Kupffer Cells from Schistosoma mansoni -Infected Mice Participate in the Prompt Type 2 Differentiation of Hepatic T Cells in Response to Worm Antigens This information is current as of September 26, 2021. Nobuki Hayashi, Kiyoshi Matsui, Hiroko Tsutsui, Yoshio Osada, Raafat T. Mohamed, Hiroki Nakano, Shin-ichiro Kashiwamura, Yasuko Hyodo, Kiyoshi Takeda, Shizuo Akira, Toshikazu Hada, Kazuya Higashino, Somei Kojima and Kenji Nakanishi3 Downloaded from J Immunol 1999; 163:6702-6711; ; http://www.jimmunol.org/content/163/12/6702 http://www.jimmunol.org/ References This article cites 52 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/163/12/6702.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Kupffer Cells from Schistosoma mansoni-Infected Mice Participate in the Prompt Type 2 Differentiation of Hepatic T Cells in Response to Worm Antigens1

Nobuki Hayashi,2* Kiyoshi Matsui,* Hiroko Tsutsui,†ʈ Yoshio Osada,¶ Raafat T. Mohamed,¶ Hiroki Nakano,† Shin-ichiro Kashiwamura,‡ Yasuko Hyodo,* Kiyoshi Takeda,§ʈ Shizuo Akira,§ʈ Toshikazu Hada,* Kazuya Higashino,*‡ Somei Kojima,¶ and Kenji Nakanishi3†‡ʈ

Infection with Schistosoma mansoni, a portal vein-residing helminth, is well known to generate life cycle-dependent, systemic immune responses in the host, type 1 deviation during the prepatent period, and type 2 polarization after oviposition. Here we investigated local immunological changes in the after infection. Unlike , hepatic lymphocytes from infected mice during the prepatent period already produced a higher amount of IL-4 and a lesser amount of IFN-␥ than those from uninfected Downloaded from mice. Hepatic lymphocytes, particularly conventional T cells, but not NK1.1؉ T cells, promptly produced IL-4 in response to worm products, soluble worm Ag preparation (SWAP), whenever presented by Kupffer cells from infected mice. The hepatic lympho- cytes that had been stimulated with SWAP presented by infected mice-derived Kupffer cells produced a huge amount of IL-4, IL-13, and IL-5 as well as little IFN-␥ in response to immobilized anti-CD3 mAb. Kupffer cells from uninfected mice produced IL-6 and IL-10, but not IL-12 or IL-18, in response to SWAP stimulation and gained the potential to additionally produce IL-4 and IL-13 after the infection. These results suggested that prompt type 2 deviation in the liver after the infection might be due to http://www.jimmunol.org/ the alteration of Kupffer cells that induces SWAP-mediated type 2-development of hepatic T cells. The Journal of Immunology, 1999, 163: 6702–6711.

acrophages are an essential component of both innate mune competent cells to type 1 shift, which is induced and aug- immunity and acquired immunity. act as mented by IL-12 and IL-18 produced by P. acnes-elicited Kupffer M effector cells by their specialized lysosomal enzymes cells, a tissue type of macrophages in the liver (12, 13). and cytotoxic molecules, such as nitric oxide, oxygen radicals, and Infection with Schistosoma mansoni results in hepatic fibrosis ␣

