IgE Enhances Parasite Clearance and Regulates Mast Cell Responses in Mice Infected with spiralis

This information is current as Michael F. Gurish, Paul J. Bryce, Hong Tao, Alison B. of October 2, 2021. Kisselgof, Elizabeth M. Thornton, Hugh R. Miller, Daniel S. Friend and Hans C. Oettgen J Immunol 2004; 172:1139-1145; ; doi: 10.4049/jimmunol.172.2.1139 http://www.jimmunol.org/content/172/2/1139 Downloaded from

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IgE Enhances Parasite Clearance and Regulates Mast Cell Responses in Mice Infected with Trichinella spiralis1

Michael F. Gurish,* Paul J. Bryce,‡ Hong Tao,* Alison B. Kisselgof,‡ Elizabeth M. Thornton,§ Hugh R. Miller,§ Daniel S. Friend,† and Hans C. Oettgen2‡

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE؊/؊) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE؊/؊ mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the , serum levels of mouse MC protease-1 also were lower in parasite-infected ؊/؊ IgE animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis Downloaded from larvae present in the of IgE؊/؊ mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo. The Journal of Immunology, 2004, 172: 1139–1145. http://www.jimmunol.org/ igorous IgE Ab production is a characteristic response infections is found not to be specific to the parasite, which has led during infection with parasitic helminths, including some authors to speculate that the IgE may in fact aid the parasite V (1, 2). IgE Abs are produced primar- by blocking IgE receptors with irrelevant IgE (directed against Ags ily in mucosal-associated lymphoid tissue and, via their interaction other than those on the parasite) (reviewed in Ref. 7). The avail- with Fc⑀RI on mucosal mast cells (MC),3 can trigger immediate ability of mice with a targeted mutation of the C⑀ exons that en- hypersensitivity reactions. In parasite endemic regions of the code the constant regions of the IgE ⑀-H chain has made it possible world, high IgE levels are associated with resistance to reinfection to examine this question directly (8). Using such mutants, we have (3–5). IgE production, however, is only one feature of a broader recently observed that IgE augments immunity to response in which type 2 Th (Th2) cells produce high levels of mansoni during primary infection (9). by guest on October 2, 2021 IL-4 and IL-5, which stimulate IgE Ab synthesis along with eo- T. spiralis infects humans, , and several other species sinophil (eos) production and activation (reviewed in Ref. 6). (10, 11). Infection is accompanied by eosinophilia, the production Thus, the associations of IgE with parasitic infection and immunity of high levels of IgE, and a MC hyperplasia in the mucosa of the could be consistent either with a critical immune function of IgE small intestine (12–14). Immediate cutaneous hypersensitivity to per se or, alternatively, with the possibility that IgE production is larval Ags is observed within the first 3 wk of infection, consistent simply a marker of a broader Th2 response in which other effectors with the production of parasite-specific IgE. Some patients also (e.g., activated eos) are the key mediators of parasite elimination. suffer from allergic symptoms including urticaria and angioedema. In fact, much of the IgE that is produced in response to Trichinosis can be investigated in rodents and the role of IgE has been studied using the passive transfer of immune IgE or by as- sessing the parasite susceptibility of relatively IgE-deficient mouse *Department of Medicine, Division of Rheumatology, Immunology and Allergy, strains. Like humans suffering from trichinosis, T. spiralis-infected Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02115; †Department of Pathology, Brigham and Womens Hos- mice produce strong IgE responses (15, 16). The infusion of pu- pital and Harvard Medical School, Boston, MA 02115; ‡Division of Immunology, rified T. spiralis immune IgE into rats has been shown to confer § Children’s Hospital, Harvard Medical School, Boston, MA02115; and Department of rapid parasite expulsion to naive animals and the suppression of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, United Kingdom active IgE responses using anti-IgE in rats has been reported to Received for publication February 19, 2002. Accepted for publication November markedly impair parasite clearance (17–19). In contrast, investi- 4, 2003. gations using mice with defects in IgE production have given in- The costs of publication of this article were defrayed in part by the payment of page consistent results. One study showed that outbred mice selected for charges. This article must therefore be hereby marked advertisement in accordance low Ab production had impaired immunity during primary infection with 18 U.S.C. Section 1734 solely to indicate this fact. while a separate investigation using relatively IgE-deficient SJA/9 in- 1 This work was supported by National Institutes of Health/National Institute of Al- lergy and Infectious Diseases Grants AI054471-01, AR/AI 47417-01, AI 07306 HL bred mice showed no defect (20, 21). These mouse studies, however, 36110-16, and AI 31599-11, Wellcome Trust Grant 060312, and Biotechnology and are limited in two ways. First, the IgE deficiency of the animals is not Biological Sciences Research Council Grant 15/S10130. absolute and, second, the basis of IgE deficiency is not understood and 2 Address correspondence and reprint requests to Dr. Hans C. Oettgen, Division of could reflect a broader defect in Th2 response. For instance STAT6- Immunology, Children’s Hospital, Boston, 320 Longwood Avenue, Boston, MA 02115. E-mail address: [email protected] deficient mice, which are IgE-deficient by virtue of impaired IL-4 3 Abbreviations used in this paper: MC, mast cell; SCF, stem cell factor; mMCP-1, signaling, have also been shown to have impaired immunity to T. mouse MC protease-1; HPF, high-power field; LPF, low-power field; eos, eosinophil. spiralis (22).

