Activation, and Cytokine Production

The Schistosome Granuloma: Characterization of Lymphocyte Migration, Activation, and Cytokine Production

This information is current as Catherine A. Rumbley, S. Ali Zekavat, Hiroko Sugaya, Peter of September 27, 2021. J. Perrin, Mohamad Ali Ramadan and S. Michael Phillips J Immunol 1998; 161:4129-4137; ; http://www.jimmunol.org/content/161/8/4129 Downloaded from

References This article cites 65 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/161/8/4129.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 27, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Schistosome Granuloma: Characterization of Lymphocyte Migration, Activation, and Cytokine Production1

Catherine A. Rumbley, S. Ali Zekavat, Hiroko Sugaya,2 Peter J. Perrin, Mohamad Ali Ramadan, and S. Michael Phillips3

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, ␥ IL-4, IFN- ), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and Downloaded from IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-␥. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes. The Journal of Immunology, 1998, 161: 4129–4137. http://www.jimmunol.org/

chistosomiasis is characterized by the formation of inflam- studies could not precisely characterize the cells which were ac- matory granulomas around deposited parasite eggs (1). tually producing cytokines in vivo. Granuloma formation is a cell-mediated immune response In this study, we examined the migration of lymphocytes to the S ϩ that is dependent on CD4 T cells sensitized to schistosomal egg granuloma, cell cycle kinetics, activation profiles, production of Ags (SEA)4 (2, 3). This T cell-mediated granulomatous response specific cytokines by lymphocytes in the spleen and granuloma, peaks between 8 and 10 wk after exposure in mice. This acute and apoptosis of lymphocytes from the spleen and granuloma us- stage granuloma is characterized by dense cellularity and maxi- ing flow cytometry. We found that lymphocytes underwent IL-2- by guest on September 27, 2021 mum cytokine production (1, 4, 5). As the infection progresses into associated division in the spleen but not in the granuloma. Gran- the chronic stage (16–20 wk postinfection (p.i.)), cytokine pro- uloma lymphocytes, when compared with splenic lymphocytes, duction and cellularity decrease while the fibrotic components of were more highly activated than their splenic counterparts, but the immunopathologic process increase (1, 4, 5). produced lower levels of cytokines and were more likely to un- The T cell responses that characterize granuloma formation are dergo apoptosis. In vitro studies showed that, based on specific very complex. The description of two subpopulations of CD4ϩ T cytokine production, lymphocytes of the spleen and granuloma do cells has augmented the analysis of the cellular regulation of gran- not appear to be the primary cells responsible for the reported Th2 uloma formation (6–10). Many investigators have studied cyto- cytokine dominance in murine schistosomiasis. kine production patterns in schistosomiasis and have demonstrated a shift from Th1 to Th2 cytokine profiles concomitant with egg Materials and Methods deposition (11–14). However, the majority of studies have relied Experimental animals and infection with Schistosoma mansoni on cell culture, including in vitro stimulation, or RNA hybridiza- cercariae tion techniques to assess cytokine production. Therefore, these Female C57BL/6 (Harlan Sprague-Dawley, Indianapolis, IN) were used in all experiments. Mice (5–6 wk of age) were infected with 30 cercariae percutaneously by tail immersion, according to the method of Bruce and Allergy and Immunology Division, University of Pennsylvania School of Medicine, Radke (15). Infected snails were obtained from Dr. Fred Lewis (Biomed- Philadelphia, PA 19104 ical Research Institute, Rockville, MD). Received for publication December 1, 1997. Accepted for publication June 9, 1998. The costs of publication of this article were defrayed in part by the payment of page Cell preparation charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Mice were sacrificed at various times p.i., and their spleens and livers were aseptically removed. Spleens were pressed through 60 mesh stainless steel 1 This work was supported by National Institutes of Health Grant AI15193. screens and the resultant cell suspension washed two times and suspended 2 Current address: Department of Parasitology, Akita University School of Medicine, in complete medium (RPMI 1640 supplemented with 1 mM sodium pyru- Akita, Japan. vate, 100 ␮g/ml streptomycin sulfate, 100 U/ml penicillin, 50 ␮g/ml gen- ϫ Ϫ5 3 Address correspondence and reprint requests to Dr. S. Michael Phillips, Allergy and tamicin, 2 mM L-glutamine, 5 10 M 2-ME, 1% nonessential amino Immunology Division, University of Pennsylvania School of Medicine, 518 Johnson acids, and 10% heat-inactivated FCS). Livers were minced in a Waring Pavilion, Philadelphia, PA 19104. E-mail address: [email protected] blender at low speed for 15 s, and granulomas were collected by sedimen- 4 Abbreviations used in this paper: SEA, schistosomal egg Ag; p.i., postinfection; tation. After extensive washing, granulomas were suspended in 10 ml com- KLH, keyhole limpet hemocyanin; DiI, 1,1Ј-dioctadecyl 3,3,3Ј,3Ј-tetramethylindo- plete medium and collagenase type IV (2.5 mg/ml; Sigma, St. Louis, MO), carbocyanine perchlorate; SB, PBS containing 2% FCS; MCF, mean channel was added to both granulomas and splenic cell suspensions with subse- fluorescence. quent incubation for1hat37°C with frequent, gentle agitation. The cell

suspensions were ﬁltered through a 100-␮m Nytex membrane (LabCor Products, Gaithersburg, MD) and washed in complete medium. Following

RBC lysis with Tris-NH4Cl, the cells were washed and viable cells were enumerated by trypan blue exclusion. Cell migration studies In vivo studies: Splenocytes from S. mansoni infected and keyhole limpet hemocyanin (KLH)-immunized mice. Donor lymphoblasts were prepared from bulk cultures of splenic suspensions using equilibrium density sedi- mentation (16). Lymphoblast separation was performed by resuspending 15 ϫ 106 cells/ml in a solution of BSA (density, 1.082) that was overlaid with a less dense solution of BSA (density, 1.060). Gradients were spun to equilibrium at 10,000 ϫ g for 30 min at 4°C. The less dense lymphoblasts were removed from the interface. B cells were depleted by a selection step with CD45R (B220) micro beads (Miltenyi Biotec, Auburn, CA). The pu- rity and viability of the lymphoblasts were determined by size, morphol- ogy, phenotypic characteristics, surface Ig staining, trypan blue exclusion, and [3H]thymidine incorporation in response to specific Ag. Using these criteria, the transferred populations were consistently Ͼ90% viable lymphoblasts. The lymphoblasts (2 ϫ 107) were labeled with the red fluorescent vital ϫ Ϫ6 dye PKH-26 (2.2 10 M) (17) (Zyn-R, Zynaxis Cell Science, Malvern, Downloaded from PA). The labeling reaction was terminated by the addition of FCS, and unbound dye was removed by density centrifugation. FIGURE 1. Cell cycle kinetics of lymphocytes obtained from spleens After washing, the cells were injected i.v. into the tails of recipient mice and granulomas. The cell cycle kinetics of lymphocytes obtained from the that had been exposed to 25 S. mansoni cercariae 10 wk before study or spleen and granulomas of 8-wk-infected animals were determined using immunized with 100 ␮g of KLH in CFA 4 wk before study. Four days propidium iodide to determine cellular DNA content as described in Ma- before transfer, 105 KLH-coated latex beads were injected into the portal terials and Methods. Data were analyzed using ModFit version 2.0 (Verity veins of the KLH-sensitized mice. Eighteen hours after cell transfer, the Software). Forty percent of the splenic lymphocytes were in S phase and http://www.jimmunol.org/ animals were sacrificed and their lymph nodes, spleens, livers, and gran- 16.5% were in G /M, whereas 47% of the granuloma lymphocytes were in ulomas were obtained. Cell suspensions were prepared from each organ, 2 S phase and 3.6% are in the G /M phase. Less than 1% of splenic lym- and the number of labeled cells was determined by FACS. 2 In vitro studies. In vitro granulomas were studied using SEA-coated latex phocytes obtained from uninfected animals were in S phase. beads. Spleen cells from animals, previously exposed to S. mansoni for 12 wk or immunized with KLH, were added to SEA or KLH Ag-coated beads to form in vitro granulomas (18). Small numbers (ϳ1000) of cloned Th1 cells, reactive to either SEA or tional 30 min on ice in the dark. The samples were washed three times with KLH (19), were labeled with the fluorescent, hydrophobic dye, 1,1Ј- SB and analyzed by flow cytometry. All data were acquired and analyzed dioctadecyl 3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate (DiI; Molecular using a FACScan or FACScalibur instrument equipped with CellQuest software, version 3.0.1 (Becton Dickinson, Mountain View, CA).