TNF- in both systems, and also play an important role as APCs and occasionally lethal liver cirrhosis in hosts, including humans by guest on September 26, 2021 in the acquired immune system (1–5). In either system, macro- and mice (14, 15). Infection with S. mansoni also induces proper phages regulate and modify the immune reaction by secretion of and unique changes in the host immune system according to the various . IL-12 and IL-18, originally designated IFN-␥- stage of its life cycle. After infection with cercariae of S. mansoni inducing factor, initiate and accelerate inflammatory reactions, in- through the skin, larvae transform to schistosomula, migrate into cluding type 1 immune responses, respectively (6–10), whereas the lungs, and eventually reach the intrahepatic portal circulation. IL-10 down-regulates them (8). Particularly, IL-12 produced by Within several weeks, the worms mate and migrate to the mesen- macrophages is demonstrated to primarily initiate type 1 T cell teric veins, and female worms lay eggs. Many investigators have differentiation in vitro and in vivo (6, 8–11). This is also the case reported that splenic T cells from S. mansoni-infected mice show for tissue-localized immunity. As previously shown, administra- life cycle-dependent changes in production profiles, in tion of heat-killed Propionibacterium acnes (3) directs hepatic im- that they become type 1 T cells during the prepatent period and change to type 2 T cells after the beginning of egg deposition (16). Type 2 deviation in the spleen has been reported to be in part *Third Department of Internal Medicine, †Department of Immunology and Medical attributable to host cell reactions to soluble egg Ag (SEA)4 (17, ‡ Zoology, and Laboratory of Host Defenses Institute for Advanced Medical Sciences, 18). In this study we investigated whether the hepatic immune Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; §Department of Host De- fense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, system shows a reaction to S. mansoni-infection similar to that in Japan; ¶Department of Parasitology, Institute of Medical Science, University of To- the spleen, particularly focusing on the role of Kupffer cells in the kyo, Tokyo, Japan; and ʈCore Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan immunological reactions. We found that hepatic T cells deviated into type 2 T cells without showing any type 1 shift. This was Received for publication May 25, 1999. Accepted for publication October 6, 1999. already observed in mice during the prepatent period. To investi- 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 gate the mechanism of how the hepatic immune system promptly with 18 U.S.C. Section 1734 solely to indicate this fact. shifts to type 2, we analyzed the responses of hepatic immune 1 This work was supported in part by a Grant-in-Aid for Scientific Research on Pri- competent cells to adolescent worm products, soluble worm Ag ority Areas (351,366); a Hitec Research Center Grant from the Ministry of Education, preparation (SWAP) (19). Kupffer cells from uninfected mice Science, Culture, and Sports; and the Osaka Foundation for Promotion of Clinical Immunology, Japan. produced IL-10 and IL-6, but do not produce IL-12 or IL-18 in 2 Current address: Laboratory of Immunology, National Institute of Allergy and In- fectious Disease, National Institutes of Health, Bethesda, MD 20892-1892 3 Address correspondence and reprint requests to Dr. Kenji Nakanishi, Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, 4 Abbreviations used in this paper: SEA, S. mansoni egg Ag; SWAP, soluble worm Nishinomiya 663-8501, Japan. E-mail address: [email protected] Ag preparation.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 6703 response to SWAP. Moreover, Kupffer cells prepared from S. man- analyzed for each assay, and data were processed with CellQuest (Becton soni-infected mice produced IL-4 and IL-13. Hepatic lymphocytes Dickinson). ϫ 6 produced IL-4, but not IFN-␥, in response to SWAP whenever For the indicated experiments, Kupffer cells (1 10 /ml) from mice infected 10 wk previously were incubated in a 24-well plate with 200 presented by Kupffer cells from S. mansoni-infected mice. We ␮g/ml of SWAP for 6 h, during the last 30 min of which the cells were show that these unique properties of SWAP, Kupffer cells, and incubated additionally with a 1/1000 volume of FluoSpheres (carboxylate- hepatic lymphocytes contribute to the prompt and accelerated type modified microspheres labeled with yellow-green fluorescence (Molecular 2 response in the liver following infection with S. mansoni. Probes, Eugene, OR)). The plate was vigorously washed to remove free beads, and the cells collected were processed for intracellular staining with PE-conjugated anti-IL-4 mAb (PharMingen), as shown previously (26). Materials and Methods Infection with S. mansoni Immunohistochemistry Female C57BL/6 mice (5–7 wk old) from Japan SLC (Sizuoka, Japan) Kupffer cells (1 ϫ 106/ml) from mice infected for 10 wk were incubated in were infected with 40 cercariae of S. mansoni via tail skin. STAT6-defi- a 24-well plate with 200 ␮g/ml of SWAP for 18 h, during the last 30 min cient mice with C57BL/6 background (female, 5–7 wk old) were used of which the cells were additionally incubated with a 1/1000 volume of (20, 21). FluoSpheres. Immunohistochemistry was performed using 10 ␮g/ml of biotinylated anti-IL-4 mAb followed by ABC kits (Vector, Burlingame, Reagents CA), as previously reported (27). SWAP was prepared from homogenized adult worms as described by T cell depletion Pearce et al. with some modification (19). Briefly, fat was removed from adult worms by homogenizing them in cold diethyl ether, and nonfat pellet Liver lymphocytes from C57BL/6 mice before or 3 wk after infection with was suspended in veronal buffered saline. Adult worm extract was prepared S. mansoni were treated with two rounds of complement-mediated lysis of Downloaded from by freezing and thawing of the pellet suspension, dialyzed against PBS, T cells with monoclonal anti-Thy-1.2 mAb (28). This procedure routinely filtered, and stored at Ϫ80°C until use as SWAP. Before use for cell stim- yields cells that are Ͻ2% CD3 positive. ulation, SWAP was preincubated with 100 U/ml polymyxin B for1hat room temperature to neutralize possibly contaminated LPS (22, 23). Anti- Cell culture IL-18 mAbs used for ELISA for mouse IL-18 were provided by Hayash- Liver lymphocytes or splenic T cells (2 ϫ 105/well/200 ␮l) were incubated ibara Biochemical Laboratories (Okayama, Japan) (12, 24). 2C11 (directed on anti-CD3-coated 96-well plates for 24 h (12). Kupffer cells or splenic against CD3⑀ chain, hamster IgG) or FITC-conjugated 2C11, FITC-con- macrophages from variously treated C57BL/6 mice (2 ϫ 106/ml) were http://www.jimmunol.org/ jugated anti-CD11b mAb (Mac-1 ␣-chain, M1/70, rat IgG2b), FITC-con- incubated with SWAP (200 ␮g/ml) or LPS (Detroit, Detroit, MI; 1 ␮g/ml) jugated anti-CD45R mAb (B220, RA3-6B2, rat IgG2a), FITC-conjugated in 24-well plates for 24 h. anti-mouse IgM mAb (R6-60.2, rat IgG2a), PE-conjugated anti-CD117 Liver lymphocytes or splenic T cells (5 ϫ 105/ml) from variously mAb (c-Kit, 2B8, rat IgG2b), biotinylated anti-NK1.1 mAb (PK136, rat treated C57BL/6 mice or uninfected STAT6-deficient mice were incubated IgG2a), FITC-conjugated anti-CD4 mAb (RM4–5, rat IgG2a), and anti- with 1 ϫ 106/ml of Kupffer cells or splenic macrophages from uninfected Fc␥R II/III mAb (2.4G2, rat IgG2b) were purchased from PharMingen C57BL/6 mice or infected C57BL/6 mice in the presence of 200 ␮g/ml of (San Diego, CA). PE-streptavidin was obtained from Becton Dickinson SWAP in a 24-well plate for 24 h. For the indicated experiments, T cell- (Mountain View, CA). Biotinylated anti-IL-4 mAb (BVD6-24G2), and PE- depleted hepatic lymphocytes (5 ϫ 105/ml) from uninfected C57BL/6 mice conjugated anti-IL-4 mAb (11B11) were purchased from PharMingen. An- were incubated with 200 ␮g/ml of SWAP in the presence of Kupffer cells ti-Thy-1.2 mAb (HO-13-4) was provided by Dr. W. E. Paul, National In- (1 ϫ 106/ml) from variously treated mice for 24 h. stitutes of Health (Bethesda, MD). Guinea pig complement was purchased by guest on September 26, 2021 from Cedarlane (Hornby, Canada). Endotoxin-free, neutralizing anti-IL-6 Secondary culture mAb (MP5-20F3), anti-IL-10 mAb (JES5-2A5), anti-B7.1 mAb (16- 10A1), and anti-B7.2 mAb (PO3.1) were purchased from PharMingen. Liver lymphocytes from uninfected mice (5 ϫ 105) were incubated for 24 h Hybridoma producing neutralizing anti-IL-4 mAb (11B11) from American with 200 ␮g/ml of SWAP in the presence of Kupffer cells (1 ϫ 106) from Type Culture Collection (Manassas, VA) was inoculated in the abdomen of mice infected with S. mansoni 10 wk previously, and nonadherent cells BALB/c-nu/nu (SLC), and purified Ig from the ascites was used for the (5 ϫ 105/ml) were vigorously washed with PBS and recultured on anti- neutralization experiments. The culture medium generally used in this CD3 mAb-coated plates for another 24 h. For the control study, the freshly study was RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 isolated liver lymphocytes were incubated on an anti-CD3 mAb-coated ␮g/ml streptomycin, 50 ␮M 2-ME, and 2 mM L-glutamine. All experi- plate for 24 h for detection of various kinds of cytokines. For the indicated ments were performed in triplicate. All data are given as the mean Ϯ SEM. experiments, the liver lymphocytes from uninfected mice (5 ϫ 105) were incubated with 200 ␮g/ml of SWAP and the infected mouse-derived Cell preparation Kupffer cells (1 ϫ 106) in the presence of anti-IL-4 mAb (100 ␮g/ml), anti-IL-10 mAb (10 ␮g/ml), anti-IL-6 mAb (10 ␮g/ml), anti-B7.1 mAb (20 Splenic T cells were prepared from C57BL/6 mice before or 4 or 16 wk ␮ ␮ after infection with S. mansoni by passing them through a nylon wool g/ml), or anti-B7.2 mAb (20 g/ml) for 24 h. Nonadherent lymphocytes column. Spleen cells from variously treated mice were incubated in plastic were collected, vigorously washed with PBS, and incubated on fresh anti- dishes for1hat37°C, and adherent cells were additionally incubated in CD3 mAb-coated plates for another 24 h. fresh plastic dishes for another 1 h. The adherent cells were used as splenic Assay for cytokines macrophages. Liver lymphocytes were prepared from STAT6-deficient mice or The concentrations of cytokines were determined by ELISA. IL-4 and C57BL/6 mice before or 2, 3, 4, 10, or 16 wk after infection with S. IFN-␥ were determined by ELISA kits from Genzyme (Boston, MA). mansoni cercariae as described previously (12). Kupffer cells were pre- IL-12 p40, IL-10, TNF-␣, and IL-6 levels were measured by ELISA kits pared from C57BL/6 mice before or 3 or 10 wk after infection with S. from BioSource (Camarillo, MA). The IL-18 level was determined by mansoni by collagenase-Pronase digestion followed by elutriation centrif- ELISA as shown previously (12). IL-5 and IL-13 levels were also mea- ugation as described previously (25). sured by ELISA kits from Endogen (Woburn, MA) andR&D(Minne- apolis, MN), respectively. FACS analysis Fluorescence staining of Kupffer cells or liver lymphocytes was performed Results after treatment with anti-Fc␥R mAb (12). Kupffer cells from C57BL/6 Direct type 2 shift in the liver of S. mansoni-infected mice mice infected with S. mansoni 10 wk previously were stained with FITC- conjugated anti-CD3 mAb, FITC-conjugated anti-11b mAb, FITC-conju- Since S. mansoni resides only in the hepatic portal vein after at- gated anti-IgM mAb, FITC-conjugated anti-CD45R mAb, or PE-conju- taining the adolescent stage 3–4 wk after the infection (14), we gated anti-CD117 mAb. Liver lymphocytes and splenic T cells from assumed that the hepatic immune system shows a unique local variously infected C57BL/6 mice or STAT6-deficient mice were stained with biotinylated anti-NK1.1 followed by incubation with FITC-anti-CD3 reaction to S. mansoni. We examined chronological changes in or FITC-anti-CD4 and PE-streptavidin. Stained cells were analyzed using cytokine production profile of hepatic T cells after S. mansoni a dual laser FACScalibur (Becton Dickinson). Ten thousand cells were infection compared with those of splenic T cells. As shown in Fig. 6704 PROMPT TYPE 2 SHIFT OF LIVER T CELLS IN SCHISTOSOMIASIS Downloaded from

