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The Journal of Immunology, 2003, 171: 1859-1867.
Copyright © 2003 by The American Association of Immunologists

Apoptosis by Neglect of CD4+ Th Cells in Granulomas: A Novel Effector Mechanism Involved in the Control of Egg-Induced Immunopathology in Murine Schistosomiasis1

Laura I. Rutitzky2, Gerardo A. Mirkin2,3 and Miguel J. Stadecker4

Department of Pathology, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In infection with Schistosoma mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammation that is mediated by CD4+ Th cells. Compared with CBA mice, C57BL/6 mice develop smaller granulomas composed of cells that exhibit reduced proliferative responses to schistosome egg Ags. In the present study, we investigated CD4+ T cell apoptosis as a possible mechanism that could account for this subdued response. We found throughout the course of several infection weeks a markedly higher proportion of apoptotic CD4+ T cells in granulomas from C57BL/6 mice than in those from CBA mice ex vivo; the apoptosis further increased upon cell cultivation in vitro. Activation-induced cell death or CD8+ T cells failed to account for the enhanced apoptosis as infected Fas-, Fas ligand,- and CD8-deficient mice exhibited similar apoptosis to that seen in wild-type counterparts. However, a strikingly lower IL-2 production by schistosome egg Ag-stimulated C57BL/6 granuloma and mesenteric lymph node cells suggested the possibility of apoptosis due to growth factor deprivation. Indeed, the CD4+ T cell apoptosis was significantly reversed by addition of rIL-2 in vitro, or by injection of rIL-2 in vivo, which also resulted in significant exacerbation of granulomatous inflammation. These findings indicate that apoptosis by neglect can represent a significant means of controlling CD4+ T cells that mediate the immunopathology in schistosomiasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with schistosome helminths causes disease in millions of people throughout tropical regions of the world. The main pathology associated with schistosomiasis is a host granulomatous and fibrosing inflammatory reaction against the parasite eggs, which in the case of the species Schistosoma mansoni takes place in the liver and intestines (1, 2). However, the magnitude of disease varies greatly, both in humans as well as among mouse strains in an experimental model of infection (3, 4). Human disease ranges from a benign symptomless intestinal form to a severe hepatosplenic form that can lead to portal hypertension, gastrointestinal hemorrhage, and death (1, 2). In the mouse, each inbred strain develops pathology of a specific intensity; polar examples are the low pathology C57BL/6 (BL/6) strain, and the high pathology CBA and C3H strains (3).

From studies in the mouse, it has been clearly demonstrated that granuloma formation is dependent on and orchestrated by MHC class II-restricted CD4+ Th lymphocytes specific for egg Ags (5, 6). Granulomas start forming shortly after oviposition at 5 wk postinfection and reach their maximal expression at 7–8 wk postinfection; thereafter, the granulomas elicited by newly arriving eggs are of a smaller size. This down-modulatory process, known as immunomodulation (7), has received considerable attention and may be mediated by a number of different mechanisms, including CD4+ T cell unresponsiveness by way of anergy (8), active suppression (9, 10, 11), anti-inflammatory cytokines (8), and idiotypic networks (12).

By comparison, much less is known about why different mouse strains reach distinct peak levels of immunopathology when challenged with identical parasitic loads. Interestingly, in the low pathology BL/6 strain, an initial, short-lived Th1-type cytokine response to a soluble schistosome egg Ag preparation (SEA)5 readily yields to a Th2-dominated response (13, 14), whereas in the high pathology CBA strain the early Th1 response lingers alongside the developing Th2 response (15). Moreover, CBA and C3H high pathology mice display a prominent CD4+ T cell response against the major Sm-p40 egg Ag (16), whereas low pathology BL/6 mice do not, and preferentially react to other SEA components (17).