Probes, Eugene, OR) and were added to the culture (20, 21). Individual cells by guest on September 27, 2021 ϫ 7 were identified by fluorescence and tracked at 4-s intervals using videomi- Intracellular cytokine staining. Cell suspensions (1 10 /ml) were in- ␮ croscopy. The identity of the labeled cell was confirmed by intermittent flu- cubated in RPMI 1640 plus 10% FCS with 10 g/ml Brefeldin A (Sigma) orescence microscopy. Images were obtained with a Dage-MTI (Michigan for3hat37°C with 5% CO2. Following incubation, the cells were har- City, IN) model 65 videocamera, a JVC (Tokyo, Japan) Br-9000 VCR, Sony vested, washed once, and surface stained as described above. After surface PVM-91 monitor (Tokyo, Japan), and Opti-quip (Highland Mills, NY) infra- staining, cells were suspended in 1 ml of freshly prepared 2.5% parafor- red stage-mounted incubator. The migratory pathway was digitized, electron- maldehyde (pH 7.4) and stored overnight at 4°C. The following day, the ically plotted, and measured with a K&E model 620005 compensating samples were washed once and permeabilized by a 10-min incubation on planimeter (Keuffel and Esser, Germany). An index of directed migration or ice in 1 ml of SB containing 0.1% Saponin (Sigma). Cells were then pel- ␮ ␮ persistence was calculated by dividing the net migration that was directed leted and suspended in 50 l SB containing 0.1% Saponin plus 10 g rat toward the central point of the bead by the total migratory path. An index of IgG and incubated an additional 10 min on ice. Without washing, FITC- ␣ ␣ ϩ1 meant that the cell migrated directly to the bead. An index of 0 meant that conjugated anti-cytokine Abs (PharMingen IL-2 clone 54B6; IL-4 clone ␣ ␥ the migration was random. An index of Ϫ1 meant that the cell migrated 11B11; IFN- clone XMG1.2) were added and samples incubated 30 min directly away from the bead. The migration index was calculated within con- on ice. Following two washes with SB plus 0.01% saponin, the samples centric rings of increasing diameter around the in vitro granuloma, i.e., were immediately analyzed by FACS. 0–25 ␮m, 25–50 ␮m, 50–100 ␮m, 100–150 ␮m, etc. Apoptosis. Apoptosis was quantitated using the terminal deoxynucleoti- dyltransferase-mediated dUTP nick end labeling (TUNEL) methodology Flow cytometry described by Gorczyca et al. (23), with modifications. Cells were prepared, surface stained, and permeabilized as above. Cells were then incubated In all flow cytometry determinations, individual isotype controls were in- with 50 ␮l of TdT reaction mixture containing 0.1 M sodium cacodylate cluded and photomultiplier tube voltage and gain settings initially adjusted (pH 7.2), 2 mM CoCl2, 0.2 mM DTT, 0.1 mg/ml BSA, 5 units TdT, and 0.3 such that isotype control values were near 10 fluorescence units. In all data nmol Cy5-dUTP (Amersham, Arlington Heights, IL) for1hat37°C. After reported here, lymphocyte gates were defined by forward and side light staining, the cells were washed three times with SB and analyzed by FACS. scatter characteristics, and background staining levels were between 5 and A control sample that contained all components of the reaction mixture 15 fluorescence units. except the TdT enzyme served as the negative control. Cell cycle kinetics. After fixation and permeabilization of cells (1 ϫ 106), cellular DNA was stained by incubating cells in staining buffer (PBS ϩ 2% FCS) containing 10 ␮g/ml propidium iodide (Sigma) and 5 ␮g/ml RNase Results A (Sigma) for 30 min (22) before analysis by flow cytometry. Data were Lymphocyte cell cycle kinetics analyzed using ModFit version 2.0 (Verity Software House, Topsham, ME) We first wished to ascertain the site of generation of the lympho- to determine the percentages of cells in G0/M,SorG2/M phases. Data shown are representative of at least three confirmatory experiments. cytes which were recovered from the spleens and granulomas of S. Surface staining. Cells (106/sample) were washed once with PBS con- mansoni-infected mice. Cell cycle kinetics of gated lymphocyte taining 2% FCS (SB), suspended in 50 ␮l of SB containing 10 ␮g rat IgG populations from 8-wk-infected animals were analyzed. Spleen (Sigma), and incubated on ice for 10 min to block nonspecific binding. and granuloma cells were sorted by flow cytometry, stained with Without washing, directly conjugated antisurface marker mAb (0.5 ␮g/106 cells) (PharMingen (San Diego, CA) ␣CD3 clone 145-2C11; ␣CD45R/ Biebrich scarlet and hematoxyline, and were 91–97% small lym- B220 clone RA3-6B2; ␣CD122 clone TM-␤1; ␣CD69 clone H1.2F3; phocytes by morphologic criteria. As shown in Figure 1, similar ␣ CD25 clone 3C7) was added and the samples were incubated an addi- percentages of lymphocytes were in G0/G1 phase from the spleen The Journal of Immunology 4131

Table I. Effect of ﬂuorochrome labeling on cell viability and function

Criteria of Evaluation

Cell Testeda Viabilityb ConAc SEA-AMBTd SEA-IVGFe IL-2Rf

PKH-2ϩ 98 Ϯ 329Ϯ 416Ϯ 2 3.5 Ϯ 0.3 347 Ϯ 26 PKH-2Ϫ 97 Ϯ 435Ϯ 519Ϯ 4 3.0 Ϯ 0.4 291 Ϯ 37

a Th1 clone G4 was labeled with PKH-2, positively selected by FACS, washed, maintained in culture with APCs, and assayed for viability and function. PKH-2Ϫ additional cells were not labeled, negatively selected by FACS, washed, and similarly assayed for function. b Percent viable cells after 48 h of incubation with SEA and APCs, using criteria of phase-contrast microscopy and trypan blue exclusion. c Response to ConA at 48 h; [3H]-thymidine incorporation: cpm ϫ 103; mean Ϯ 2 SEM. d Ag-mediated proliferative response to SEA at 72 h; [3H]-thymidine incorporation: cpm ϫ 103; mean Ϯ 2 SEM. e In vitro granuloma formation around SEA-coated beads: granuloma index Ϯ 2 SEM (18) f IL-2R expression: photometric Units Ϯ 2 SEM. There is no signﬁcant difference between the value shown by PKH-2ϩ and PKH-2Ϫ cells for any criteria of evaluation: p Ն 0.05: Student’s t test. Downloaded from