FIGURE 2. Type 2 dominant immune responses in the liver after S. mansoni infection. Liver lymphocytes from uninfected or S. mansoni-in- http://www.jimmunol.org/ fected mice at the indicated time points after infection were incubated with immobilized anti-CD3 mAb for 24 h. Culture supernatants were harvested and tested for their concentrations of IL-4 (hatched column) and IFN-␥ (closed column). The data are the mean Ϯ SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments. ND, not detected.

naive T cells into type 2 T cells directly (29–32). As shown in Fig. ϩ ϩ 1b, the proportion of CD4 NK1.1 T cells increased in splenic T by guest on September 26, 2021 cells after infection. By contrast, the proportion of this population was continuously reduced in liver after infection. The cell yield of FIGURE 1. Straightforward type 2 polarization in the liver after S. man- hepatic lymphocytes from infected mice was, at most, about 1.5- soni infection. a, Splenic T cells (Spleen) and hepatic lymphocytes (Liver) fold more than that from uninfected mice (data not shown). These were isolated from mice at the indicated time points after the infection with differences and changes in composition of both populations before S. mansoni. The lymphocytes were cultured on anti-CD3 mAb-coated 96- and after the infection did not allow us to determine whether dif- ␥ well microtiter plates for 24 h. IL-4 (hatched column) and IFN- (closed ferent cytokine profiles of splenic and hepatic T cells reflected ␥ column) in each resulting supernatant were measured by ELISA. IFN- their different cell compositions or their functional capacities. and IL-4 were not observed in the supernatant of splenic T cells or liver A more detailed kinetic study revealed an exponential type 2 lymphocytes incubated in the absence of anti-CD3 mAb. The data are the ␥ mean Ϯ SEM of triplicate samples from one experiment. b, The surface polarization of hepatic lymphocytes with down-regulated IFN- phenotypes of the cells prepared in a were determined by FACS after production after the infection (Fig. 2). Histological study disclosed staining them with FITC-conjugated anti-CD4 mAb and biotinylated anti- that accumulated around the worm in liver of mice at NK1.1 mAb followed by PE-streptavidin. The results shown are represen- 4 wk postinfection, giving another clue to early type 2 shift in liver tative of three independent experiments. ND, not detected. after infection (data not shown). Thus, hepatic T cells directly and promptly developed into type 2 T cells without showing a remark- able type 1 shift after S. mansoni infection. 1, like splenic T cells, hepatic T cells prepared from uninfected mice produced both IL-4 and IFN-␥ in response to immobilized Unique cytokine production profile of SWAP-stimulated Kupffer anti-CD3 mAb. As expected (16), splenic T cells showed type 1 cells shift at the adolescent worm stage and at 4 wk after infection, and Since hepatic immune system shows a unique type 2 immune re- then turned into type 2 T cells after S. mansoni began to lay eggs sponse upon S. mansoni infection at adolescent worm stage (Figs. at 8 wk and later after the infection (Fig. 1a, upper panel). In 1 and 2), we investigated the regulatory role of Kupffer cells in this contrast, hepatic T cells already showed type 2 shift at the ado- type 2 deviation by examining their cytokine production profile in lescent worm stage, and their type 2 polarization was further fa- response to stimulation with SWAP compared with that to LPS, a cilitated after starting egg deposition (Fig. 1a, lower panel). potent, bacteria-derived stimulus for macrophages (25). As shown To investigate whether the different cytokine profiles between in Table I, Kupffer cells from uninfected mice produced almost the spleen and hepatic T cells was due to the difference in the com- same level of IL-10 in response to both stimuli, but they produced position of the cell population, we analyzed cell proportions, par- 8-fold more IL-6 in response to LPS than in response to SWAP ticularly focusing on that of CD4ϩNK1.1ϩ T cells because of their (33, 34). In contrast, the same cells produced a much lesser amount prompt production of IL-4, an essential cytokine to differentiate of type 1 driving cytokines, IL-12 and IL-18 (7–9, 11, 24), in The Journal of Immunology 6705