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1140 IgE PROMOTES WORM EXPULSION IN T. spiralis-INFECTED MICE

IgE produced during infection with T. spiralis is primarily were washed twice with PBS containing 0.05% Tween 20 (Sigma- bound to mucosal MC in the gut (1). Studies using neutralizing Aldrich). Serum was added at dilutions ranging from 1/50 to 1/1000. Pu- Abs to the MC growth factor, c-kit ligand, or the MC-deficient rified IgE was used as standard, ranging in concentration from 12.5 to 200 W W-v ng/ml. Standards and sera were diluted in PBS/1% BSA (Sigma-Aldrich) mouse strains WBB6F1/J-Kit /Kit (W/Wv) and WCB6F1/J- and left on plates overnight at 4°C. Plates were then washed four times and Sl Sl-d Kitl /Kitl (Sl/Sld), which lack fully functional c-kit or its ligand incubated for 45 min at room temperature with biotinylated anti-IgE at 1 stem cell factor (SCF), respectively, have shown a critical effector ␮g/ml. After five washes, plates were given avidin conjugated to HRP function for MC in immunity to T. spiralis (23–25). Neither MC- (Zymed Laboratories, San Francisco, CA) and incubated for 30 min at room temperature. Plates were washed six times before adding substrate, deficient mice nor animals treated with blocking Abs to SCF man- 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (Zymed Laborato- ifested the mastocytosis associated with T. spiralis infection or ries). ODs of wells were read 10–20 min after addition of substrate at 405 effective rejection of the worms. Recently, IgE has been shown to nm. Concentrations of IgE in sera were extrapolated from a graph of stan- be a survival factor for MC in culture (26, 27). For us, this obser- dard ODs vs concentrations. vation raised the possibility that IgE might function not only in the Immunohistochemistry specific immune response to T. spiralis, but also by regulating MC homeostasis and maintaining this population of cells that are crit- Detection of IgE on muscle larvae was performed using tissue sections from the tongue of 28-day infected mice. These were prepared for immu- ical to parasite elimination. nostaining by successive washes through xylene, 100% ethanol, 90% eth- To examine the roles of IgE in immunity to T. spiralis, espe- anol, 80% ethanol, and PBS. Ag unmasking was achieved by microwaving cially in regard to MC and eos responses provoked by this parasite, the slides at full power for 15 min in 10 mM Tris/1 mM EDTA (pH 9.0). we infected IgEϪ/Ϫ BALB/c mice and compared them to normal Endogenous biotin was blocked using an endogenous avidin/biotin block- BALB/c mice. Wild-type animals, but not the mutants, showed a ing kit (Zymed Laboratories) as per the manufacturer’s instructions. The sections were then incubated at 37°Cfor2hinahumidified chamber with Downloaded from vigorous IgE response peaking on day 14 of primary infection. A 5 ␮g/ml biotin-labeled anti-mouse IgE (BD PharMingen) diluted in 5% peripheral blood eosinophilia, jejunal mastocytosis, and eos influx BSA/PBS. Following repeated washes in 5% BSA/PBS, the sections were occurred in both wild-type and IgE-deficient mice. However, MC incubated with 1.25 ␮g/ml streptavidin-HRP (Zymed Laboratories) at numbers in the spleen of IgEϪ/Ϫ mice were markedly diminished 37°C for 30 min. Finally, a diaminobenzidine-nickel cobalt substrate kit (Zymed Laboratories) was used as per the manufacturer’s instructions and and serum levels of the jejunal-specific secretory granule protease, the slides were dehydrated and mounted. mouse MC protease (mMCP)-1 were reduced. The intestinal worm burden of IgEϪ/Ϫ mice was elevated throughout the period of in- Cellular concentrations and densities http://www.jimmunol.org/ fection with the mutant animals harboring detectable numbers of The enumeration of blood eos was done directly on whole blood. Blood intestinal worms for Ͼ1 wk longer than wild-type mice. The num- was diluted 1/2 with PBS containing 100 U/ml heparin. This mixture was ber of encysted larvae in the muscle of IgEϪ/Ϫ mice was also further diluted 1/10 in Discombe’s fluid (0.05% Eosin Y) and eos were increased compared with BALB/c controls and there was less ev- counted directly in a hemocytometer (29). idence of larval necrosis in the absence of IgE. The larvae in wild- ELISA for mMCP-1 serum concentrations type but not in IgE-deficient mice were found to be coated with IgE, suggesting that this isotype may participate in the reduction of Serum mMCP-1 levels were determined as previously described (30). A sandwich ELISA was performed using affinity-purified rat monoclonal anti- larval burden. These findings provide evidence that IgE plays a mMCP-1 clone RF6.1 (31) for capture (5 ␮g/ml) and polyclonal sheep by guest on October 2, 2021 role in parasite clearance and regulates the splenic MC responses anti-mMCP-1 (32) for detection (2 ␮g/ml). A donkey anti-sheep IgG HRP during T. spiralis infection. conjugate (Sigma-Aldrich) was used at 1/8000 to develop the plates.