Although the different responses to egg Ags or dissimilar cytokine profiles most likely play a decisive role in directing the outcome of the granulomatous inflammation, the possible effector mechanisms used still remain unclear. One of these mechanisms is apoptosis of the CD4+ T cells that mediate the immunopathology. Largely depending on local levels of IL-2, apoptotic T cell death can occur either actively, by receptor-initiated signal transduction (death by instruction or activation-induced cell death (AICD)), which operates by signaling through specific cell surface receptors, or passively, by loss of growth factor(s) that promotes cell survival (death by neglect or by growth factor deprivation) (18, 19, 20, 21). Apoptosis of both kinds in lymphoid cells has been shown to represent a critical means to remove unwanted T cells in context with thymic selection or following clonal expansion induced by Ag. Several pathogens (22, 23, 24, 25, 26, 27, 28), including schistosomes (29, 30, 31, 32), have also been shown to manipulate apoptosis to control host cell death.

In the present study, we examined different immunological parameters of CD4+ T cells present in the egg granulomas and found that in the low pathology BL/6 strain these pathogenic cells exhibited high levels of apoptosis, whereas in the high pathology CBA strain the levels of apoptosis were strikingly lower. We also identified a mechanism that accounts for the observed difference in CD4+ T cell apoptosis between these two polar strains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, infection, and SEA

Female wild-type (WT) BL/6 and CBA/J (CBA) mice as well as Fas-deficient (FaslprB6.MRL), Fas ligand (FasL)-deficient (B6Smn.C3H-Faslgld), and CD8-deficient (B6.129P2-{beta}2mtm1Unc) mice, 6–8 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the Animal Facility at Tufts University School of Medicine. Mice were infected by i.p. injection of 80 cercariae of S. mansoni (Puerto Rico strain), which were obtained from infected Biomphalaria glabrata snails, provided to us by the Biomedical Research Institute (Rockville, MD), through National Institutes of Health/National Institute of Allergy and Infectious Diseases Contract N01-AI-55270. SEA from S. mansoni was purchased from the Biomedical Research Institute and prepared as described before (33).

Assessment of liver histopathology

At 8 wk postinfection, liver samples were fixed in 10% buffered Formalin and processed for routine histopathologic analysis. Sections (5 µm), stained with H&E, were examined for qualitative and quantitative changes. The area of hepatic granulomatous inflammation around schistosome eggs was measured by computer-assisted morphometric analysis using Image-Pro Plus (Media Cybernetics, Silver Spring, MD) software. The lesions were assessed on coded slides by an observer unaware of the experimental setting. A minimum of 15 granulomas per liver section was evaluated.

Cell preparations

Granuloma cells were obtained from livers from 5–15 mice per group and were homogenized in a Waring blender, and granulomas were isolated by several steps of 1 x g sedimentation and extensive washing. Cells in granulomas were freed after enzymatic digestion with 1 mg/ml of collagenase type H, from Clostridium histolyticum (Sigma-Aldrich, St. Louis, MO). For proliferation assays, T cells were enriched by removal of nylon wool-adherent granuloma cells during 45-min incubation at 37°C. In some experiments, CD8+ granuloma T cells were depleted by incubation in the presence of anti-CD8 mAb (clone TIB 211), followed by two cycles of lysis with complement. Less than 1% CD8+ T cells remained present, as determined by FACS analysis. Single cell suspensions from pooled mesenteric lymph nodes (MLN) were prepared by teasing the tissues in complete RPMI medium (34). Erythrocytes were lysed with Tris ammonium chloride buffer (pH 7.2) for 15 min on ice. All preparations were composed of >95% viable cells, as determined by trypan blue exclusion.

Cell culture supernatants and cytokine determinations

Bulk cell suspensions (5 x 106 cells/ml) from pooled hepatic granuloma or MLN were incubated in the presence or absence of 20 µg/ml of SEA for 24, 36, and 48 h. At these times, the culture supernatants were removed, filtered, and stored at -36°C until analysis. The cytokines IL-2, IFN-{gamma}, IL-5, and IL-10, present in the supernatants, were measured by ELISA, using Abs, standard cytokines, and protocols obtained from BD PharMingen (San Diego, CA).