Table I. Comparison of cytokine production profiles between LPS- and SWAP-stimulated Kupffer cellsa

Cytokines Produced (pg/ml)

Stimuli IL-6 IL-10 IL-12 p40 IL-18 TNF-␣ IL-4 IL-13

Uninfected mice Medium 238 Ϯ 10 NDb ND ND ND ND ND LPS 9720 Ϯ 263 269 Ϯ 21 341 Ϯ 24 27.3 Ϯ 0.1 148 Ϯ 22 ND ND SWAP 1242 Ϯ 20 283 Ϯ 16 ND ND ND ND ND Infected mice Medium 216 Ϯ 85 ND ND ND ND ND ND LPS 11593 Ϯ 1110 179 Ϯ 9 367 Ϯ 16 18.2 Ϯ 0.1 133 Ϯ 54 ND ND SWAP 2176 Ϯ 127 321 Ϯ 24 ND ND ND 628 Ϯ 51 1568 Ϯ 88

a Kupffer cells from three uninfected mice or S. mansoni-infected mice (10 wk) were pooled and incubated with or without LPS (1 ␮g/ml) or with SWAP (200 ␮g/ml) for 24 h. Concentration of cytokines were determined by ELISA. Data are shown as mean Ϯ SEM. The results shown are representative of three independent experiments with similar results. b ND, not detected. response to SWAP stimulation than in response to LPS. Thus, contained 95% Kupffer cells, as determined by of

SWAP appears to stimulate Kupffer cells to fail to induce type 1 latex beads and by morphological property (12, 25). Downloaded from immune responses.

IL-4 production by Kupffer cells from infected mice in response Prompt IL-4 production by hepatic lymphocytes in response to to SWAP SWAP presented by Kupffer cells from the infected mice during Next, we examined whether S. mansoni infection changes the cy- the prepatent period

tokine production profile of Kupffer cells in response to SWAP. As Next, we investigated whether SWAP can stimulate hepatic T cells http://www.jimmunol.org/ shown in Fig. 3a, Kupffer cells from the mice in the prepatent to produce IL-4 directly or with help from Kupffer cells. To test period remained to produce IL-6 and IL-10 in response to stimu- this, we prepared hepatic lymphocytes or splenic T cells from un- lation with SWAP. Kupffer cells still failed to secrete TNF-␣, IL- infected mice or mice infected with S. mansoni 3 wk previously, 12, or IL-18 in response to SWAP (data not shown). To our sur- incubated them with SWAP together with Kupffer cells or splenic prise, at 3 wk after the infection they started to produce IL-4 in macrophages from uninfected or infected mice for 24 h, and mea- response to SWAP (Fig. 3a). As shown in Table I and Fig. 3a, the sured IL-4 in each resulting supernatant. Infected mouse-derived capacity for IL-4 production was tremendously increased as infec- Kupffer cells incubated with SWAP produced 80 pg/ml of IL-4 in tion progressed to the egg deposit stage (describe below). To con- the culture supernatant (Fig. 4a). Hepatic lymphocytes from unin- firm that infected mouse-derived Kupffer cells can produce IL-4, fected or infected mice produced much more IL-4 in response to by guest on September 26, 2021 we examined intracellular IL-4 staining of the Kupffer cell frac- SWAP presented by Kupffer cells from infected mice than the tion. As shown in Fig. 3b, left, in the cell preparation Kupffer cells themselves produced in response to SWAP, indicat- were able to be stained with anti-IL-4 mAb directly, indicating that ing that hepatic lymphocytes can respond to SWAP presented by Kupffer cells, at least hepatic adherent phagocytes, produced IL-4 the appropriate APCs, infected mouse-derived Kupffer cells. The in response to SWAP. The specificity of PE-conjugated mAb same hepatic lymphocytes, however, did not produce IL-4 in re- against IL-4 was proven by competitive inhibition by an excess of sponse to SWAP in the absence of the Kupffer cells from infected unconjugated mAb in a separate experiment. This was also proven mice, indicating that hepatic lymphocytes absolutely require APCs by immunohistochemistry. As shown in Fig. 3b, right, Kupffer to respond to SWAP (data not shown). The hepatic T cells did not cells from infected mice that has a property common to mono- produce IL-4 even in the presence of infected mouse-derived cytes-macrophages of large cytoplasm with relatively small nu- Kupffer cells whenever SWAP was not added to the culture (data cleus, ingest beads and also have cytoplasm stained with anti-IL-4 not shown). Furthermore, hepatic lymphocytes from either group mAb. The Kupffer cell fraction contained many cells similar to the of mice did not produce IL-4 in response to SWAP stimulation in cell shown in Fig. 3b, right. The cells were not stained when in- the presence of Kupffer cells from uninfected mice (Fig. 4a), in- cubated with control Ab instead of anti-IL-4 mAb (data not dicating that only Kupffer cells from infected mice have the ability shown). Moreover, the SWAP-stimulated Kupffer cells from the to present SWAP to hepatic lymphocytes. IFN-␥ was not detect- infected mice also produced IL-13, a second, potent, type 2-induc- able in resulting supernatants of any reconstitution mixture in re- ing cytokine (Table I) (35, 36). Interestingly, Kupffer cells from S. sponse to SWAP (data not shown). mansoni-infected mice (at 10 wk) did not lose the potential to In contrast to hepatic lymphocytes, splenic lymphocytes from produce IL-12, IL-18, and TNF-␣, because Kupffer cells produced uninfected or infected mice did not produce IL-4 in response to all the cytokines except IL-4 and IL-13 in response to LPS (Table SWAP, even when infected mouse-derived Kupffer cells were I). Thus, Kupffer cells gained the capacity to produce IL-4 and used as APCs (Fig. 4a). As shown in Fig. 4b, hepatic lymphocytes IL-13 uniquely in response to adult worm products during produced IL-4, but much less, in response to stimulation with infection. SWAP in the presence of splenic macrophages isolated from in- Next, we confirmed that the Kupffer cell fraction from infected fected mice than in that of Kupffer cells from the same infected mice was not contaminated with IL-4-producing cells, such as T mice. Unlike Kupffer cells, splenic macrophages from infected cells and mast cells (37), by FACS analysis. As shown in Fig. 3c, mice did not produce IL-4 in response to SWAP (Fig. 4b). These the Kupffer cell fraction isolated from infected mice did not con- stimulation conditions also did not induce IFN-␥ production from tain T cells, B cells, or c-Kitϩ cells. Mac1ϩ cells in the cell prep- either hepatic or splenic lymphocytes (data not shown). Thus, only aration appeared to be about 70%. A similar staining pattern was hepatic lymphocytes showed potency to produce IL-4 in response observed in the Kupffer cell fraction from uninfected mice, which to stimulation with SWAP, and Kupffer cells from the infected 6706 PROMPT TYPE 2 SHIFT OF LIVER T CELLS IN SCHISTOSOMIASIS Downloaded from