FIGURE 2. SEA-reactive lymphoblasts specifically traffic to S. mansoni (44%) and granuloma (50%). Forty percent of splenic lymphocytes granulomas. Mice were exposed to S. mansoni or immunized against KLH (Fig. 1A) and 46% of granuloma lymphocytes (Fig. 1B) were in S and injected i.p. with 105 KLH-latex-coated beads to produce S. mansoni phase. In contrast a large difference was observed in the numbers and KLH granulomas, respectively. The mice were then injected via their tail veins with 107 PKH26-labeled lymphoblasts with specificity for SEA of lymphocytes in the G2/M phase of the cell cycle. When cells, (anti-SEA LB) or KLH (anti-KLH LB) or with lymphoblasts that had been obtained from spleens and granulomas, were compared, over 16% http://www.jimmunol.org/ of splenic lymphocytes were in G /M, whereas Ͻ4% of granuloma heat killed (HK) (20 min at 56°C). After 18 h the mice were sacrificed and 2 cell suspensions prepared from their combined axillary, inguinal, medias- lymphocytes were in G /M ( p Ͻ 0.01). These findings, coupled 2 tinal, and periaortic lymph nodes, spleen, livers, or granulomas. The results with the observation that there were many more lymphocytes per are expressed as the percent of cells obtained from each organ Ϯ SEM. 10,000 total FACS events in spleens (3,857 Ϯ 1,340) than in granulomas (403 Ϯ 308) suggested that the vast majority of lymphocyte division was occurring in the spleen while few if any lym- labeled cells in each organ (Fig. 2). When anti-SEA lymphoblasts phocytes were dividing in the granulomas. Moreover, this division were used as the source of transferred cells, Ͼ15% of the recov- was contingent on infection by S. mansoni since Ͼ98% of spleen ered labeled cells were found in the SEA granulomas compared by guest on September 27, 2021 cells from naive animals were resting in the G0/G1 stage of the cell with Ͻ3% in the KLH granulomas. When anti-KLH lymphoblasts cycle (data not shown). were the source of transferred cells, ϳ12% of the recovered cells were located in the KLH granulomas and Ͻ3% were in the S. Cell trafficking mansoni granulomas. Using heat-killed anti-SEA cells as the In vivo studies. Since our initial studies suggested that lympho- source of transferred cells resulted in the recovery of Ͻ5% of the cyte division was occurring in the spleen, we next sought direct cells in the S. mansoni or KLH granulomas and nearly 75% of the evidence for specific migration of Ag-reactive lymphocytes from cells from the liver. Significant numbers of heat-killed cells were the spleen to the granulomas. also seen in histologic sections of the lung; however, for technical In vivo trafficking experiments were performed using SEA-re- reasons, these cells could not be recovered efficiently for FACS active lymphocytes labeled with the red fluorescent vital dye quantitation. The distribution of SEA-reactive lymphoblasts in PKH-26 (17) to determine whether these cells could be actively normal, unsensitized animals were similar to those observed in recruited to the schistosome granuloma. We first confirmed that the KLH-sensitized mice and for KLH-reactive lymphoblasts in nor- labeling and flow cytometry sorting procedures had no significant mal and S. mansoni-infected mice (data not shown). These studies deleterious effects on the function of the transferred cells. To de- clearly demonstrated the preferential accumulation of SEA-reac- termine whether cell labeling altered cell function, an SEA-reactive lymphoblasts in S. mansoni granulomas due to active recruit- tive Th1 clone, G4 (24), was labeled with PKH-26. Viability, pro- ment and/retention within S. mansoni granulomas. liferation in response to Con A or SEA stimulation, IL-2R In vitro studies. If lymphocytes were generated systemically and expression, and in vitro granuloma formation around SEA coated migrated to the granulomas, wherein the granuloma might exert beads were examined (Table I). The viability of G4 cells was un- effects on these cells, then the granulomas could become potential changed as a result of PKH labeling. Both labeled and unlabeled regulatory organelles. In vivo studies to assess recirculation and G4 cells responded to Con A or SEA stimulation with similar residence time were not feasible since mice have recirculation levels of proliferation. Similar IL-2R expression was observed in times of less than 4 s. Therefore, in vitro studies were performed labeled and nonlabeled cells and the in vitro granuloma index was to see if cells could migrate specifically to the granuloma and not affected by labeling with PKH. subsequently leave these granulomas. Cloned T lymphocytes re- After in vitro experiments had demonstrated that both labeling active to SEA or KLH (19) were labeled with DiI and followed of cells with PKH and FACS sorting had no deleterious effects, in with time-lapsed videomicroscopy to assess their directed migra- vivo trafficking experiments were performed. Donor lymphoblasts tion toward, residence within, and egress from in vitro granulomas. were prepared from S. mansoni or KLH-immunized mice and An index of directed migration was determined for cells as they transferred i.v. to mice 10 wk after exposure to S. mansoni. After approached the in vitro granuloma. SEA-reactive T clones dem- 18 h, the recipient mice were sacrificed. Cell suspensions were onstrated dramatically increased directed migration as they neared prepared and analyzed by FACS to determine the number of PKH- the source of SEA; the SEA-coated bead (Fig. 3). The index of 4132 CHARACTERIZATION OF LYMPHOCYTES IN SCHISTOSOMIASIS

FIGURE 4. Cloned SEA-reactive T lymphocytes demonstrate directed egress from in vitro granulomas. C57BL/6 mice were exposed to 25 S. mansoni cercaria. Twelve weeks after exposure their spleen cells were Downloaded from obtained and used to produce in vitro granulomas around SEA-coated latex FIGURE 3. Cloned SEA-reactive T lymphocytes demonstrate increased beads. DiI-labeled cloned anti-SEA or anti-KLH lymphocytes were added directed ingress toward in vitro granulomas. C57BL/6 mice were exposed to the cultures after 3 days of culture and traced by videomicroscopy. to 25 S. mansoni cercaria. Twelve weeks after exposure their spleen cells Granulomas were observed until a DiI-labeled cell entered a granuloma. were obtained and used to produce in vitro granulomas around SEA-coated That cell was tracked by videomicroscopy. An index of directed migration latex beads. DiI-labeled cloned anti-SEA or anti-KLH lymphocytes were was measured in finite concentric rings around the granulomas. http://www.jimmunol.org/ added to the cultures after 3 days and traced by videomicroscopy. An index of directed migration was measured in finite concentric rings around the granulomas. KLH granulomas (Table II). Therefore, the SEA-reactive lymphocytes could be specifically sequestered and retained in SEA granulomas ( p Ͻ 0.01). directed migration approached 1.0; i.e., the SEA-reactive T clones The cells were next studied for their ability to leave the gran- moved directly toward the SEA-coated bead. Conversely, T ulomas. An index of directed migration was calculated as cells left clones, which were reactive to KLH, showed more random migra- the granuloma. The initial egress index for SEA-reactive cloned T tory patterns until they came very close to the SEA-coated bead cells from SEA granulomas was slow, suggesting that egress of (Ͻ100 ␮). In addition, in vitro granulomas formed around KLH- SEA-reactive T clones from SEA granulomas was initially re- by guest on September 27, 2021 coated beads, attracted cloned anti-KLH cells from KLH-sensi- tarded. Egress accelerated as the cell moved away from the gran- tized mice but did not attract the cloned anti-SEA cells as effec- uloma (Fig. 4) and migration was random after the cell had moved tively (Table II). Therefore, SEA-reactive T cells selectively over 300 ␮m from the SEA-coated bead. During the course of the migrated toward an SEA nidus such as an in vitro granuloma. egress studies, the granulomas were actually growing in size with The labeled cells in the granulomas were next studied. Granu- the majority of cells moving toward the granulomas (18) (data not lomas were produced in cultures containing 1000 unlabeled spleen shown). Therefore, the egress of SEA-reactive lymphocytes from cells per labeled, SEA-reactive, cloned T cell. In vitro granulomas SEA granulomas was a true directed migration and not simply the were studied as soon as a single labeled cell had entered the gran- result of the granulomas disintegrating. Unlike the SEA-reactive uloma to determine the mean residence time of the labeled cell clones, KLH-reactive clones showed more random migratory pat- within the in vitro granuloma (Table II). The mean residence time terns away from the SEA-coated bead. Reciprocal egress migration of the SEA-reactive Th1 cells within the SEA granuloma was patterns were observed for SEA-reactive cells in KLH granulomas 38.4 Ϯ 7.4 h. The mean residence time of the anti-KLH cells and for KLH-reactive cells in KLH granulomas (Table II). These which entered the SEA granuloma was 6.2 Ϯ 3.8 h. Analogous data indicated that the migration and residence patterns of the reciprocal differences in residence times were observed for SEA- SEA- and KLH-reactive clones were contingent upon the antigenic reactive cells in KLH granulomas and for KLH-reactive cells in composition of the granulomas.

