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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 lympho- cytes 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, g 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-g. 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 1 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 CD41 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 mg/ml streptomycin sulfate, 100 U/ml penicillin, 50 mg/ml gen- 3 25 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, 1,19-dioctadecyl 3,3,39,39-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 Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 4130 CHARACTERIZATION OF LYMPHOCYTES IN SCHISTOSOMIASIS suspensions were filtered through a 100-mm 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 3 106 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 3 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 .90% viable lymphoblasts. The lymphoblasts (2 3 107) were labeled with the red fluorescent vital 3 26 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
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