FIGURE 4. IL-4 production by hepatic lymphocytes in response to SWAP presented by infected mouse-derived macrophages. a, Liver lym- phocytes or splenocytes from uninfected mice (hatched column) or S. man- soni-infected mice at 3 wk (closed column) were incubated with Kupffer cells from uninfected mice or S. mansoni-infected mice at 3 wk in the http://www.jimmunol.org/ presence or the absence of 200 ␮g/ml of SWAP for 24 h. IL-4 in each resulting supernatant was measured. b, Liver lymphocytes or splenocytes from uninfected mice (hatched column) or S. mansoni-infected mice at 3 wk (closed column) were incubated with splenic macrophages from unin- fected mice or S. mansoni-infected mice at 3 wk in the presence or the absence of 200 ␮g/ml of SWAP for 24 h. IL-4 in each supernatant was measured. Dotted horizontal lines indicate the mean amount of IL-4 pro- duced by infected mouse-derived macrophages in response to SWAP

(Kupffer cells, 40 pg/ml; splenic macrophages, undetectable). The data are by guest on September 26, 2021 the mean Ϯ SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments with similar results. ND, not detected. FIGURE 3. Cytokine production profile of SWAP-stimulated S. man- soni-infected Kupffer cells. a, Kupffer cells started to produce IL-4 after S. mansoni infection. Kupffer cells prepared from the uninfected mice (open IL-4 production by conventional hepatic T cells in response column) or S. mansoni-infected mice at 3 wk (closed column) or 10 wk to SWAP (hatched column) were incubated with or without 200 ␮g/ml of SWAP for 24 h. Culture supernatants were harvested and tested for their concentra- We investigated what cell type in the liver produces IL-4 in re- tions of IL-4, IL-6, and IL-10. ND, not detected. The data shown are the sponse to SWAP presented by infected mouse-derived Kupffer mean Ϯ SEM of triplicate samples of one experiment. b, IL-4 production cells. To test the contribution of T cells to this phenomenon, we by Kupffer cells. Kupffer cells from infected mice at 10 wk were incubated depleted T cells from hepatic lymphocytes in vitro and incubated with SWAP for 5.5 h and additionally with fluorescence-labeled beads for them under the same conditions as those described in Fig. 4a.As 30 min. The cells were then intracellularly stained with PE-conjugated shown in Fig. 5a, both CD3ϩNK1.1Ϫ T cells and CD3ϩNK1.1ϩ T anti-IL-4 mAb. The intensity of intracellular IL-4 and the level of phago- cells were eliminated after T cell depletion. Infected mouse-de- cytosis were determined by FACS (left). SWAP-stimulated Kupffer cells rived Kupffer cells produced 80 pg/ml of IL-4 in response to were also immunohistochemically stained using biotinylated anti-IL-4 SWAP (Fig. 4a). Hepatic lymphocytes from infected or uninfected mAb followed by ABC kit (right). Kupffer cells ingested beads as indicated by arrowheads, and IL-4 was homogeneously detected in its cytoplasm. mice did not produce IL-4 in response to SWAP even in the pres- The photo is representative of three independent experiments with similar ence of Kupffer cells, once T cells were depleted (Fig. 5b). They results. c, Lack of CD3ϩ, CD45Rϩ, IgMϩ, or c-Kitϩ cells in the Kupffer did not produce IFN-␥ under any stimulation condition (data not cell fraction. Kupffer cells from S. mansoni-infected mice at 10 wk were shown). Thus, T cells of hepatic lymphocytes have the potential to stained with FITC-anti-CD3, FITC-anti-11b, FITC-anti-IgM, FITC- anti- produce IL-4 in response to SWAP presented by Kupffer cells CD45R, or PE-anti-CD117 after Fc␥R blocking. The surface phenotypes from infected mice. were determined by FACS analysis. The results shown are representative Since the liver has many more NK1.1ϩ T cells, particularly of three independent experiments with similar results. NK1.1ϩCD4ϩ T cells, than other tissues (12, 29), we investigated which cell type of hepatic T cells, NK1.1ϩ T cells and/or NK1.1Ϫ T cells, produces IL-4 in response to SWAP, although the propor- tion of NK1.1ϩCD4ϩ T cells decreased after the infection (Fig. mice had a much higher capacity to present SWAP than splenic 1b). As STAT6Ϫ/Ϫ mice have impaired type 2 development of macrophages, presumably due in part to IL-4 production by them- conventional T cells, NK1.1Ϫ T cells, but intact IL-4-producing selves in response to SWAP; this will be examined later (Fig. 6). NK1.1ϩCD4ϩ T cells (38, 39), we examined IL-4 production by The Journal of Immunology 6707 Downloaded from http://www.jimmunol.org/

