture conditions
Female inbred BALB/c mice, originally purchased from Jackson Laboratories (Bar Harbor, Me.) in 1982, were used in this study; they have been maintained and reproduced in our animal facilities for more than 20 generations.
Mice were bled by cardiac punction and subsequently killed by cervical dislocation. Spleens were removed under sterile conditions from infected and control mice, and splenic cells were obtained by mincing and ®ltering, washed, and resuspended in culture medium made of RPMI 1640 (Gibco BRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS, Gibco BRL), 100 units of penicillin/streptomycin, 2 mM glutamine, 25 mM HEPES buffer, and 1% nonessential amino acids (Gibco BRL). Splenocytes were adjusted to 5 ´ 106 cells/ml in this medium. Next 100-ll volumes of the cell suspensions were placed into 96-well ¯at-bottom culture plates (Costar, Cambridge, Mass.) and stimulated with concanavalin A (Con-A, 2 lg/ml; sigma, St. Louis Mo.); plates were incubated at 37 °C and 5% CO2 for 72 h. At 18 h prior to culture termination, 0.5 lCi tritiated thymidine (methyl-[3H]-TDR, sp. act. 247.9 GBq/mmol; NEN, Boston, Mass.) was added to each well. After further incubation for 18 h, splenocytes were harvested onto glass ®lter papers and processed for liquid scintillation counting. Cells from the same animals were processed by antigeninduced proliferation, antigen being added in 20 ll of RPMI-1640 to give a ®nal concentration of 50 lg/ml (optimal concentration), and cultures were incubated for 6 days and processed as described above.
Parasites
Evaluation of cytokine production in vitro
Metacestodes of Taenia crassiceps (ORF) used in this study were harvested from the peritoneal cavity of female BALB/c mice after 2±4 months of infection. The cysticerci were washed four times in phosphate-buered saline (PBS; 0.15 M NaCl, 0.01 M sodium phosphate buer, pH 7.2) and selected for infection of mice or were immediately centrifuged at 45,700 g for 1 h at 4 °C to obtain their vesicular ¯uid as a source of antigen. The supernatant fraction, mainly consisting of vesicular ¯uid, was collected; its protein concentration was measured by Lowry's method (Lowry et al. 1951); and it was frozen at )70 °C until used. This supernatant fraction served as the antigen for serum antibody detection by enzymelinked immunoabsorbent assay (ELISA) or for antigen-induced proliferation.
Single-cell suspensions of splenocytes prepared as described above were diluted in 10% FBS-supplemented RPMI-1640 to give 5 ´ 106 cells/ml. A 1-ml volume of ®nal cell suspension was placed in each well of a 24-well plate (Costar) and incubated with Con-A at 2 lg/ ml for 24 h at 37 °C and 5% CO2. After centrifugation the supernatants were collected, aliquoted, and stored at )20 °C until used. An aliquot of the same cell suspension was stimulated with speci®c antigen at 50 lg/ml for 72 h, and supernatants were processed as described above. Basal levels (nonstimulated) of all cultures were measured. The cytokines IFN-c, IL-2, IL-4, and IL-10 were measured by sandwich ELISA using methods according to the manufacturer's instructions (PharMingen). The pairs of cytokine-speci®c monoclonal antibodies and recombinant cytokines were obtained from PharMingen.
Materials and methods Mice
Treatments At the moment of infection, each of ®ve mice per group was inoculated i.p. only once with (a) 50 lg anti-IFN-c monoclonal antibody (Genzyme, Cambridge, Ma.), (b) 50 lg anti-IL-10, or (c) 50 lg anti-IL-4 (Genzyme). These monoclonal antibodies have a halflife of 2 weeks, according to the manufacturer. Rat IgG (Sigma
Antibody subclass detection Sera obtained from normal and infected mice were diluted 1:100 and analyzed for speci®c anti-cysticercal antibodies by ELISA. IgG isotypes were detected using monoclonal antibodies to IgG2a,
137 IgG2b, and IgG1 diluted 1:1,000 (Zymed, San Diego, Calif.). Reactions were revealed with ABTS solution (Zymed). Statistical analysis The statistical signi®cance of the eects of the experimental variables on parasite intensities and immunologic parameters were determined by the nonparametric Mann-Whitney U-test (Wilcoxon rank).
Results Eect of anticytokine monoclonal antibodies on susceptibility and immune response to Taenia crassiceps As shown in Fig. 1, the parasite burden in mice treated with anti-IL-10 was signi®cantly reduced (P