Table II. In vitro ingress, residence, and egress patterns

SEA Granulomasb KLH Granulomasc

Aga Ingressd Residencee Egressf Ingressd Residencee Egressf

SEA 0.79 Ϯ 0.09g 38.4 Ϯ 7.4g Ϫ0.73 Ϯ 0.14g 0.37 Ϯ 0.12g 10.7 Ϯ 4.9g Ϫ0.28 Ϯ 0.19g KLH 0.35 Ϯ 0.13 6.2 Ϯ 3.8 Ϫ0.28 Ϯ 0.04 0.74 Ϯ 0.11 31.5 Ϯ 8.6 Ϫ0.62 Ϯ 0.21

a Ag specificity of T clone. b Ag specificity of in vitro granuloma: SEA-coated beads. c Ag specificity of in vitro granuloma: KLH-coated beads. d Index of directed ingress migration Ϯ SEM measured 0–100 ␮ from the surface of the bead. e Average residence time in granuloma: hours Ϯ SEM. f Index of directed egress migration Ϯ SEM measured 0–100 ␮ from the surface of the bead. g Value differs significantly from that of KLH clone cultured with SEA-coated beads and from that of SEA clone cultured with KLH-coated beads. p Յ 0.05; Student’s t test; n Ն 8. The Journal of Immunology 4133

FIGURE 6. Granuloma lymphocytes express higher levels of IL-2R during S. mansoni infection. Expression of the IL-2R ␤-chain (CD122) (A) and IL-2R ␣-chain (CD25) (B) on lymphocytes from the spleen (f) and granuloma (छ) at various weeks p.i. was measured by FACS. Data are Downloaded from expressed as the percentage of lymphocytes expressing the IL-2R ␤-chain or IL-2R ␣-chain (CD25). Collectively, these two chains constitute the high afﬁnity IL-2 receptor. FIGURE 5. Granuloma lymphocytes express higher levels of CD69 during the course of S. mansoni infection. Expression of the very early activation marker CD69 on lymphocytes from the spleen (Ⅺ) or granulomas (f) at various weeks p.i. was measured by FACS. The fold change in p.i., CD69 expression steadily declined on splenic lymphocytes http://www.jimmunol.org/ MCF intensity was determined by dividing the MCF of CD69-stained cells whereas CD69 expression on granuloma lymphocytes remained by the MCF of an isotype-matched control tube. The data are expressed as relatively constant. Since 10,000 events were counted in each the fold change in that ratio. Each data point represents the mean Ϯ SEM FACS determination, a 1% change in MCF is signiﬁcant. These of at least three observations. observations suggested that lymphocytes were more highly activated in the granuloma.

Characterization of lymphocyte activation Expression of IL-2R Initial studies showed that cells could be obtained from spleens After examination of CD69 expression, we studied the expression injected into animals and subsequently recovered in vivo from of the IL-2R, a more specific marker of immune activation. Ag by guest on September 27, 2021 granulomas. These trafficking patterns were confirmed by in vitro activation of T lymphocytes results in synthesis of the ␣-chain and experiments. We next wished to determine whether any functional up-regulation of the ␤- and ␥-chains to form the high affinity, changes occurred in these lymphocytes as a consequence of their heterotrimeric IL-2R complex (29, 30). We determined the per- migration to and residence within granulomas. We first assessed centage of splenic and granuloma lymphocytes which were posi- the state of activation of lymphocytes obtained from spleens and tive for the IL-2R ␣-or␤-chain as a function of weeks p.i. A single granulomas of S. mansoni-infected mice. We used three criteria of group of animals was infected with S. mansoni and serially sacri- activation: expression of the early activation marker CD 69 (25, ficed. IL-2R ␤-chain (CD122) and IL-2R ␣-chain (CD25) expres- 26); expression of a later, more functional activation marker, the sion was examined on splenic and granuloma lymphocytes (Fig. 6, IL-2R (CD25) (27, 28); and production of IL-2. A and B). The data shown in Figure 6 are representative of at least three separate groups of animals that were infected and serially Expression of the early activation Ag CD69 examined at various weeks p.i. The percentage of IL-2R ␤-chain- Lymphocytes were assessed by multiparameter FACS for quanti- positive lymphocytes in the spleen remained relatively constant, tative phenotypic evidence of activation. The kinetics of surface around 2%, during the course of infection. A much different result CD69 expression by spleen and granuloma lymphocytes obtained was observed in the granuloma. At 6.5 wk p.i., ϳ4.5% of the from animals 6 to 26 wk p.i. are shown in Figure 5. Data are granuloma lymphocytes were IL-2R ␤-chain positive. This per- expressed as the fold change in mean channel fluorescence (MCF) centage increased dramatically with increasing weeks p.i., reach- of CD69-stained samples over isotype-matched control samples. A ing 15% at 9 to 10 wk p.i. After 10 wk pi., the percentage of MCF of 1 indicates no significant CD69 expression. At 7 wk p.i., granuloma lymphocytes bearing the IL-2R ␤-chain began to de- granuloma lymphocytes expressed ϳ1.5 ϫ higher levels of CD69 cline and returned to ϳ4%. The patterns of IL-2R ␤-chain expres- than splenic lymphocytes. CD69 expression on granuloma lym- sion paralleled the kinetics of granuloma formation, which also phocytes increased in subsequent weeks p.i. and was maximal 10 peaked at 10 wk p.i. (31). wk p.i. when a 4.5-fold change in MCF was observed. Examina- Expression of CD25 (IL-2R ␣-chain) was also examined at var- tion of CD69 expression on splenic lymphocytes showed a 2-fold ious weeks p.i. CD25 expression on splenic lymphocytes peaked increase in CD69 MCF very early in granuloma formation (6–8 around 7 wk p.i., at which point ϳ25% of the lymphocytes were wk p.i.). The relatively low level of CD69 expression observed on positive for CD25 (Fig. 6B). After 8 wk p.i., CD25 expression on splenic lymphocytes from 7–9 wk p.i. corresponded with the splenic lymphocytes decreased and remained relatively low during higher levels of CD69 expression in granuloma lymphocytes. the remainder of the infection period studied. In the granuloma at From 8 to 10 wk p.i., CD69 expression increased on splenic lym- 7 wk p.i., nearly 50% of the lymphocytes were positive for CD25, phocytes and reached a peak level of expression around 10.5 wk and maximal CD25 expression occurred around 9 wk p.i. After 9 p.i., at the time of maximum granuloma formation. After 11 wk wk p.i., the percentage of CD25ϩ lymphocytes decreased slightly 4134 CHARACTERIZATION OF LYMPHOCYTES IN SCHISTOSOMIASIS

Table III. Percentages of CD25ϩ lymphocytes

Weeks p.i. Spleena Granulomaa p valueb

7–8 21.54 Ϯ 2.81c 48.43 Ϯ 4.3 Ͻ0.01 10–11 12.02 Ϯ 1.14 46.3 Ϯ 4.3 Ͻ0.01 p valued Ͻ0.01 0.35

a Cells obtained from spleen or granuloma. b Significance of difference between spleen and granuloma. Statistics were calculated by paired Student’s t test. c Data compiled from five separate experiments; mean Ϯ SEM. d Significance of difference between wk 7–8 and wk 10–11 spleen or granuloma cells. Statistics were calculated by paired Student’s t test. but remained Ͼ40%, which was ϳ3–4 times the percentages observed in the spleen at the same time. Table III compares the percentages of CD25ϩ lymphocytes from the spleen and granuloma at 7 to 8 wk p.i. vs 10 to 11 wk p.i. At both time points, significantly greater percentages of granuloma lymphocytes were CD25ϩ than were splenic lymphocytes. In addition, Table III shows that while Downloaded from the number of CD25ϩ lymphocytes remains relatively unchanged in the granuloma from 7 to 8 wk vs 10 to 11 wk, in the spleen approximately twice as many lymphocytes were CD25ϩ at7to8 wk p.i. vs 10 to 11 wk p.i., suggesting that lymphocytes may be initially activated in the spleen from 7–8 wk p.i. and then are

recruited to the granuloma. Since both the IL-2R ␤- and ␣-chains http://www.jimmunol.org/ were expressed by granuloma lymphocytes, these cells expressed the high afﬁnity IL-2R that characterizes activated Ag-reactive lymphocytes.