Materials and Methods Histology Mice Animals were sacrificed at the indicated time points and tissue samples were obtained and immediately fixed in 4% paraformaldehyde as previ- IgE-deficient mice were generated by gene targeting as previously de- ously described (28). The tissues were embedded in JB4 glycolmethacry- scribed and have been backcrossed through 10 generations onto the BALB/ late, sectioned at 2-␮m thickness, and placed on glass slides. eos were cAnTac (Taconic Farms, Germantown, NY) background (8). BALB/cAn- enumerated after staining the tissues with Congo Red. MC were enumer- Tac mice were used as controls. All mice were maintained in the animal ated after staining the tissues for chloroacetate esterase. At least 20 high- facility at Children’s Hospital, Harvard Medical School. Mice used in these power (ϫ50 objective) fields (HPF) were counted for at least three mice experiments were between 8 and 16 wk old. All animal protocols have been each for each genotype at each of the seven time points. reviewed and approved by the Dana-Farber Cancer Institute and Children’s Larval and degenerate cysts containing necrotic larvae were as- Hospital Animal Care and Use Committees in accordance with the Public sessed histologically in sections of the tongue after staining with DiffQuik Health Service Policy and provisions of the Animal Welfare Act. (Dade-Behring, Newark, DE) as previously described (33). Briefly, ne- crotic cysts were identified by the loss of the wall and infiltration of T. spiralis infection and worm burden determination granulocytes into the cyst. Thirty low-power fields (LPF, ϫ10 objective) Trichinella larvae were obtained from BALB/c mice infected Ͼ 1mo were counted for each animal, and a mean number of cysts/LPF was cal- before. The larvae were obtained by digesting the skeletal muscle in the culated. Cysts with necrotic larvae were counted simultaneously and were presence of 1% (Sigma-Aldrich, St. Louis, MO) and 1% HCl in expressed as the percentage of cysts with necrotic larvae in the tongue for each animal. Values are the mean Ϯ SEM from five to six animals. water as previously described (28). Larvae were washed in dH2O by low- speed centrifugation after being passed through a screen (60 gauge) to get rid of large pieces of undigested tissue and bone. The released larvae were Statistical analysis counted microscopically. Mice were infected with ϳ450 larvae in 200 ␮l Data sets for wild-type and IgEϪ/Ϫ animals were analyzed for variance by by direct gastric installation. Worm burden was determined at the times two-way ANOVA using GraphPad Prism software (GraphPad, San Diego, indicated by excising the small intestine, slicing it open longitudinally, and CA). Some data sets, as indicated in the text, were also subjected to Stu- then into 4-mm-long sections. Each intestine was incubated in PBS for 3 h dent’s two-tailed t test (assuming equal variances) using Microsoft Excel at 37°C with gentle agitation and the released worms were enumerated (Redmond, WA). using an inverted microscope. Results were obtained using four to six animals per time point for days 4–14 and 28 and three mice for day 21. Results IgE ELISA and immunocytochemistry T. spiralis elicits a vigorous IgE response and peripheral blood Serum IgE levels were determined using the standard protocol provided by eosinophilia. Mice infected with T. spiralis displayed a dramatic BD PharMingen (San Diego, CA.). Briefly, 96-well plates were coated rise in IgE levels, increasing to 53.3 Ϯ 3.9 ␮g/ml on day 14 and, Ϫ Ϫ with purified anti-IgE at 1 ␮g/ml overnight at 4°C. The next day, plates as expected, IgE / animals did not produce any IgE. Consistent The Journal of Immunology 1141 with our previous observations (8), the IgEϪ/Ϫ mice had no defect in IgG responses. T. spiralis infection caused IgG1 levels to in- crease by Ͼ10-fold in both wild-type and IgEϪ/Ϫ animals (15.8- fold vs 21.0-fold for the BALB/c and IgEϪ/Ϫ mice, respectively). Peripheral blood eosinophilia was also monitored following T. spiralis infection and showed a peak at day 21 (Fig. 1A). There was no impairment of eos production in the IgEϪ/Ϫ mice; in fact, their eos mobilization was actually significantly greater ( p Ͻ 0.05). Blood eos counts in IgEϪ/Ϫ mice peaked at 617,000 Ϯ 73,000 eos/ml, while the maximum for BALB/c controls was 383,000 Ϯ 93,000 eos/ml. Tissue recruitment of eos to the jejunum of IgEϪ/Ϫ animals was intact and similar in both strains, with an initial peak at day 11 followed by a second wave of influx from days 21 to 28 (Fig. 1B).