Lymphoproliferative responses

Nonadherent granuloma cells (4 x 105 cells/well) plus 4 x 105 syngeneic irradiated splenic APC or bulk MLN cells (2.5 x 105 cells/well) were cultured together with the indicated amounts of SEA in a volume of 200 µl of medium for 96 h. Control cultures excluded either Ag or APC. Proliferating cells were labeled with 0.5 µCi/well of [3H]thymidine (New England Nuclear, Boston, MA) during the last 18 h of culture. Radioactive label incorporation into DNA was measured by liquid scintillation spectroscopy.

Flow cytometry analysis

Single cell suspensions from granuloma and MLN cells were prepared, as described above. Either ex vivo or after 36 h in culture with 20 µg/ml of SEA, cells were washed twice with PBS containing 1% BSA and 0.1% sodium azide (FACS buffer). Washed cells were resuspended at 2 x 107/ml in FACS buffer containing 300 µg/ml rat IgG (Sigma-Aldrich) (blocking buffer) and incubated 15 min on ice to block nonspecific binding of mAb. One million cells (50 µl) were added to test tubes containing 50 µl of a combination of mAb (BD PharMingen) specific for mouse PerCP-labeled anti-CD3 (clone 145-2C11), PE-labeled anti-CD4 (clone GK1.5), PE-labeled anti-CD8 (clone 53-6.7), FITC-labeled anti-Fas (clone Jo2), biotin-labeled anti-FasL (clone MFL3), FITC-labeled anti-CD25 (clone 7D4), and FITC-labeled anti-CD69 (clone H1.2F3), at the optimal dilutions (between 2 and 10 µg/ml) in blocking buffer. Cells were incubated 30 min on ice in the dark and washed twice with FACS buffer. For the detection of FasL, FITC-labeled steptavidin (BD PharMingen) was used as a second step. Cells were fixed with freshly prepared 1% paraformaldehyde (PFA) in PBS.

For the detection of intracellular cytokines in live or apoptotic CD4+ T cells, granuloma cells were cultured at 2 x 106/ml (2 ml final volume) in 24-well plates for 24–36 h, in the presence of SEA (20 µg/ml). Thereafter, cells were harvested, washed, and restimulated for 5 h in the presence of PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin at 2 µg/ml (all from Sigma-Aldrich) to inhibit the secretory pathway. Detection of apoptosis in cultured cells was performed, as described by Ledru et al. (35), with minor modifications. Briefly, granuloma cells were washed twice in FACS buffer, exposed to blocking buffer to prevent nonspecific binding of Abs, and stained at 4°C for 30 min in the dark with PE-labeled anti-CD4 and the DNA intercalating agent 7-amino actinomycin D (7-AAD, 20 µg/ml; Sigma-Aldrich). After labeling, the cells were washed again twice with FACS buffer containing actinomycin D (AD, 20 µg/ml; Sigma-Aldrich) (FACS-AD), and fixed overnight in freshly prepared 2% PFA containing 20 µg/ml of AD (PFA-AD). The labeled, fixed cells were washed with FACS-AD, resuspended for 15 min at room temperature in 50 µl of permeabilization buffer (0.1% saponin in FACS buffer containing 300 µg/ml rat IgG and 20 µg/ml AD, SAP-BLOCK-AD), and further incubated for 30 min at 4°C in the dark with an additional 50 µl of biotinylated rat mAbs against IL-2 (JES6-5H4), IFN-{gamma} (XMG1.2), IL-5 (TRFK4), or IL-10 (JES5-16E3), each added at optimal concentrations between 2 and 5 µg/ml in SAP-BLOCK-AD. After washing twice with SAP-AD, the cells were further incubated for 30 min at 4°C in the dark in 100 µl of FITC-labeled streptavidin at 1 µg/ml in SAP-AD. After two washes in SAP-AD and FACS-AD, cells were fixed in 1% PFA-AD. Controls included cells stained with anti-cytokine Abs without prior permeabilization, and permeabilized cells stained with isotype control Ab. Control intensity never exceeded the first log.

Cells were acquired on a FACSCalibur flow cytometer using CellQuest software version 3.2.1 (BD Biosciences, Franklin Lakes, NJ). The lymphocytes within the granuloma or MLN cell preparations were gated on the basis of forward and side light scatter parameters. Thirty thousand events within this lymphocyte gate were acquired. Data were analyzed using the WINLIST 3D 4.0 software (Verity Software, Topsham, MA).