FIGURE 5. Conventional T cells promptly produced IL-4 in response to SWAP presented by infected mouse-derived Kupffer cells. a, T cell depletion from hepatic lymphocytes. T cells were depleted from liver lymphocytes from uninfected mice or S. mansoni-infected mice at 3 wk using anti-Thy-1.2 mAb followed by incubation with complement for two cycles. The effectiveness of depletion was monitored by FACS analysis of cells stained with biotinylated anti-NK1.1 mAb followed by PE-streptavidin and FITC-conjugated anti-CD3 mAb. The results shown are representative of three independent experiments with similar results. b, Lack of IL-4 production by T cell-depleted hepatic lymphocytes in response to SWAP. Untreated (Whole) or T cell-depleted (T depleted) hepatic lymphocytes from uninfected mice (hatched column) or infected mice at 3 wk (closed column) were incubated with Kupffer cells prepared by guest on September 26, 2021 from mice infected with S. mansoni at 3 wk in the presence of SWAP for 24 h to determine the IL-4 response. c, Surface phenotypes of hepatic lymphocytes from STAT6-deficient mice similar to those from wild-type mice. Hepatic lymphocytes from uninfected STAT6-deficient mice or wild-type mice (C57BL/6) were stained with biotinylated anti-CD3 mAb followed by PE-streptavidin and FITC-conjugated anti-NK1.1 mAb. d, Impaired IL-4 production by hepatic lymphocytes from STAT6-deficient mice in response to SWAP. Hepatic lymphocytes from uninfected STAT6-deficient mice (hatched column), uninfected wild-type mice (hatched column), or S. mansoni-infected wild-type mice at 3 wk (closed column) were incubated under the conditions described in b. Dotted horizontal lines in b and d indicate the mean amount of IL-4 produced by infected mouse-derived Kupffer cells in response to SWAP. The data are the mean Ϯ SEM of triplicate samples from one experiment. The results shown are representative of three independent experiments. ND, not detected. hepatic lymphocytes from STAT6Ϫ/Ϫ mice in response to SWAP of Kupffer cells from infected mice, the secondary culture revealed in the presence of Kupffer cells from infected wild-type mice. The that hepatic T cells had the same cytokine production profile as profile of these cells in the liver is almost same in wild-type and that observed in the primary culture (data not shown). During the STAT6-deficient mice (Fig. 5c). As shown in Fig. 5d, STAT6- stimulation with SWAP presented by infected mouse-derived deficient hepatic lymphocytes produced much less IL-4 than wild- Kupffer cells, hepatic T cells rapidly differentiated into type 2 T type controls. Therefore, conventional T cells are a major source of cells. IL-4 produced by SWAP-stimulated hepatic lymphocytes. Since Kupffer cells from the infected mice produced a consid- erable amount of IL-4, IL-6, and IL-10 in response to SWAP (Fig. Differentiation into type 2 hepatic T cells by stimulation 6b), and these cytokines have been reported to be involved in type with SWAP 2 differentiation of T cells (30–34), we analyzed whether these To confirm whether prompt IL-4 production by SWAP-activated cytokines derived from Kupffer cells contribute to the SWAP-in- hepatic T cells is due to their rapid differentiation into type 2 T duced prompt development of hepatic T cells into type 2 T cells. cells, we examined their cytokine production profile in response to The amount of anti-IL-4 mAb used was 10-fold that which inhib- anti-CD3 challenge. Hepatic T cells from uninfected mice that had ited 10 ng/ml of IL-4 (data not shown). The amounts of anti-IL-10 been stimulated with SWAP in the presence of infected mouse- and anti-IL-6 mAbs were dependent on the protocol. As shown in derived Kupffer cells in vitro were stimulated with immobilized Fig. 6b, any treatment with anti-cytokine mAb did not remarkably anti-CD3 mAb. As shown in Fig. 6a, they produced much more down-regulate type 2 T cell differentiation of hepatic T cells de- IL-4, IL-5, and IL-13, but much less IFN-␥, than control hepatic T termined by IL-4 production. These treatments also did not reduce cells (Figs. 1 and 2). When the hepatic T cells were incubated with the level of their type 2 differentiation measured by IL-13 or IL-5 SWAP in the presence of splenic macrophages from infected or production (data not shown). In addition, we examined the effects uninfected mice or uninfected mouse-derived Kupffer cells instead of anti-B7 mAbs on their quick type 2 differentiation, because B7.2 6708 PROMPT TYPE 2 SHIFT OF LIVER T CELLS IN SCHISTOSOMIASIS

FIGURE 7. Ultimate IL-4 production by Kupffer cells from S. mansoni- infected mice (egg deposit stage) in response to SWAP. Kupffer cells were prepared from uninfected mice or S. mansoni-infected mice at 3 or 10 wk.

They were incubated with (hatched, closed, and dotted columns) or without Downloaded from (open column) hepatic lymphocytes from uninfected mice (hatched col- umn), infected mice at 3 wk (closed column), or infected mice at 10 wk (dotted column) in the presence of SWAP for 24 h. IL-4 in each superna- tant was measured. The data are the mean Ϯ SEM of triplicate samples from one experiment. The results shown are representative of three inde- pendent experiments. ND, not detected. http://www.jimmunol.org/