Cytokine production by lymphocytes Our previous study using enzyme-linked immunospot (ELISPOT) FIGURE 7. Splenic and granuloma lymphocytes produce low levels of ␥ analysis showed that high levels of cytokines were produced in the IL-4 and IFN- while splenic lymphocytes produce higher levels of IL-2. Lymphocytes were isolated from spleens and granulomas of acutely in- granuloma (32). Numerous other studies using ELISA assays and

fected animals (10–12 wk p.i.) and stained for intracellular cytokine. ---, by guest on September 27, 2021 in situ hybridization have ascribed similar cytokine production pri- Isotype control; ––, anti-cytokine mAb. marily to Th2 lymphocytes and have suggested that cytokine production has significant effects on both local and systemic immunity (13, 33, 34). Therefore, we wished to determine the exact cytokine production profiles of splenic and granuloma-derived lymphocytes with low levels of IL-2 production in the granuloma lymphocytes. in S. mansoni-infected mice and to assess if the lymphocytes per se Therefore, we examined the survival of lymphocytes in granulo- expressed a Th1 to Th2 profile, as defined by IFN-␥ or IL-4 pro- mas by measuring lymphocyte apoptosis. Table IV shows the per- duction. We used FACS to measure intracellular cytokine produc- centage of apoptotic splenic and granuloma lymphocytes, the ex- tion by cells within the lymphocyte gate from acutely infected pression of IL-2R, and the production of IL-2, measured 10 to 12 (10–12 wk p.i.) mice. Figure 7 shows typical cytokine staining wk after exposure. In the spleen, where IL-2 production was high ϳ patterns from 10 wk p.i. We measured intracellular IL-2, IL-4, and and IL-2R expression was relatively low, 6% of the lymphocytes Ͼ IFN-␥ in freshly isolated splenic and granuloma lymphocytes were apoptotic (n 5). In the granuloma, where IL-2R expression without further in vitro stimulation. The results showed relatively was high and IL-2 protein was low, creating a microenvironment ϳ high levels of IL-2 in the spleen, with significantly lower levels of relatively deficient in IL-2, 50% of the lymphocytes were apo- IL-2 in the granuloma. Low but similar levels of IFN-␥ were pro- ptotic. These data suggested that lymphocytes in the granuloma duced by both splenic and granuloma lymphocytes. IL-4 production was minimally elevated in granuloma lymphocytes compared with splenic lymphocytes. However, when data were pooled from Table IV. Comparison of IL-2, IL-2R, and apoptosis in splenic and multiple experiments (n Յ 5) examining lymphocytes from 10 to granuloma lymphocytes 12 wk p.i., the only statistically significant difference in cytokine production observed was greater IL-2 production by splenic lym- % IL-2Rϩ a b c phocytes compared with granuloma lymphocytes. No statistically Source of Cells IL-2 MCF (CD25) % Apoptosis significant differences were measured for IL-4 or IFN-␥. There- Spleen 3.25 Ϯ 0.38 14.3 Ϯ 2.50d 6.00 Ϯ 2.15d fore, we observed no detectable Th1 or Th2 cytokine production Granuloma 1.83 Ϯ 0.28 47.3 Ϯ 3.54d 53.7 Ϯ 4.26d preference by the unstimulated splenic or granuloma lymphocytes. a Cells were obtained from infected C57BL/6 mice 10–12 wk p.i. and prepared as described in Materials and Methods. Apoptosis of lymphocytes b The fold change in MCF was determined by dividing the mean fluorescence of gated lymphocytes stained for intracellular IL-2 by MCF of gated lymphocytes If the granuloma is an immunoregulatory organelle, it may act stained with an isotype control Ab. through the elimination of Ag-reactive lymphocytes. IL-2 with- c Apoptosis was quantitated by FACS using TUNEL methodology with Cy5- dUTP plus TdT enzyme. A negative control without TdT enzyme but with Cy5-dUTP drawal with resultant IL-2 desaturation is known to induce apo- was used. ptosis. We had observed high levels of IL-2R expression along d Statistical accuracy, Ϯ1%. The Journal of Immunology 4135 were more likely to undergo apoptosis than were lymphocytes in Based on the expression of the activation Ags CD69 and the the spleen. IL-2R, and the production of IL-2, our studies showed that lymphocytes were more highly activated in the granuloma than in the spleen. This preferential activation might be due to selective mi- Discussion gration of the most highly activated cells from the spleen to the granulomas or due to augmented activation in the granuloma per The role(s) of granulomas in schistosomiasis have been the focus se. Since cells demonstrating high levels of CD69 expression were of intense study. Many functions have been ascribed to this lesion. never found in the spleen, the latter explanation is favored. These On one hand, granulomas may play a protective role by seques- data further suggested that lymphocyte activation was being reg- tering hepatotoxins secreted from eggs (35, 36). On the other hand, ulated in the granuloma. The increased levels of lymphocyte ac- the granulomatous response is a cell-mediated inflammatory re- tivation observed in the granuloma along with the presence of sponse that may result in tissue fibrosis and pathology (37–40). granuloma lymphocytes in the S phase but not in G2/M suggested Indeed, our earlier studies in athymic mice (41) have shown that that granuloma lymphocytes may be more susceptible to apoptosis, immunopathology is actually less in athymic mice than in het- since prior activation and cell cycle entry are known criteria for erozygotes and thymic-reconstituted mice. Additional studies have apoptosis (46–48). In this context, the higher local Ag concentra- shown that the granuloma may be essential for conveying viable tion and greater resultant T cell activation in the granulomas may eggs through tissues to complete the parasite life cycle (42). In this be the stimulus for this apoptosis. study, we propose an additional potential role of the granuloma The host immune response to S. mansoni infection has been may be to function as an immunoregulatory antipathology or- shown to be a T cell-dependent process (1, 2, 24, 49). Classically, Downloaded from ganelle. As an immunoregulatory organelle, the granuloma may the host initially responds with a Th1-type response which has function to both activate and/or destroy Ag-reactive cells during been shown to be directed against early stages of the parasite and the course of S. mansoni infection. Thus the granuloma may be a to be important for the induction of cell-mediated protective im- site of activation and immunopathogenesis or a site for inactivation munity to S. mansoni (50–52). As the disease progresses, schis- of potentially immunopathogenic cells. In the latter context one of tosomal SEA become recognized by specific granuloma forming ϩ the primary immunoregulatory roles of the granuloma would be to CD4 T lymphocytes (53, 54). Disease progression is also char- http://www.jimmunol.org/ sequester and/or eliminate Ag-reactive lymphocytes in an attempt acterized by an increasing dominance of Th2 cytokines (IL-4, to protect host tissues from the pathologic consequences of over- IL-5, granulocyte-macrophage CSF, IL-10) relative to Th1 (IFN- production of inflammatory cytokines by Ag-reactive lympho- ␥)-associated cytokines. This transition has been attributed to a cytes. Our findings support such a hypothesis. change in Th1 to Th2 lymphocyte dominance (12, 13, 55, 56). The Our initial studies were aimed at defining the site of generation balance of Th1 to Th2 cytokines is important in the regulation of of SEA-reactive lymphocytes. While it is true that granulomas pathology, in particular granuloma formation and hepatic fibrosis form around eggs located in the liver, schistosome Ag and circu- (12, 13, 49, 55, 57–61), and may have systemic consequences as lating immune complexes exist in the bloodstream and Ag-reactive well (33, 34, 62, 63). lymphocytes can be found in the spleen (43–45). Therefore, it is Other recent studies which have suggested non-CD4ϩ sources by guest on September 27, 2021 possible that Ag-driven cell division could occur at any site. When of IL-4 (64, 65) underscore the importance of precisely defining spleen cells from naive animals were examined, Ͼ98% of the lym- the cellular source(s) of cytokines in the particular experimental phocytes were resting in the G0/G1 stage of the cell cycle (data not model being examined. In the current study we assessed lympho- shown). Our cell cycle kinetics data obtained from S. mansoni- cyte cytokine production on a single cell basis using FACS to infected animals showed that ϳ40% of splenic and granuloma precisely define the cellular source(s) of the reported Th1 and Th2 lymphocytes were in S phase but only splenic lymphocytes were cytokines at the level of 99% accuracy. We examined spleen and capable of reaching the G2/M phase of the cell cycle. These results granuloma lymphocytes from acutely infected animals, immedi- implied that lymphocytes were undergoing division in the spleen ately upon recovery, without further in vitro stimulation in an at- but not in the granuloma. The absence of lymphocytes in the G2/M tempt to ascertain the true in vivo cytokine profiles as closely as phase of the cell cycle in the granuloma suggested that the gran- possible. Our findings showed that the lymphocytes per se were uloma microenvironment was not conducive to successful cell di- not responsible for the reported Th2 cytokine dominance found in vision. Cells which were in S phase in the granulomas might either granulomas in S. mansoni-infected mice. However, we did observe leave the granuloma to complete division elsewhere or may follow a dominant Th2 cytokine profile in a nonlymphocyte population, a pathway of development which preferentially leads to their elim- and the results of those studies are the subject of a different report ination, perhaps through Ag-driven cell death (apoptosis). (32). The studies that have reported Th1 to Th2 cytokine domi- If the granuloma is a regulatory organelle, it might influence nance in lymphocytes employed artificial immunization models pathology by locally sequestering and destroying pathogenic cells and/or in vitro stimulation in their cytokine assays. The signifi- or by promoting the development of tolerogenic cells, which cance of the potential for Th2 cytokine production after in vitro would leave the granuloma and act peripherally. Therefore, we (12) stimulation and the strength of the analogy between findings examined the recruitment and migration of lymphocytes to the based on the lung and liver granuloma models (66, 67) and in vivo granuloma by means of in vivo and in vitro trafficking studies. Our events occurring as a consequence of infection, are problematic. trafficking data showed the preferential recruitment of SEA-reac- In our determinations, the profiles of cytokine production by tive splenic lymphocytes to the granulomas in an antigenically splenic and granuloma lymphocytes were very similar with the specific manner. Since the circulation time in a mouse is less than exception of IL-2 production. Granuloma lymphocytes appeared to 4 s, we were unable to assess directly the ability of activated lym- be relatively deficient in IL-2 production when compared with phocytes to reenter the periphery or to undergo multiple recircu- spleen-derived lymphocytes. IL-2 withdrawal with resultant IL-2R lation cycles in vivo. However, our in vitro studies confirmed that desaturation is known to be one mechanism whereby apoptosis is cloned SEA-reactive lymphocytes migrated into granulomas, were induced. In addition to IL-2 withdrawal, cellular activation and specifically retained within granulomas, and subsequently mi- entry into the cell cycle are prerequisites for apoptosis (68–70). grated out of SEA granulomas. Since we initially observed a relative deficiency of IL-2 in the 4136 CHARACTERIZATION OF LYMPHOCYTES IN SCHISTOSOMIASIS granuloma accompanied by higher levels of IL-2R expression and 18. Doughty, B. L., and S. M. Phillips. 