Alterations in tissue mastocytosis in T. spiralis-infected mice We and others have observed that infection with T. spiralis stim- ulates a large increase in the number of MC and eos residing in the small intestine (16, 28). The normal expulsion of adult worms is Downloaded from dependent upon this mastocytosis (22–25, 34, 35). Recently pub- lished data have suggested that IgE is a survival factor for cultured MC but the role of IgE in MC differentiation in vivo has not yet been clearly defined (26, 27). To investigate the relationship be- FIGURE 2. Gut and spleen MC numbers following T. spiralis infection. tween IgE and tissue MC numbers during the course infection with MC numbers/HPF (ϫ50 objective) were determined by the chloroacetate T. spiralis, we examined the gut and spleen of infected mice esterase reaction in sections of jejunum (A) or spleen (B) at the indicated Ϫ/Ϫ F E http://www.jimmunol.org/ The most dramatic MC hyperplasia after T. spiralis infection time points in wild-type ( ) and IgE ( ) mice infected with T. spiralis .p Ͻ 0.0001, NS, Not significant ,ءءء .(MC/HPF Ϯ SEM, n ϭ 3/point) occurs in the small intestine (Fig. 2). Increased MC numbers were evident in the jejunum as early as 7 days post-infection and peaked Ϫ/Ϫ at 14 days with 20.5 Ϯ 4.2 MC/HPF in wild-type mice and 20.6 Ϯ IgE animals throughout the 4-wk course of the study. These 1.6 in IgEϪ/Ϫ mice. findings indicate that IgE supports the splenic MC hyperplasia ob- As previously reported (28), a marked increase in MC numbers served after T. spiralis infection but is not critical for the jejunal was observed in the spleen as well, beginning 4 days after infection MC response. and persisting throughout the experiment. In contrast to the jejunal Diminished MC secretory granule protease release is associated

MC hyperplasia, the splenic mastocytosis was substantially re- Ϫ/Ϫ by guest on October 2, 2021 Ϫ Ϫ Ϫ Ϫ with impaired worm clearance in T. spiralis-infected IgE duced in IgE / mice (Fig. 2B). At the peak (day 11), IgE / mice mice had 0.9 Ϯ 0.1 MC/HPF compared with 1.9 Ϯ 0.2 in the control animals and the reduced MC numbers persisted in the We have previously observed that T. spiralis infection elicits not only increased MC numbers but also the release of mediators, in- cluding mMCP-1. Mice deficient in mMCP-1 were recently used to show that this protease plays a direct role in parasite immunity; mMCP-1Ϫ/Ϫ mice exhibit normal mastocytosis but delayed worm expulsion following primary T. spiralis infection (30). Since our current findings indicate a role for IgE in the splenic MC hyper- plasia in this model and also since parasite-specific IgE, via Fc⑀RI, could be critical for MC activation, we sought to establish whether normal levels of MC activation would be induced by T. spiralis infection in the absence of IgE. To investigate these questions, the release of mMCP-1 into plasma was monitored. Serum mMCP-1 levels did increase in infected IgEϪ/Ϫ mice but reached peak levels less than half of those observed in wild-type control animals (4.4 Ϯ 0.4 vs 11.9 Ϯ 0.7 ␮g/ml, mean Ϯ SEM, n ϭ 2 with four to six animals per time point, respectively; Fig. 3). To determine whether these altered MC responses in IgEϪ/Ϫ mice are associated with an impaired ability to eliminate worms, we counted intestinal worms throughout the course of infection (Fig. 4). Wild-type and IgEϪ/Ϫ mice had similar worm burdens when first assessed at day 4 (281 Ϯ 40 and 285 Ϯ 17 worms, respectively). However, the groups diverged soon thereafter. From day 7 through the completion of the experiment at day 28, con- FIGURE 1. Blood and tissue eos responses following T. spiralis infec- sistently greater worm numbers were recovered from the T. spi- tion. A, Total blood eos counts were determined at the indicated time points Ϫ/Ϫ in wild-type (F) and IgEϪ/Ϫ (E) mice infected with T. spiralis (eos/ml ralis-infected IgE mice. Although wild-type mice had already ϮSEM). B, Congo Red-stained 2-␮m jejunal sections were prepared and completely rid themselves of worms by day 21 (none of eight eos/HPF (ϫ50 objective) enumerated by light (mean eos/ BALB/c mice evaluated in two separate experiments had any re- p Ͻ 0.05, NS, Not significant. maining worms), the IgE-deficient mice still had residual parasite ,ء .(HPF Ϯ SEM, n ϭ 3/point 1142 IgE PROMOTES WORM EXPULSION IN T. spiralis-INFECTED MICE