IL-4 after the egg deposition stage (Table I and Fig. 3a), allowing us to investigate whether the main cell type producing IL-4 in response to SWAP changes from hepatic T cells to Kupffer cells after the egg deposition stage. As shown in Fig. 7, hepatic lym- phocytes from the infected mice appeared to produce a tremendous FIGURE 6. Prompt type 2 differentiation of hepatic T cells after stim- amount of IL-4 upon stimulation with SWAP presented by Kupffer ulation with SWAP. a, SWAP presented by Kupffer cells induced type 2 differentiation in hepatic T cells. Hepatic lymphocytes from uninfected cells from infected mice at the egg deposit stage. However, this by guest on September 26, 2021 Ͼ mice were first incubated for 24 h with SWAP in the presence of Kupffer reflected the fact that 65% of the amount of IL-4 observed was cells from infected mice. After being vigorously washed, lymphocytes produced by SWAP-stimulated infected mouse-derived Kupffer were incubated on anti-CD3 mAb-coated plates for another 24 h, and the cells by themselves (10 wk; Table I and Fig. 7). Hepatic lympho- concentration of each cytokine was measured (closed columns). For the cytes from any mice did not produce IL-4 under the same stimu- control study, hepatic lymphocytes freshly isolated from the same unin- lation conditions, except for SWAP or Kupffer cells (data not fected mice were incubated on anti-CD3 mAb-coated plates for 24 h, and shown). Thus, Kupffer cells acquired a much greater potential to cytokine levels were determined (open columns). b, IL-4-independent type produce IL-4 in response to SWAP in the egg deposit phase than 2 differentiation of hepatic T cells by SWAP presented by Kupffer cells. in the prepatent phase, whereas hepatic T cells from mice in the Hepatic lymphocytes from uninfected mice were also incubated with egg deposit phase showed an equivalent level of IL-4 production SWAP in the presence (␣-series) or the absence (Control) of various kinds of neutralizing mAbs as well as infected mouse-derived Kupffer cells for as those in the prepatent phase. 24 h, and the secondary culture was performed according to the same protocol as that shown in a. Cytokines in each supernatant were measured Discussion Ϯ by ELISA. The data are the mean SEM of triplicate samples from one This study clearly demonstrated for the first time that Kupffer cells experiment. The results shown are representative of three independent ex- have the capacity to produce both IL-4 and IL-13. To date, only T periments with similar results. ND, not detected. cells, particularly type 2 T cells and NK1.1ϩ CD4ϩ T cells, and myeloid cells, such as , mast cells, and eosinophils, can produce IL-4 after the appropriate activation (29, 37, 42, 43). and B7.1 are relevant surface molecules for the development of Kupffer cells also have capacity to produce IL-4 in response to the Th2 and Th1 cells, respectively (40, 41). However, the develop- appropriate in vivo and in vitro stimulation (Table I and Figs. 3 and ment of hepatic T cells into type 2 T cells was not dramatically 7). The mechanism by which Kupffer cells change to produce IL-4 affected by these treatments (Fig. 6b). and IL-13 is unclear, although a unique property of SWAP appears to play a critical role (Table I). A subpopulation of Kupffer cells Ultimate IL-4 production by hepatic lymphocytes induced by predominantly producing IL-4 and IL-13 may proliferate after in- SWAP-activated Kupffer cells from infected mice in the egg fection with S. mansoni. During the infection, Kupffer cells may deposit phase gain a new signal transducing pathway to induce IL-4 and IL-13 Next, we investigated immunological properties of the hepatic im- production in response to SWAP. Kupffer cells may produce a mune system of S. mansoni-infected mice at the egg deposit phase. new, unknown cytokine critical to the induction of IL-4 and IL-13 In the prepatent phase, hepatic T cells produced more IL-4 in re- production. However, it is noted that these type 2-related cytokine- sponse to SWAP than infected mouse-derived Kupffer cells (Figs. producing Kupffer cells are able to quickly change to produce 3–5). However, Kupffer cells began to produce a huge amount of IL-12 and IL-18 when stimulated with LPS (Table I), suggesting The Journal of Immunology 6709 that S. mansoni infection does not delete the Kupffer cell popula- cate that prompt IL-4 production in response to SWAP stimulation tion that preferentially produces IL-12 and IL-18 in response is mainly attributable to type 2 differentiated conventional T cells to LPS. in the liver. We demonstrated that SWAP, a crude mixture of worm-derived S. mansoni infection has been reported to result in the accumu- Ags, has potent activity to induce prompt IL-4 production and lation of IL-4-producing cells. Non-T, non-B, myeloid cells, in- rapid type 2 differentiation in hepatic T cells isolated from healthy cluding mast cells and basophils, have been demonstrated to be mice if presented by infected mouse-derived Kupffer cells (Figs. involved in type 2 polarization in the spleen (17, 18). Non-T, 4–6). Macrophages from only S. mansoni-infected mice have the non-B cells produce IL-4 in response to IL-3 and/or SEA (17, 18). potential to present SWAP to T cells, while those from uninfected We observed that the Kupffer cell population mainly consisted of mice did not induce any IL-4 or IFN-␥ production by T cells in mononuclear phagocytes, not T cells, B cells, or mast cells (c-Kitϩ response to SWAP (Fig. 4). Moreover, Kupffer cells from infected cells), and produced IL-13 as well as IL-4 in response to SWAP mice have much greater ability to induce IL-4 production by lym- (Table I and Figs. 3 and 7). In addition, their capacity to produce phocytes than splenic macrophages from the same mice (Fig. 4). IL-4 was tremendously enhanced as the infection was prolonged Several possibilities account for these differences. First, as adoles- (Table I and Figs. 3 and 7). This may not exclude the possibility cent worms reside in portal veins, Kupffer cells may receive par- that a minor population composed of contaminated myeloid cells ticular and proper influences by interaction with adolescent worms such as basophils in the Kupffer cell fraction may contribute to this and/or their products, which may not reach splenic macrophages. phenomenon. Recently, eosinophils have been demonstrated to be Second, as the liver is thoroughly the circulatory system directly major source of Th2 cytokines in hepatic granuloma at egg deposit connecting with the intestine, which is characterized to be almost phase (45). The Kupffer cell fraction and hepatic lymphocyte frac- Downloaded from equivalent to an outer environment, Kupffer cells may have more tion from the infected mice at the egg deposit phase (Fig. 7) were activity to respond to foreign molecules than splenic macrophages. not obviously contaminated with eosinophils as determined by Interestingly, hepatic lymphocytes from uninfected mice can pro- morphological study (data not shown). Therefore, we can only duce much more IL-4 and IFN-␥ in response to immobilized anti- conclude that Kupffer cells, at least liver adherent cells, except T CD3 mAb than splenic lymphocytes from the same mice (Fig. 1). cells and mast cells, are able to promptly produce IL-4 in response