1982. Delayed hypersensitivity granuloma activation, as well as the lack of successful lymphocyte division in formation around Schistosoma mansoni eggs in vitro. I. Definition of the model. J. Immunol. 128:30. the granuloma, we subsequently examined lymphocyte apoptosis 19. Linette, G. P., P. J. Lammie, and S. M. Phillips. 1987. Allogeneic substitution for as a possible mechanism whereby the granuloma might regulate nominal antigen-specific T cell clone reactivity in schistosomiasis. Immunology cell survival and subsequent immunopathology. Our initial studies 57:567. 20. Pomeranz, H. D., J. P. Rothman, and M. D. Gershon. 1991. Colonization of the clearly showed that a higher percentage of granuloma lymphocytes post-umbilical bowel by cells derived from the sacral neural crest: direct tracing were apoptotic than splenic lymphocytes. We are currently inves- of cell migration using an intercalating probe and a replication-defective virus. tigating the possibility that cytokine production may effect subse- Development 111:647. 21. Toes, R. E., I. M. den Haan, M. C. Feltkemp, R. I. Blom, C. I. Melief, quent apoptosis by specific subpopulations of T cells. E. Claassen, and W. M. Kast. 1993. In vivo detection of fluorescent tumor- In summary, these data suggest that the granuloma recruits and specific cytotoxic T cell clones. J. Immunol. Methods 163:23. activates lymphocytes. Moreover, the granuloma may function as 22. Schmid, I., C. H. Uittenbogaart, and J. V. Giorgi. 1991. A gentle fixation and permeabilzation method for combined cell surface and intracellular staining with an immunoregulatory organelle that regulates Ag-specific lympho- improved precision in DNA quantification. Cytometry 15:12. cyte activation, cytokine production, and apoptosis. Splenic and 23. Gorczyca, W., J. Gong, and Z. Darzynkiewicz. 1993. Detection of DNA strand granuloma lymphocytes did not exhibit a dominant Th2 cytokine breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl trans- ferase and nick translation assays. Cancer Res. 53:1945. production profile in these experiments, which examined cells 24. Chikunguwo, S. M., T. S. Harris, P. H. Brodeur, D. A. Harn, and M. J. Stadecker. without in vitro stimulation. We did observe a nonlymphocyte 1992. The cell-mediated response to schistosomal antigens at the clonal level: population that produced large amounts of Th2 cytokines in the development and characterization of a panel of egg antigen-specific murine T cell clones. Eur. J. Immunol. 22:917. granuloma, and these data will be the subject of a future report. 25. Yokoyama, W. M., F. Koning, P. J. Kehn, G. M. B. Pereira, G. Stingl, Granuloma lymphocytes were more likely to undergo apoptosis J. E. Coligan, and E. M. Shevach. 1988. Characterization of a cell surface-ex- Downloaded from than splenic lymphocytes, and we are investigating this phenom- pressed disulfide-linked dimer involved in murine T cell activation. J. Immunol. 141:369. enon in greater detail to determine whether the granuloma might 26. Ziegler, S. F., F. Ramsdell, and M. R. Alderson. 1994. The activation antigen eliminate resident, potentially pathogenic, lymphocytes by IL-2- CD69. Stem Cells 12:456. dependent, Fas/Fas ligand-mediated apoptotic mechanisms. The 27. Nakanishi, K., S. Hirose, T. Yoshimoto, K. Ishizashi, K. Hiroishi, T. Tanaka, precise mechanism(s) of the induction of apoptosis in SEA-reac- T. Kono, M. Miyasaka, T. Taniguchi, and K. Higashino. 1992. Role and regulation of interleukin (IL)-2 receptor ␣ and ␤ chains in IL-2 driven B-cell growth. tive lymphocytes sequestered in the granulomas and the systemic Proc. Natl. Acad. Sci. USA 89:3551. http://www.jimmunol.org/ implications of that apoptosis are the subjects of ongoing studies. 28. Malek, T. R., R. K. Furse, M. L. Fleming, A. J. Fadell, and Y.-W. He. 1995. Biochemical identity of and characterization of the mouse Interleukin-2 receptor ␤ ␥ and c subunits. J. Interferon Cytokine Res. 15:447. References 29. Minami, Y., T. Kono, T. Miyazaki, and T. Taniguchi. 1993. The IL-2 receptor complex: its structure, function and target genes. Annu. Rev. Immunol. 11:245. 1. Boros, D. L. 1989. Immunopathology of Schistosoma mansoni infection. Clin. 30. Smith, K. A. 1988. The interleukin 2 receptor. Adv. Immunol. 42:165. Microbiol. Rev. 2:250. 31. Bentley, A. G., S. M. Phillips, R. J. Kaner, V. J. Theodorides, G. P. Linette, and 2. Mathew, R. C., and D. L. Boros. 1986. Anti-L3T4 antibody treatment suppresses B. L. Doughty. 1985. In vitro delayed hypersensitivity granuloma formation: hepatic granuloma formation and abrogates antigen-induced interleukin-2 pro- Development of an antigen-coated bead model. J. Immunol. 134:4163. duction in Schistosoma mansoni infection. Infect. Immun. 54:820. 32. Phillips, S. M., A. Zekavat, C. Rumbley, H. Sugaya, B. Hilliard, and J. S. Moore. ␥␦ 3. Iacomini, J., D. E. Ricklan, and M. J. Stadecker. 1995. T cells expressing the 1997. The granuloma is an antigen specific organelle which regulates immuno- by guest on September 27, 2021 T cell receptor are not required for egg granuloma formation in schistosomiasis. pathology in schistosomiasis. J. Allergy Clin. Immunol. 99:S254 (Abstr. 1038). Eur. J. Immunol. 25:884. 33. Actor, J. K., M. A. Marshall, I. A. Eltoum, R. M. Buller, J. A. Berzofsky, and 4. Boros, D. L., R. P. Pelley, and K. S. Warren. 1975. Spontaneous modulation of A. Sher. 1994. Increased susceptibility of mice infected with Schistosoma man- granulomatous hypersensitivity in schistosomiasis mansoni. J. Immunol. 114: soni to recombinant vaccinia virus: association of viral persistence with egg gran- 1437. uloma formation. Eur. J. Immunol. 24:3050. 5. Doughty, B. L., and S. M. Phillips. 1982. Delayed hypersensitivity granuloma 34. Sher, A., P. Florentino, P. Caspar, E. Pearce, and T. Mosmann. 1991. Production formation and modulation around Schistosoma mansoni eggs in vitro. of IL-10 by CD4ϩ T lymphocytes correlates with down-regulation of TH1 cy- II. Regulatory T cell subsets. J. Immunol. 128:37. tokine synthesis in helminth infection. J. Immunol. 147:2713. 6. King, C. L., J. Xianli, C. H. June, R. Abe, and K. P. Lee. 1996. CD28-deficient 35. Phillips, S. M., and D. G. Colley. 1978. Immunologic aspects of host responses mice generate an impaired Th2 response to Schistosoma mansoni infection. Eur. to schistosomiasis: resistance, immunopathology, and eosinophil involvement. J. Immunol. 26:2448. Prog. Allergy 24:49. 7. King, C. L., and T. B. Nutman. 1992. Biological role of T cell subsets in helminth 36. von Lichtenberg, F. 1964. Studies on granuloma formation. III. Antigen Seques- infections. Chem. Immunol. 54:136. tration and destruction in the schistosome pseudotubercle. Am. J. Pathol. 45:75. 8. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. 37. Lukacs, N. W., S. W. Chensue, R. M. Strieter, K. Warmington, and S. L. Kunkel. 1986. Two types of murine helper T cell clone. I. Definition according to profiles 1994. Inflammatory granuloma formation is mediated by TNF-alpha-inducible of lymphokine activities and secreted proteins. J Immunol. 136:2348. intercellular adhesion molecule-1. J. Immunol. 152:5883. 9. Mosmann, T. R., and R. L. Coffman. 1987. Two types of mouse helper T cell clones: implications for immune regulation. Immunol. Today 3:223. 38. Mathew, R. C., and D. L. Boros. 1986. Regulation of granulomatous inflamma- tion in murine schistosomiasis. III. Recruitment of antigen-specific IJϩ suppres- 10. Reiner, S. L., and R. M. Locksley. 1993. The worm and protozoa: Stereotyped sor cells of the granulomatous response by an IJϩ soluble suppressor factor. J. responses or distinct antigens? Parasitol. Today 9:258. Immunol. 136:1093. 11. Stadecker, M. J., and D. G. Colley. 1992. Report from a Symposium: the role of Immunological mechanisms in the induction and regulation of granulomatous 39. Olds, G. R., and A. A. Mahmoud. 1980. Role of host granulomatous response in hypersensitivity to egg antigens of Schistosoma mansoni. Parasitol. Today. murine schistosomiasis mansoni. eosinophil-mediated destruction of eggs. J. 8:218. Clin. Invest. 66:1191. 12. Wynn, T. A., and A. W. Cheever. 1995. Cytokine regulation of granuloma for- 40. Phillips, S. M., and P. J. Lammie. 1986. Immunopathology of granuloma forma- mation in schistosomiasis. Curr. Opin. Immunol. 7:505. tion and fibrosis in schistosomiasis. Parasitol. Today 2:296. 13. Pearce, E. J., P. Caspar, J. M. Grzych, F. A. Lewis, and A. Sher. 1991. Down- 41. Phillips, S. M., J. J. DiConza, J. A. Gold, and W. A. Reid. 1977. Schistosomiasis regulation of Th1 cytokine production accompanies induction of Th2 responses in the congenitally athymic (nude) mouse. I. Thymic dependency of eosinophilia, by a parasitic helminth, Schistosoma mansoni. J. Exp. Med. 173:159. granuloma formation, and host morbidity. J. Immunol. 118:594. 14. Pearce, E. J., and S. L. Reiner. 1995. Induction of Th2 responses in infectious 42. Doenhoff, M., R. Musallam, J. Bain, and A. McGregor. 1978. Studies on the diseases. Curr. Opin. Immunol. 7:497. host-parasite relationship in Schistosoma mansoni-infected mice: the immuno- 15. Bruce, J. L., and M. G. Radke. 1971. I. Cultivation of Biomphalaria glabrata and logical dependence of parasite egg excretion. Immunology 35:771. maintenance of S. mansoni in the laboratory. In Culturing Biomphalaria glabrata 43. Osman, A., R. el Ridi, N. Guirguis, and D. A. Dean. 1994. Identification of and Oncomelania (Gastropoda) for Large Scale Studies of Schistosomiasis. Bio- Schistosoma mansoni antigens recognized by T cells of C57BL/6 mice immu- medical Department, 406th Medical Laboratory, San Francisco, CA, pp. 1–84. nized with gamma-irradiated cercariae. J. Parasitol. 80:421. 16. Phillips, S. M., P. J. Perrin, D. J. Walker, N. G. Fathelbab, G. P. Linette, and 44. Lawley, T. J., E. A. Ottesen, R. A. Hiatt, and L. A. Gazze. 1979. Circulating M. A. Idris. 1988. The regulation of resistance to Schistosoma mansoni by auto- immune complexes in acute schistosomiasis. Clin. Exp. Immunol. 37:221. anti-idiotypic immunity. J. Immunol. 141:1728. 45. Reiner, N. E., R. Kamel, G. I. Higashi, A. el-Naggar, M. Aguib, J. J. Ellner, and 17. Teare, G. F., D. K. Horan, C. Smith, and J. B. Hay. 1991. Long term trafficking A. A. Mahmoud. 1979. Concurrent responses of peripheral blood and splenic of lymphocytes in vivo: the migration of PKH-labeled lymphocytes. Cell. Im- mononuclear cells to antigenic and mitogenic stimulation in human hepatosplenic munol. 134:157. schistosomiasis. J. Infect. Dis. 140:162. The Journal of Immunology 4137