Detection of apoptotic CD4+ T cells

To detect apoptotic CD3+CD4+ T cells, dispersed lymphoid granuloma or MLN cell populations were first stained with PerCP-labeled anti-CD3 and PE-labeled anti-CD4 mAb. The cells were then fixed with 4% PFA in PBS pH 7.4 (30 min at room temperature), and permeabilized by exposure to 0.1% Triton X-100 in citrate buffer for 2 min on ice. DNA breaks were detected by the TUNEL assay (36) using the fluorescein-labeled in situ cell death kit from Roche Diagnostic (Indianapolis, IN), in accordance with the manufacturer’s instructions.

In vitro and in vivo IL-2 treatments

For the purpose of treatment with IL-2 in vitro, granuloma cells were isolated, as described above. Cells in the process of apoptosis were first eliminated after staining the granuloma cells with 7-AAD (Sigma-Aldrich) (37) at 20 µg/ml for 20 min on ice, followed by two washes with 1x PBS/1% BSA, and sorting on a MoFlo instrument (Cytomation, Fort Collins, CO) on the basis of forward and side scatter characteristics. Nonapoptotic (7-AAD-negative) cells were cultured at a concentration of 2 x 106 cells/ml in medium containing the indicated amounts of mouse rIL-2 (rmIL-2; BD PharMingen) and/or purified anti mouse IL-2 mAb (clone S4B6; BD PharMingen) for 48 h, at which time apoptosis of CD4+ T cells was measured by the TUNEL assay, as described above. For the purpose of assessing the effect of IL-2 in vivo, 5-wk-infected BL/6 mice were injected i.p. with increasing amounts (10,000–100,000 IU) of human rIL-2 (kind gift from Chiron, Emeryville, CA) in 500 µl of PBS, every other day for 2 wk, and every day during an additional week. At 8 wk postinfection, mice were sacrificed, and the effect of the IL-2 treatment on CD4+ T cell apoptosis and hepatic histopathology was assessed, as described above.

Statistical analysis

The ANOVA test was used to determine the statistical significance of the differences in cytokine levels between groups. The Mann-Whitney test was used to determine the statistical significance of the differences in granuloma areas. Differences in the proliferative responses between groups were assessed using multiple linear or nonlinear regression analysis and Student’s t test. FACS data were also analyzed using the Student’s t test. Results differing with a p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dissimilar immunopathology and T cell parameters in schistosome-infected BL/6 and CBA mice

The BL/6 and CBA strains of mice are known polar examples of immunopathology in experimental murine schistosomiasis, for which the magnitude of hepatic granulomatous inflammation is commonly used as an indicator of disease severity. From data of multiple experiments, we confirmed that, at 8 wk postinfection, granulomas are significantly smaller in BL/6 mice than in CBA mice (Fig. 1A). There were no obvious differences in granuloma cell composition, and the amount of collagenized extracellular matrix present in the granulomas was proportional to their size. However, cytometric analysis of dispersed granuloma cells revealed a smaller proportion of CD4+ T cells in BL/6 mice (Fig. 1B); moreover, granuloma cell proliferative responses to SEA stimulation following the necessary depletion of inhibitory adherent cells (38, 39) were also markedly lower in BL/6 mice (Fig. 1C). By comparison, the MLN cells from both strains contained comparable percentages of CD4+ T cells ex vivo (Fig. 1D), but the BL/6 cells still displayed relatively lower proliferative responses to SEA (Fig. 1E). To explain the difference in granuloma size, the lower percentage of granuloma CD4+ T cells, and the disproportionally low proliferative responses displayed by cells from the BL/6 mice, we investigated CD4+ T cell apoptosis as a possible underlying mechanism that could operate differently in the low and high responder strains.