FIGURE 4. Expulsion of T. spiralis from the jejunum. Wild-type (F) and IgEϪ/Ϫ (E) mice received 450 worms on day 0. Five to six mice of FIGURE 3. mMCP-1 serum levels following T. spiralis infection. Ϫ Ϫ each strain were analyzed for jejunal worm burden on the indicated days Wild-type (F) and IgE / (E) mice were infected with T. spiralis. Serum .p ϭ 0.01 ,ءء .(mean worms recovered per mouse Ϯ SEM) samples were collected at the indicated time points and total mMCP-1 was determined by ELISA (mean Ϯ SEM, micrograms per milliliter, n ϭ 2). .p Ͻ 0.0001 ,ءءء in the regulation of MC responses in vivo. In our experiments, infection with T. spiralis elicited the expected sharp increase in counts through day 28 at the termination of the experiment (seven IgE levels in wild-type but not IgEϪ/Ϫ mice. Peripheral blood eo-

Ϫ Ϫ Downloaded from of nine IgE / mice had detectable worms on day 28). The mean sinophilia and migration of eos into the gut also was seen in both Ϫ Ϫ intestinal worm number per IgE / mouse was 7.7 Ϯ 1.5 on day mutant and control animals. Both groups of animals displayed sim- 21 (mean Ϯ SE of three mice, n ϭ 1) and 2.2 Ϯ 0.6 on day 28 ilar kinetics and levels of jejunal mastocytosis. Strikingly however, (mean of nine mice, n ϭ 2). The differences in worm burdens over the MC number in the spleen was markedly diminished in IgEϪ/Ϫ time were significant ( p ϭ 0.01) as assessed by ANOVA. In ad- mice. The IgE-null mice also had significantly lower levels of cir- dition, worm counts at individual time points, assessed by Stu- culating mMCP-1 and persistently greater worm burdens through- dent’s t test, differed significantly ( p Ͻ 0.05) at all time points out the course of infection and retained detectable worms in their http://www.jimmunol.org/ except days 4 and 11. These findings provide strong support for a intestines for a week longer than did wild-type controls. The num- protective role of IgE in mice in intestinal worm expulsion fol- ber of larvae in the skeletal muscle was significantly less in wild- lowing primary infection with T. spiralis. type BALB/c mice than in IgEϪ/Ϫ mice and this was associated To examine the role of IgE Abs in the response to muscle larvae, with binding of IgE to the muscle larvae and evidence of increased we examined the numbers and morphology of T. spiralis cysts in cyst necrosis. These findings provide strong support for a role for Ϫ Ϫ the tongues of BALB/c and IgE / mice 28 days following in- IgE in immunity to primary infection with T. spiralis and indicate fection (Fig. 5). Three weeks after infection, T. spiralis larvae are that IgE Abs play a prominent role in jejunal MC activation and observed encysting the skeletal muscle. We have previously re- regulation of splenic MC numbers in vivo. by guest on October 2, 2021 ported that assessment of larval cyst numbers in the tongue pro- Previous investigations on the role of IgE in the clearance of T. vides a good indication of encystment throughout the skeletal mus- spiralis have given varied results. Naive rats have been reported to cle and used tongue as the reference organ for monitoring larval exhibit enhanced parasite clearance after passive immunization encystment at the conclusion of this study (33). Because we have with