Proc. Nail. Acad. Sci. USA Vol. 88, pp. 5513-5517, July 1991 Immunology Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice (interleukin 4/interleukin 5/IgE/eosinophil/HeLigmosomoides polygyrus) JOSEPH F. URBAN, JR.*t, ILDY M. KATONAt§, WILLIAM E. PAUL¶, AND FRED D. FINKELMAN§ *Helminthic Diseases Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705-2350; Departments of tPediatrics and §Medicine, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 208884799; and 1Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Contributed by William E. Paul, March 28, 1991 ABSTRACT Parasitic helminths typically induce compo- with the nematode Nippostrongylus brasiliensis (20) and nents of immediate-type hypersensitivity, including elevated from liver granulomas induced by eggs of the trematode serum IgE, eosinophilia, and mucosal mast cells. These re- Schistosoma mansoni, without affecting lesion size (15). sponses are T-cell-dependent and associated with rapid expul- However, neutralization of endogenously produced IL-4 or sion of parasitic worms from a sensitized host; existing exper- IL-5 fails to measurably compromise host immunity in mice imental systems have failed to derme the precise role of infected with S. mansoni (21) or N. brasiliensis (ref. 20; cytokines in these responses. We report here that anti- J.F.U., I.M.K., and F.D.F., unpublished data). interleukin 4 or anti-interleukin 4 receptor antibodies block the The absence of a definitive role for IL-4 and IL-5 in host polyclonal IgE response to a parasitic nematode, Heligmo- responses to these parasites prompted the study of a nema- somoides polygyrus, and abrogate protective immunity to the tode parasite with a completely enteric and more chronic infection. In contrast, anti-interleukin 5 antibody prevented H. infection than N. brasiliensis to determine whether mucosal polygyrus-induced eosinophilia but did not prevent protection. elements important in protective immunity were regulated by These data provide evidence that a specific cytokine affects the IL-4 or IL-5. Parasitic third-stage larvae ofHeligmosomoides physiology and survival of a parasitic nematode in the host. polygyrus (Hp) invade the intestinal mucosa after oral inoc- ulation and molt twice within 8 days before emerging into the Resolution of gastrointestinal helminthiasis is a T-cell- intestinal lumen as adults that persist for several weeks (22). dependent process. Parasite expulsion mechanisms do not The results of the studies reported here show that antibodies function in congenitally athymic nude mice or in mice that against IL-4 or the IL-4 receptor (IL-4R), but not antibodies have been depleted of T cells (1-3). Host responses that against IL-5, block host protective mechanisms that normally modulate parasite physiology, such as worm fecundity, are limit parasite egg production and reduce the development of also known to be T-cell-dependent (4, 5). Because there is no new worms inoculated into previously infected mice. evidence that T cells directly kill nematode worms, immune and inflammatory responses that combine to eliminate par- MATERIALS AND METHODS asites or impair their activity might result from T-cell-derived cytokines. Interleukin 4 (IL-4) and interleukin 5 (IL-5) are Animals. Female BALB/c mice were obtained from the two cytokines that could potentially affect immunity to Small Animal Division of the National Cancer Institute helminths. (Frederick, MD) and were used for experimentation at 8-12 IL-4 has multiple immunoregulatory functions, including weeks ofage. All experiments were conducted with five mice autocrine T-cell growth factor activity, regulation of B-cell per group. Blood samples were obtained from the orbital IgE isotype expression, and stimulation ofthe growth and/or plexus. Serum was separated from clotted blood by centrif- differentiation of macrophages, hematopoietic cells, and ugation and stored at -80'C until used. An anthelminthic mast cells (6-12). The complexing of IgE receptors on drug, pyrantel pamoate (Strongid T, Pfizer Diagnostics), was mucosal mast cells with IgE antibody specific for parasite- administered orally at a dose of 1-2 mg per mouse to some derived antigens is postulated to be important in protective parasite-infected mice to remove adult worms from a primary immunity because of the subsequent release of mediators of infection. inflammation that could adversely affect parasite survival Parasitological Parameters. Infective, ensheathed third- (13). IL-5 regulates the generation of eosinophil myelocyte stage larvae of Hp (specimens on file at the U. S. National precursors in bone marrow and the development of mature Parasite Collection, U. S. National Museum Helmintholog- eosinophils after helminth infection (14, 15), and there is ical Collection no. 81930, Beltsville, MD) were propagated evidence that eosinophils