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  		FIGURE 1. Granulomatous inflammation and lymphoid cell parameters in 8-wk-infected BL/6 and CBA mice. A, Granuloma size. Granuloma areas on H&E-stained liver sections were measured by computer-assisted morphometric analysis. Results shown, expressed in mean surface units ± SEM, are from a representative experiment of multiple performed. Seventy-five to one hundred granulomas from 5–10 mice per group were evaluated in this experiment. Granulomas from BL/6 mice were significantly smaller than granulomas from CBA mice (p < 0.001). B, CD3+CD4+ cells in granulomas. Granuloma cells from 8-wk-infected mice were stained ex vivo with anti-CD3 and anti-CD4 mAb, as described in Materials and Methods. Shown are percentages of CD3+CD4+ cells within the lymphocyte gate, which was set on the basis of the cells’ forward and side light scatter characteristics. GC, granuloma cells. C, Proliferation of nonadherent granuloma cells. Adherent cell-depleted granuloma cells from 8-wk-infected mice were incubated with irradiated normal splenic APC and SEA for 96 h, and lymphoproliferation, expressed as means of triplicate determinations ± SEM, was assessed, as described in Materials and Methods. Data shown are from a representative experiment of two using pooled cells from 10–15 mice from each strain. Proliferative responses were significantly lower in BL/6 than CBA mice (p = 0.0175). D, CD3+CD4+ cells in MLN. MLN cells from 8-wk-infected mice were stained ex vivo with anti-CD3 and anti-CD4 mAb, as described in Materials and Methods. Shown are percentages of CD3+CD4+ cells within the lymphocyte gate, which was set on the basis of the cells’ forward and side light scatter characteristics. MLNC, MLN cells. E, Proliferation of bulk MLN cells. Bulk MLN cells from 8-wk-infected mice were incubated with SEA for 96 h, and lymphoproliferation, expressed as means of triplicate determinations ± SEM, was assessed, as described in Materials and Methods. Data shown are from a representative experiment of three using pooled cells from 5–10 mice from each strain. Proliferative responses were significantly lower in BL/6 than CBA mice (p < 0.001).

 
CD4+ T cell apoptosis is significantly higher in schistosome-infected, low pathology BL/6 mice

Analysis by the TUNEL assay of CD4+ T cells ex vivo revealed a significantly higher proportion of apoptotic cells in hepatic egg granulomas from BL/6 mice than those from CBA mice. This difference was consistent during the seventh to ninth infection weeks, which are characterized by vigorous granuloma formation, and persisted through wk 11, during the early phase of immunomodulation (Fig. 2A). The percentage of apoptotic granuloma CD4+ T cells further increased during in vitro cultivation (Fig. 2C). Both ex vivo and cultured MLN from BL/6 mice also contained more apoptotic CD4+ T cells, albeit to a somewhat lesser extent than the granulomas (Fig. 2, B and D). Remarkably, antigenic stimulation in vitro had no influence on the results, as the levels of CD4+ T cell apoptosis in the granuloma or MLN cell cultures were essentially the same in the presence or absence of SEA at several times tested (data not shown).



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  		FIGURE 2. Apoptotic CD4+ T cells in granulomas and MLN from infected BL/6 and CBA mice. Granuloma cells (A) and MLN cells (B) from BL/6 and CBA mice were stained ex vivo with anti-CD3 and anti-CD4 mAb and subjected to the TUNEL assay, as described in Material and Methods, at the indicated times after infection. CD3+CD4+ T cells from BL/6 mice displayed significantly higher levels of apoptosis at all time points (all p values at least <0.05). Data shown are mean percentages ± SEM of 2–11 independent experiments per time point. C, Granuloma cells from 7-, 8-, 9-, or 11-wk-infected BL/6 and CBA mice, and D, MLN cells from 7- or 8-wk-infected BL/6 and CBA mice were cultured in the presence of 20 µg/ml SEA for 36 h, stained with anti-CD3 and anti-CD4 mAb, and subjected to the TUNEL assay, as described in Materials and Methods. CD3+CD4+ T cells from BL/6 mice displayed significant higher levels of apoptosis at all time points in granuloma cells (all p values <0.01) and MLN cells (both p < 0.05). Data shown are mean percentages ± SEM of two to three independent experiments per time point.