T. spiralis-immune IgE (17, 18). Active suppression of IgE also previously observed that there is evidence of necrosis and cyst responses during primary infection using anti-IgE Abs has been infiltration with granulocytes at this stage of infection, we exam- shown to markedly impair parasite elimination (19). Using outbred ined the histological appearance of the T. spiralis cysts. mice selected for impaired IgE production, Perrudet-Badoux et al. Ϫ Ϫ The number of cysts in the tongues of IgE / mice (3.6 Ϯ (20) observed impaired resistance in the low-responder mice. 0.5/LPF) was more than twice that in control BALB/c mice (1.7 Ϯ However, these genetically heterogeneous mice likely differed 0.1/LPF, p ϭ 0.01, Fig. 5A). The percentage of cysts containing from controls at many loci. Since the basis of their depressed IgE Ϫ Ϫ necrotic larvae in the IgE / mice was reduced (1.5 Ϯ 0.4%) response was not characterized, the possibility that low IgE per se compared with that observed in BALB/c mice (4.0 Ϯ 1.1%, p ϭ increased their susceptibility to T. spiralis could not be distin- 0.05, Fig. 5B). Because these results suggested that the IgE par- guished from the alternative that more global immunological de- ticipates in larval toxicity, we sought the presence of IgE in and fects affecting T cell responses, Ab isotype switching, MC activa- around the larvae. Sections of tongue from 28-day T. spiralis- tion, and/or other immunological functions accounted for their infected mice were stained with anti-IgE Ab. Only the muscle altered response to the parasite. To focus the analysis on IgE in the larvae from wild-type mice stained intensely for IgE (Fig. 6), dem- current study, we used IgE-deficient BALB/c mice generated by onstrating significant binding of IgE to the worms at this stage in gene targeting. These mice are genetically very similar to the con- their life cycle. These findings indicate that IgE may facilitate not trol animals, likely differing only at a very few tightly linked loci only expulsion of worms from the intestine but may also contribute around the C⑀ gene. Thus, our findings provide direct evidence for to the immune response to T. spiralis larvae encysted in skeletal the importance of the IgE isotype in T. spiralis clearance. We have muscle. previously observed that these mice also have impaired responses to S. mansoni (9) and now demonstrate that their response to T. Discussion spiralis is impaired as well (Fig. 4). BALB/c mice typically reject T. spiralis infection in humans and rodents has long been known the parasite burden we use after 14–17 days. Yet the IgE-null mice to elicit a vigorous IgE response. In mice, infection is correlated still had detectable worms 28 days postinfection. Thus, a signifi- with a dramatic MC hyperplasia in the intestine and spleen. Using cant protective benefit is afforded by the IgE response even in a mice with a targeted disruption of the IgE H chain gene and con- primary infection. genic wild-type BALB/c controls, we have now been able to di- Our examination of cyst numbers in the tongues of BALB/c and rectly assess the role of IgE both in elimination of the worms and IgEϪ/Ϫ mice revealed that significantly fewer cysts were present in The Journal of Immunology 1143