and IgE may constitute an impor- and stored at 40C until used (23). Mice were inoculated orally tant defense mechanism in rats infected with schistosomes with 200 larvae by using a ball-tipped feeding tube. Adult (16) or the nematode Trichinella spiralis (17). Treatment of worms and total parasite eggs in the intestinal contents were mice with monoclonal antibody (mAb) against IL4 (llB11) determined at necropsy (23). Worm fecundity was expressed in vivo completely suppresses polyclonal IgE responses (18, by dividing the total number of eggs recovered from each 19) and limits the expansion of mucosal mast cells (K. B. mouse by the total number of female worms. Madden, J.F.U., H. J. Ziltener, J. W. Schrader, F.D.F., and Antibodies. The preparation and specificity testing of the I.M.K., unpublished work). Anti-IL-5 (TRFK-5) suppresses following antibodies have been previously described: affini- eosinophilia and eliminates eosinophils from the cellular ty-puriflied rabbit anti-mouse IgG1 (24) and anti-mouse IgE infiltrates responding to larvae in the lungs of mice infected Abbreviations: IL-4, interleukin 4; IL-5, interleukin 5; IL-4R, inter- The publication costs of this article were defrayed in part by page charge leukin 4 receptor; mAb, monoclonal antibody; Hp, Heligmo- payment. This article must therefore be hereby marked "advertisement" somoides polygyrus. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5513 Downloaded by guest on September 23, 2021 5514 Immunology: Urban et al. Proc. Natl. Acad. Sci. USA 88 (1991) (25); monoclonal rat IgG2b anti-mouse CD4 (GK1.5) (26); monoclonal rat IgG2a anti-mouse IgE (EM95) (27); mono- Hp + uNP clonal mouse IgE anti-dinitrophenyl (SPE-iv-7) (28); mono- N Secondary Infection clonal rat IgGl anti-mouse IL-4 (ilB11) (29) (Verax, Leba- Hp + AIL-4/5 * Primary Infection non, NH); monoclonal rat IgG2a anti-mouse IL-4R (ml) (30); monoclonal rat IgG1 anti-4-hydroxy-3-nitrophenylacetyl Hp + rAL-5 (J4-1) (31); monoclonal rat IgGl anti-mouse IL-5 (TRFK-5) (20); alkaline phosphatase-conjugated goat anti-rabbit immu- noglobulin (APGaRIg) (32). Hp + ct IL-4 Quantitation of Serum Immunoglobulin. Serum levels of mouse IgGl were determined by radial immunodiffusion with Hp -I02 standards and materials purchased from Hazelton Laborato- ries (Rockville, MD). Serum IgE levels were determined by Uninfected a micro ELISA (33). Peripheral Blood Analysis. Eosinophils were counted from 0 11000 2000 fresh blood samples by using the Unopette Test (Becton Eosinophils/mm3 Dickinson). Total peripheral blood leukocyte counts of ci- trated blood collected at were FIG. 2. Combined effects ofanti-IL-4 and anti-IL-5 on eosinophil the same time determined by responses to Hp. Mice were the same as those described for Fig. 1. using a ZM Coulter Counter. Eosinophils were counted from fresh blood samples and expressed as arithmetic means and SEs on day 21 after primary inoculation RESULTS (broad peak response between day 7 and 21) and day 11 after secondary inoculation (peak response between day 7 and 12). Total The kinetics of the serum IgE response to parasite infection peripheral blood leukocyte counts of citrated blood collected con- was followed to evaluate mAb treatment in vivo (Fig. 1). currently were similar among all groups at times examined. aNP, Serum IgE levels rose 4100-fold in response to a primary Hp anti-4hydroxy-3-nitrophenylacetyl. infection in mice treated with anti-IL-5 mAb, control mAb, or no antibody; a second inoculation with Hp produced a further The role ofIL-4 and IL-5 in protective immunity to Hp was 5-fold increase in serum IgE levels in these mice. Treatment evaluated by measuring adult worm survival and fecundity of Hp-infected mice with anti-IL-4 or a combination of (eggs per female worm) in mice cleared ofa primary infection anti-IL4 and anti-IL-5 mAb inhibited the Hp-induced rise in with an anthelminthic drug and then reinoculated with Hp serum IgE by a factor of 100 (Fig. 1). (Fig. 3). Previously uninfected mice had an average of 131 Although anti-IL-4 blocked the IgE response to infection, adult worms at necropsy 17 days after inoculation with Hp, it did not inhibit all T-dependent antibody responses to Hp whereas previously infected mice given a secondary infection because the IgG1 response was not blocked (34). Anti-IL-5 alone or after treatment with anti-IL-5 or control mAb mAb, which totally blocked eosinophilia (Fig. 2), had no averaged <10 adult worms. Mice infected similarly but given effect on either IgG1 or IgE responses (34). Interestingly, anti-IL-4 or anti-IL-4 plus anti-IL-5 averaged 46 and 54 adult anti-IL4 treatment considerably enhanced the eosinophil worms, respectively. Anti-IL-4 suppression ofthe protective response to Hp, especially after a secondary challenge in- immunity resulted in increased worm fecundity as well as fection (Fig.