46. Munn, D. H., J. Pressey, A. C. Beall, R. Hudes, and M. R. Alderson. 1996. 59. Kullberg, M. C., E. J. Pearce, S. E. Hieny, A. Sher, and J. A. Berzofsky. 1992. Selective activation-induced apoptosis of peripheral T cells imposed by macro- Infection with Schistosoma mansoni alters Th1/Th2 cytokine responses to a non- phages: a potential mechanism of antigen-specific peripheral lymphocyte dele- parasite antigen. J. Immunol. 148:3264. tion. J. Immunol. 156:523. 60. Chensue, S. W., K. S. Warmington, J. Ruth, P. M. Lincoln, and S. L. Kunkel. 47. Green, D. R., R. P. Bissonnette, J. M. Glynn, and Y. Shi. 1992. Activation- 1994. Cross-regulatory role of interferon-␥ (IFN-␥), IL-4, and IL-10 in schisto- induced apoptosis in lymphoid systems. Semin. Immunol. 4:379. some egg granuloma formation: in vivo regulation of Th activity and inflamma- 48. Van Parijs, L., A. Ibraghimov, and A. K. Abbas. 1996. The roles of costimulation tion. Clin. Exp. Immunol. 98:395. and Fas in T cell apoptosis and peripheral tolerance. Immunity 4:321. 61. Cheever, A. W., Y. Xu, J. G. Macedonia, T. Cox, S. Hieny, and A. Sher. 1992. 49. Chensue, S. W., K. S. Warmington, S. D. Hershey, P. D. Terebuh, M. Othman, The role of cytokines in the pathogenesis of hepatic granulomatous disease in and S. L. Kunkel. 1993. Evolving T cell responses in murine schistosomiasis: Th2 Schistosoma mansoni infected mice. Mem. Inst. Oswaldo Cruz 87(Suppl 4):81. cells mediate secondary granulomatous hypersensitivity and are regulated by ϩ 62. King, C. L., A. Medhat, I. Malhotra, M. Nafeh, A. Helmy, J. Khaudary, CD8 T cells in vivo. J. Immunol. 151:1391. S. Ibrahim, M. El-Sherbiny, S. Zaky, R. J. Stupi, K. Brustoski, M. Shehata, and 50. Lukacs, N. W., and D. L. Boros. 1992. Utilization of fractionated soluble egg M. T. Shata. 1996. Cytokine control of parasite-specific anergy in human urinary antigens reveals selectively modulated granulomatous and lymphokine responses schistosomiasis. IL-10 modulates lymphocyte reactivity. J. Immunol. 156:4715. during murine schistosomiasis mansoni. Infect. Immun. 60:3209. 63. Gazzinelli, R. T., I. P. Oswald, S. L. James, and A. Sher. 1992. IL-10 inhibits 51. Zhu, Y., N. W. Lukacs, and D. L. Boros. 1994. Cloning of TH0- and TH2-type parasite killing and nitrogen oxide production by IFN-␥-activated macrophages. helper lymphocytes from liver granulomas of Schistosoma mansoni-infected J. Immunol. 148:1792. mice. Infect. Immun. 62:994. ϩ 64. Sabin, E. A., and E. J. Pearce. 1995. Early IL-4 production by non-CD4 cells at 52. Mountford, A. P., S. Anderson, and R. A. Wilson. 1996. Induction of Th1 cell- the site of antigen deposition predicts the development of a T helper 2 cell re- mediated protective immunity to Schistosoma mansoni by co-administration of sponse to Schistosoma mansoni eggs. J. Immunol. 155:4844. larval antigens and IL-12 as an adjuvant. J. Immunol. 156:4739. 65. Sabin, E. A., M. A. Kopf, and E. J. Pearce. 1996. Schistosoma mansoni egg- 53. Chikunguwo, S. M., J. J. Quinn, D. A. Harn, and M. J. Stadecker. 1993. The induced early IL-4 production is dependent upon IL-5 and eosinophils. J. Exp. cell-mediated response to schistosomal antigens at the clonal level. III. Identifi- Med. 184:1871. cation of soluble egg antigens recognized by cloned specific granulomagenic murine CD4ϩ Th1-type lymphocytes. J. Immunol. 150:1413. 66. Eltoum, I. A., T. A. Wynn, R. W. Poindexter, F. D. Finkelman, F. A. Lewis, 54. Cai, Y., J. G. Langley, D. I. Smith, and D. L. Boros. 1996. A cloned major A. Sher, and A. W. Cheever. 1995. Suppressive effect of interleukin-4 neutral- Downloaded from Schistosoma mansoni egg antigen with homologies to small heat shock proteins ization differs for granulomas around Schistosoma mansoni eggs injected into elicits Th1 responsiveness. Infect. Immun. 64:1750. mice compared with those around eggs laid in infected mice. Infect. Immun. 55. Chensue, S. W., K. Warmington, J. Ruth, P. Lincoln, M. C. Kuo, and 63:2532. S. L. Kunkel. 1994. Cytokine responses during mycobacterial and schistosomal 67. Wynn, T. A., I. Eltoum, A. W. Cheever, F. A. Lewis, W. C. Gause, and A. Sher. antigen-induced pulmonary granuloma formation: production of Th1 and Th2 1993. Analysis of cytokine mRNA expression during primary granuloma forma- cytokines and relative contribution of tumor necrosis factor. Am. J. Pathol. 145: tion induced by eggs of Schistosoma mansoni. J. Immunol. 151:1430. 1105. 68. Radvanyi, L. G., Y. Shi, G. B. Mills, and R. G. Miller. 1996. Cell cycle pro-