FIGURE 5. Quantitation of cysts and necrotic larvae in IgEϪ/Ϫ and BALB/c control mice after T. spiralis infection. The number of cysts per LPF (A) and the percentage of degenerating cysts with ne- crotic larvae (B) were enumerated in the tongues of IgEϪ/Ϫ and wild-type BALB/c mice. Sections of tongue were stained with DiffQuik (reference bar in D is 20 ␮m). A representative intact is shown in C and a necrotic larva, with loss of cyst wall (white arrow) and infil- trating granulocytes, including eos (black arrows), is shown in D. The mean cyst number was obtained by counting at least Downloaded from 30 LPF from each animal. Values are the means Ϯ SEM from five to six animals. Two-tailed t tests were used to assess sta- tistical significance of differences be- tween data sets: total cysts, p ϭ 0.01; percent necrotic cysts, p ϭ 0.05. http://www.jimmunol.org/

the muscle of wild-type mice and that the extent of necrosis was The effective recruitment of granulocytes to muscle cysts in by guest on October 2, 2021 increased in the presence of IgE (Fig. 5). Furthermore, tissue stain- wild-type but not IgEϪ/Ϫ mice and the increased rate of cyst ne- ing for IgE revealed the presence of IgE in and around the encysted crosis in wild-type animals implicates IgE Abs in supporting an larvae (Fig. 6). Previously published reports on the kinetics of cyst effective cytotoxic response. Neither Fc⑀RI nor CD23 (high- and rejection have shown that cyst destruction can occur during this low-affinity IgE receptors, respectively) are believed to be ex- acute phase in miniature swine (36). Studies in rats have suggested pressed by eos or other cytotoxic effector cells in mice. However, that IgE Abs are important in the early response to cysts, mediating activation of tissue MC by parasite-specific IgE Abs is likely to both the recruitment of eos to encysted larvae in muscle and re- elicit the release of an array of cytokines, chemokines, and lipid ducing the numbers of muscle cysts (19). In recent studies of T. mediators capable of recruiting an acute inflammatory response. It spiralis-infected mice, we have observed that the chemokine re- ceptor CCR3 is important for eos recruitment and acute larval is less likely, but also possible, that IgE Abs directly target larvae cytotoxicity (33). Thus, our observations indicate that the role of for destruction by effector cells, including eos and NK cells. It has IgE in immunity to T. spiralis is not restricted to facilitating the been reported that Fc␥RIII can bind IgE (37, 38), This receptor is expulsion of adult worms from the intestine but also includes kill- expressed by eos and thus provides a potential mechanism ing of larval stages of the parasite. whereby eos-mediated larval killing is enhanced in the presence of IgE (39). Capron et al. (40) reported that IgE Abs can opsonize parasites for killing by eos in rodents. Our observations contrast with those of Watanabe et al. (21) who found that SJA/9 mice, which have a markedly depressed IgE response to nematode parasites, had no defect in the clearance of T. spiralis compared with wild-type controls. Two possible expla- nations for this discrepancy exist. First, these mice do not have an inherent defect in IgE biosynthesis per se but rather have abnormal IL-4 production (41). They do produce small amounts of IgE and it is possible that this modest response (in contrast to the total lack of IgE in the IgEϪ/Ϫ mice) was sufficient to support elimination of FIGURE 6. Anti-IgE immunostaining of skeletal muscle larvae in T. spiralis. Second, the SJA genetic background is different from IgE Ϫ/Ϫ and BALB/c control mice. Sections of tongue from 28-day post-T. spiralis-infected BALB/c (A)orIgEϪ/Ϫ (B) mice were stained with a rat BALB/c and it is possible that alternative effector mechanisms, not anti-mouse IgE Ab. No staining was observed in control sections of operative in BALB/c mice, compensate for the low IgE response in BALB/c mice not incubated with the primary Ab. the SJA/9 strain. 1144 IgE PROMOTES WORM EXPULSION IN T. spiralis-INFECTED MICE

The absence of an IgE response did not impair the ability of that in the case of T. spiralis, IgE clearly does enhance parasite IgEϪ/Ϫ mice to mobilize eos to the gut in response to T. spiralis clearance at two different stages in its life cycle. Our finding that infection in our study. In fact, the mutant animals had the same MC homeostasis and mMCP-1 release are altered in IgEϪ/Ϫ mice degree of eos influx into infected jejunum as wild-type mice and provides some clues as to possible mechanisms for its effects on peripheral blood eos counts were actually significantly elevated the jejunal worm burden and along with past studies suggest a (Fig. 1). One possible explanation for the more robust response possible mechanism for the larval cytotoxicity. Furthermore, the observed in IgEϪ/Ϫ mice is that their heavier parasite burden decreased numbers of splenic MC in IgE-null mice following T. through the course of infection provided a stronger stimulus for spiralis infection is the first evidence to our knowledge that in vivo eosinophilopoiesis and recruitment. Alternatively, IgE might affect IgE may be enhancing MC survival. Further studies will be nec- eos development, migration, or life span (as indicated for splenic essary to clarify the connections among IgE, MC turnover, and T. MC, see below). spiralis clearance. T. spiralis infection leads to marked increases in MC numbers in the gut and spleen and a number of studies have established that References MC are critical in the process of parasite expulsion (23–25). Wild- Ϫ Ϫ 1. Alizadeh, H., J. F. Urban, Jr., I. M. Katona, and F. D. Finkelman. 1986. Cells type and IgE / mice both showed jejunal mastocytosis following containing IgE in the intestinal mucosa of mice infected with the nematode par- T. spiralis infection in our study, indicating that this Ab does not asite Trichinella spiralis are predominantly of a mast cell lineage. J. Immunol. 137:2555. play a role in the recruitment and expansion of mucosal MC in the 2. Capron, A., J. P. Dessaint, M. Capron, J. H. Ouma, and A. E. Butterworth. 1987. gut following (Fig. 2). However, the splenic Immunity to schistosomes: progress toward vaccine. Science 238:1065. mastocytosis was markedly attenuated in IgEϪ/Ϫ animals. We pre- 3. Dunne, D. W., A. E. Butterworth, A. J. C. Fulford, H. C. Kariuki, J. G. Langley,