This may also reflect the anatomical condition of the liver, in that to SWAP, which may in part contribute to the differentiation of http://www.jimmunol.org/ the hepatic immune system is endogenously activated to be more hepatic T cells into type 2 T cells in vitro and possibly in vivo. We sensitive to exogenous stimulation than the splenic one. Further- need further study to know whether SWAP-stimulated Kupffer more, Kupffer cells from infected mice have the capacity to induce cells or egg-elicited eosinophils are the major source of IL-4. type 2 differentiation in hepatic T cells (Fig. 6a). This type 2 dif- APCs are potent cells to determine the immune response to Ags. ferentiation was not down-regulated by the neutralization of IL-4, Macrophages produce IL-12 when stimulated with microbes or IL-6, or IL-10 (Fig. 6b), which Kupffer cells produce in response microbe products (9, 25). As previously reported, administration of to the same stimulation (Fig. 3a), indicating that type 2 differen- heat-killed P. acnes, intracellular bacteria, render Kupffer cells to tiation-inducing activity of the Kupffer cells is independent of produce IL-12, which then induces type 1 differentiation in hepatic these cytokines. In addition, treatment of Kupffer cells with neu- T cells (12). LPS also stimulates Kupffer cells to produce IL-12 as by guest on September 26, 2021 tralizing anti-B7.2 did not result in inhibition of type 2 develop- well as other proinflammatory cytokines directly (25). Dendritic ment of hepatic T cells, indicating that B7.2 is not involved in this cells have been shown to produce IL-12 when presenting Ag to type 2 differentiation. IL-13, presumably in collaboration with naive CD4ϩ T cells (46, 47). In the case of S. mansoni infection, IL-4, produced by SWAP-stimulated Kupffer cells may largely either uninfected or infected mouse-derived Kupffer cells prefer- contribute to induce type 2 T cell differentiation. Other unknown entially produce both IL-6 and IL-10, but not IL-12 or IL-18, in surface molecules that Kupffer cells become able to express after response to SWAP. IL-12 is an essential factor for type 1 differ- infection may contribute to induce this rapid type 2 T cell differ- entiation, and IL-18 markedly enhances it in collaboration with entiation. Recently, it has been shown that chemo- IL-12 (6–11). IL-6 and IL-10 play some role in direction of T cells attractant protein-1, a member of the C-C-chemokines, up-regu- to type 2 cells (33, 34) and may exert their actions much more lates IL-4 production by spleen cells from SEA-sensitized mice efficiently in the absence of IL-12 and/or IL-18 than in their pres- in response to SEA, suggesting that our Kupffer cells might pro- ence. However, these characteristics are still inadequate to induce duce such a chemokine to help type 2 differentiation of hepatic differentiation of naive T cells into type 2 T cells (Fig. 6). Rather, T cells (44). it may be important that infected mice-derived Kupffer cells be- Many investigators have tried to identify the cell type that ini- come able to produce IL-4 and IL-13 in response to SWAP (Fig. tiates differentiation of naive T cells into type 2 T cells. IL-4 is 4 and Table I). In contrast, S. mansoni-infected mouse-derived widely accepted as a prototype of Th2 cytokines (30–32). splenic macrophages did not produce IL-4 (Fig. 4) or IL-13 (data NK1.1ϩCD4ϩ T cells have been shown to produce IL-4 promptly not shown) in response to the same stimulation. Recently, dendritic in response to anti-CD3 mAb or anti-IgD Ab (29). However, cells, professional APCs, were functionally divided into two pop- NK1.1ϩCD4ϩ T cells are not required for S. mansoni infection- ulations according to their ability to induce Th1 and Th2 differ- ␤ induced type 2 polarization in vivo, because 2-microglobulin- entiation, and D2 cells help Th2 differentiation (48). Like dendritic deficient mice, lacking NK1.1ϩ T cells, showed type 2 polarization cells, Kupffer cells may be divided into two populations, and after the infection equal to that observed in wild-type mice (42). Kupffer cells from infected mice may belong to a type 2 T cell The liver contains many more NK1.1ϩCD4ϩ T cells than the differentiation-inducing subset. Thus, Kupffer cells acquire IL-4- spleen (12, 29), suggesting that NK1.1ϩCD4ϩ T cells might re- and IL-13-producing activity after infection with S. mansoni, pre- spond to SWAP stimulation with prompt IL-4 production. How- sumably due to interaction with adolescent worm products, possi- ever, hepatic lymphocytes from uninfected STAT6-deficient mice, bly leading to the unique feature of the S. mansoni-infected liver. composed of NK1.1ϩCD4ϩ T cells with intact IL-4 production The infected mouse-derived Kupffer cells do not solely contrib- and conventional T cells impaired in type 2 differentiation (38, 39), ute to the prompt type 2 response during infection. Because of the produced much less IL-4 in response to SWAP presented by in- migration pattern exhibited by the parasite, skin draining lymph fected mouse-derived Kupffer cells (Fig. 5d). These results indi- nodes and lungs could contribute to a rapid Th2 response. Wilson 6710 PROMPT TYPE 2 SHIFT OF LIVER T CELLS IN SCHISTOSOMIASIS et al. demonstrated that irradiated cercariae are able to promote a 16. Pearce, E. J., P. Caspar, J.-M., Grzych, F. A. Lewis, and A. Sher. 1991. Down- protective Th1 response, while normal unattenuated parasites elic- regulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J. Exp. Med. 173:159. ited higher IL-4 and IL-5 expression upon both primary and sec- 17. Kullberg, M. C., J. A. Berzofsky, D. Lj. Jankovic, S. Barbieri, M. E. Williams, ondary stimulations (49). These findings were documented in skin P. Perlmann, A Sher, and M. Troye-Blomberg. 1996. T cell-derived IL-3 induces the production of IL-4 by non-B, non-T cells to amplify the Th2-cytokine re- draining lymph nodes, very early after exposure to the parasites. sponse to a non-parasite antigen in Schistosoma mansoni-infected mice. J. Im- Similar findings were reported in a study by Wynn et al., in which munol. 156:1482. the cytokine response to both irradiated and normal parasites was 18. Falcone, F. H., C. A. Dahinden, B. F. Gibbs, T. Noll, U. Amon, H. Hebestreit, O. Abrahamsen, J. Klauche, M. Schlaak, and H. Haas. 1996. Human basophils studied in the lung 6–28 days after exposure to parasites (50). release interleukin-4 after stimulation with Schistosoma mansoni egg antigen. Here, as in the study by Wilson et al., normal parasites stimulated Eur. J. Immunol. 26:1147. a rapid type 2 response in the lung, which was obvious as early as 19. Pearce, E. J., S. L. James. S. Hieny, D. E. Lnar, and A. Sher. 1988. 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