 
Fas and FasL are not involved in the apoptosis of CD4+ T cells from BL/6 mice

AICD may be triggered through several receptors, but in peripheral CD4+ T cells this most likely involves Fas (CD95, APO/1), which is a member of the TNFR family (19). Fas is engaged by FasL, which eventually results in the activation of certain caspases and ultimately in the fragmentation of nuclear DNA. However, the failure of SEA to influence CD4+ T cell apoptosis in vitro, together with the lower expression of the activation markers CD69 (40) and CD25 (41, 42) in the granuloma and MLN cells (Table I), suggested that the observed apoptosis in the BL/6 mice was not a manifestation of AICD. Nevertheless, we formally investigated the Fas/FasL pathway, particularly because it had been previously implicated in activation-induced CD4+ T cell apoptosis in context with the schistosome infection (29, 30). Analysis after a 36-h culture period, indeed, revealed a significantly higher proportion of Fas on granuloma CD4+ T cells, and FasL on bulk granuloma cells from the CBA mice with low apoptosis (Fig. 3A). Furthermore, the assessment of CD4+ T cell apoptosis in granulomas from infected Fas-deficient BL/6 lpr mice and FasL-deficient BL/6 gld mice revealed no differences with respect to the WT controls (Fig. 3B), and their level of hepatic granuloma formation was identical as well (Fig. 3C). Taken together, these results clearly indicate that neither Fas nor FasL mediates the apoptosis of CD4+ T cells from the BL/6 mice.


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  		Table I. Ex vivo expression of CD25 and CD69 on CD4+ T cells from 8-wk-infected BL/6 and CBA micea

 


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  		FIGURE 3. Fas and FasL expression in granuloma CD4+ T cells from 8-wk-infected BL/6 and CBA mice, and the role of Fas/FasL on CD4+ granuloma T cell apoptosis and granuloma size in 8-wk-infected BL/6 WT, BL/6 lpr, and BL/6 gld mice. A, Granuloma cells from 8-wk-infected BL/6 and CBA mice were triple stained with anti-CD3, anti-CD4, and anti-Fas, or with anti-FasL mAb after 36 h of culture in the presence of 20 µg/ml SEA, as described in Materials and Methods. CD3+CD4+ granuloma T cells from BL/6 mice expressed significantly less Fas, and total granuloma cells expressed significantly less FasL than those from CBA mice (both p < 0.01). Results are expressed as percentage of cells expressing Fas or FasL, and represent the means ± SEM of three to six independent experiments. B, Granuloma cells from 8-wk-infected BL/6 WT, BL/6 lpr (Fas-deficient), and BL/6 gld (FasL-deficient) mice were stained with anti-CD3 and anti-CD4 mAb and then subjected to the TUNEL assay, as described in Materials and Methods. Shown are percentages of CD3+CD4+ granuloma T cells positive for the TUNEL assay after 36 h of culture in the presence of 20 µg/ml of SEA; they were not significantly different from one another. Dashed line is the negative control for the TUNEL assay performed without TdT enzyme. Results are representative of two independent experiments. C, Granuloma size. Granuloma areas were evaluated, as described in Fig. 1. Results are from a representative experiment of two performed. Twenty to sixty granulomas from five mice per group were evaluated in this experiment. Differences between groups are not statistically significant.