J. H. Ouma, A. Capron, R. J. Pierce, and R. F. Sturrock. 1992. Immunity after Downloaded from viously reported that many of the splenic MC in T. spiralis-in- treatment of human : association between IgE antibodies to adult fected mice are undergoing apoptosis (42). We have hypothesized worm antigens and resistance to reinfection. Eur. J. Immunol. 22:1483. that the spleen represents a site of MC clearance during the reso- 4. Hagan, P., U. J. Blumenthal, D. Dunn, A. J. G. Simpson, and H. A. Wilkins. 1991. Human IgE, IgG4 and resistance to reinfection with Schistosoma haema- lution of parasitic infections of the gut as many of the splenic MC tobium. Nature 349:243. appeared to have migrated back to the spleen from the small in- 5. Rihet, P., C. E. Demeure, A. Bourgois, A. Prata, and A. J. Dessein. 1991. Evi- testine during the resolution of the mastocytosis. In this context, dence for an association between human resistance to and high anti-larval IgE levels. Eur. J. Immunol. 21:2679. Ϫ/Ϫ http://www.jimmunol.org/ our finding that T. spiralis-infected IgE mice have fewer MC in 6. Oettgen, H. C., and R. S. Geha. 2001. IgE regulation and roles in asthma patho- the spleen than the infected wild-type controls suggests that signals genesis. J. Allergy Clin. Immunol. 107:429. that support MC survival, or prevent their apoptosis, may be lack- 7. Pritchard, D. I., C. Hewitt, and R. Moqbel. The relationship between immuno- logical responsiveness controlled by T- helper 2 lymphocytes and infections with ing in these mutant mice. A number of factors including SCF, IL-3, parasitic helminths. Parasitology 115:S33, 1997. IL-4, IFN-␥, and TNF-␣ have been reported to play a role in the 8. Oettgen, H. C., T. R. Martin, A. Wynshaw-Boris, C. Deng, J. M. Drazen, and mastocytosis (24, 43–46). In addition, two groups have identified P. Leder. 1994. Active anaphylaxis in IgE-deficient mice. Nature 370:367. 9. King, C. L., J. Xianli, I. Malhotra, S. Liu, A. A. Mahmoud, and H. C. Oettgen. monomeric IgE itself as an anti-apoptotic factor for cultured MC 1997. Mice with a targeted deletion of the IgE gene have increased worm burdens (26, 27). In these studies, IgE altered MC survival in the absence and reduced granulomatous inflammation following primary infection with Schis- of specific Ag. Cultured MC deprived of growth factors normally tosoma mansoni. J. Immunol. 158:294. 10. Despommier, D. 1977. Immunity to Trichinella spiralis. Am. J. Trop. Med. Hyg. by guest on October 2, 2021 undergo apoptosis (47, 48); however, in the presence of mono- 26:68. meric IgE, but without Ag, the cells were protected from death. 11. Most, H. 1978. Trichinosis–preventable yet still with us. N. Engl. J. Med. 298:1178. The mechanism of this effect remains unsettled. Although Kale- 12. Matossian, R. M., I. Salti, and E. Stephan. 1977. Variation in serum immuno- snikoff et al. (27) reported that monomeric IgE induced the pro- globulin levels in acute trichinosis. J. 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Katona, D. A. Dean, and F. D. Finkleman. 1984. The cellular IgE response of rodents to infection with Nippostrongylus brasiliensis, MC to exocytose granule-associated proteases is activation by Trichinella spiralis and Schistosoma mansoni. Vet. Parasitol. 14:193. Fc⑀RI-bound IgE cross-linked by polyvalent (parasitic) Ags, the 17. Ahmad, A., C. H. Wang, and R. G. Bell. 1991. A role for IgE in intestinal diminished mMCP-1 levels likely reflect diminished MC activa- immunity: expression of rapid expulsion of Trichinella spiralis in rats transfused Ϫ/Ϫ with IgE and thoracic duct lymphocytes. J. Immunol. 146:3563. tion via this Ag-specific pathway. Low mMCP-1 levels in IgE 18. Bell, R. G., J. A. Appleton, D. A. Negrao-Correa, and L. S. Adams. 1992. Rapid mice might contribute to persistence of T. spiralis since the secre- expulsion of Trichinella spiralis in adult rats mediated by monoclonal antibodies tory granule ␤-chymase is specifically expressed in mucosal MC of distinct IgG isotypes. Immunology 75:520. 19. Dessein, A. J., W. L. Parker, S. L. James, and J. R. David. 1981. IgE antibody and (31), mMCP-1 levels peak at the time of worm expulsion (49), and resistance to infection. I. Selective suppression of the IgE antibody response in Ϫ Ϫ mMCP-1 / animals have a significant delay in worm expulsion rats diminishes the resistance and the eosinophil response to Trichinella spiralis during T. spiralis infection (30). Like IgEϪ/Ϫ animals, mMCP-1- infection. J. Exp. Med. 153:423. 20. Perrudet-Badoux, A., R. A. Binaghi, and Y. Boussac-Aron. 1978. Trichinella deficient mice (which are also on a BALB/c background) have gut spiralis infection in mice: mechanism of the resistance in animals genetically worm burdens similar to those of controls early in the course of selected for high and low antibody production. Immunology 35:519. infection but begin to show higher worm counts at the later time 21. Watanabe, N., K. Katakura, A. Kobayashi, K. Okumura, and Z. Ovary. 1988. 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A critical role for stem cell factor and c-kit in protective immunity to ing the direct role of this Ab isotype in immunity. Our data show an intestinal helminth. Int. Immunol. 8:559. The Journal of Immunology 1145

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