56. Grzych, J. M., E. Pearce, A. Cheever, Z. A. Caulada, P. Caspar, S. Heiny, gression out of G1 sensitizes primary-cultured nontransformed T cells to TCR- http://www.jimmunol.org/ F. Lewis, and A. Sher. 1991. Egg deposition is the major stimulus for the pro- mediated apoptosis. Cell. Immunol. 170:260. duction of Th2 cytokines in murine schistosomiasis mansoni. J. Immunol. 146: 69. Ucker, D. S., L. D. Hebshi, J. F. Blomquist, and B. E. Torbett. 1994. Physiolog- 1322. ical T-cell death: susceptibility is modulated by activation, aging, and transfor- 57. Pearce, E. J., and A. Sher. 1991. Functional dichotomy in the CD4ϩ T cell mation, but the mechanism is constant. Immunol. Rev. 142:273. response to Schistosoma mansoni. Exp. Parasitol. 73:110. 70. Walker, P. R., J. Kwast-Welfeld, H. Gourdeau, J. Leblanc, W. Neugebauer, and 58. Xu, Y. H., J. Macedonia, A. Sher, E. Pearce, and A. W. Cheever. 1991. Dynamic M. Sikorska. 1993. Relationship between apoptosis and the cell cycle in lym- analysis of splenic Th1 and Th2 lymphocyte functions in mice infected with phocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1. Exp. Cell Schistosoma japonicum. Infect. Immun. 59:2934. Res. 207:142. by guest on September 27, 2021