 
Apoptosis of BL/6 granuloma CD4+ T cells is not dependent on CD8+ T cells

CD8+ T cells have been widely implicated in the down-regulation of the immune response and immunopathology in schistosomiasis (9, 10, 11). We therefore considered the possibility that CD8+ T cells could exert their function by promoting apoptosis of the pathogenic CD4+ T cells, a consideration that was further bolstered by the observation that CD8+ T cells were significantly overrepresented in granulomas from BL/6 mice (Fig. 4A). However, in vitro depletion of CD8+ T cells had no effect on the proportion of granuloma CD4+ T cells undergoing apoptosis after 36 h of culture in either the BL/6 or the CBA mice (Fig. 4B). A possible effect of CD8+ T cells on CD4+ T cell apoptosis in the BL/6 mice was further investigated in vivo with the use of {beta}2-microglobulin ({beta}2m) (MHC class I)-deficient mice, in which egg granuloma formation is unaltered (6), even though CD8+ T cells fail to develop (43). The results indicated that the level of granuloma CD4+ T cell apoptosis after 36 h of culture seen in 8-wk-infected CD8-deficient mice was no different from that in the WT BL/6 controls, and in both cases was significantly higher than in the CBA mice (Fig. 4C). These findings demonstrate that CD8+ T cells do not mediate the apoptosis of CD4+ T cells from the BL/6 mice.



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  		FIGURE 4. CD8+ T cells in granulomas from BL/6 and CBA mice and their effect on CD4+ T cell apoptosis in vitro and in vivo. A, Granuloma cells from 8-wk-infected BL/6 and CBA mice were stained with anti-CD3 and anti-CD8 mAb after 36 h of culture in the presence of 20 µg/ml of SEA. There were more CD3+CD8+ T cells in the granulomas from BL/6 mice. B, Granuloma cells from 8-wk-infected BL/6 and CBA mice were, or were not, depleted of CD8+ T cells (CD8 depl.), as described in Materials and Methods, and cultured in the presence of 20 µg/ml of SEA. After 36 h, cells were stained with anti-CD3 and anti-CD4 mAb and subjected to the TUNEL assay, as described in Materials and Methods. The percentage of apoptotic CD3+CD4+ T cells was significantly higher in the BL/6 mice than in the CBA mice (p < 0.01), but neither was significantly affected by the depletion of CD8+ T cells. Results are mean percentages ± SEM of two separate experiments. C, Granuloma cells from 8-wk-infected BL/6 WT, BL/6 {beta}2m knockout (CD8-deficient), and CBA mice were cultured in the presence of 20 µg/ml of SEA for 36 h. Cells were stained with anti-CD3 and anti-CD4 mAb and subjected to the TUNEL assay, as described in Materials and Methods. The percentage of apoptotic CD3+CD4+ T cells was significantly higher in the BL/6 WT mice than in the CBA mice (p < 0.01), but not significantly different from the BL/6 {beta}2m knockout mice.

 
Stimulated granuloma and MLN cells from BL/6 mice produce significantly lower amounts of the Th1-type cytokines IL-2 and IFN-{gamma} than those from CBA mice

The availability of growth factors is essential to sustain cell viability; in the case of T cells, cytokines are critical survival factors that promote the expression of the antiapoptotic molecules Bcl-2 and Bcl-xL, and thus, uphold mitochondrial homeostasis (18). Given that the previous results seemed to exclude AICD as a cause for the enhanced CD4+ T cell apoptosis in BL/6 mice, we investigated whether this could be related to inadequate cell cytokine production. To this effect, granuloma and MLN cells from 8-wk-infected BL/6 and CBA mice were stimulated with SEA, and 24-, 36-, or 48-h culture supernatants were assayed by ELISA for the presence of the Th1-type cytokines IL-2 and IFN-{gamma} and the Th2-type cytokines IL-5 and IL-10. As shown in Fig. 5A, the granuloma cells from BL/6 mice produced significantly lower amounts of IL-2 and IFN-{gamma} than those from the CBA mice, whereas relative differences in the production of IL-5 and IL-10 were not as pronounced. In MLN cells, the production of the Th1-type cytokines IL-2 and IFN-{gamma} by SEA-stimulated cells was dramatically lower in the BL/6 mice relative to the CBA mice; this difference was not seen in the case of the Th2-type cytokines IL-5 and IL-10 (Fig. 5B).



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  		FIGURE 5. Cytokine production by bulk granuloma and MLN cells from BL/6 and CBA mice. A, Pooled granuloma cells from 5–10 BL/6 and 5–10 CBA mice were cultured in the presence of 20 µg/ml SEA for 36 h. Cytokines in the supernatants were measured by ELISA, and levels are expressed as means of triplicate determinations ± SEM. The levels of IL-2 and IFN-{gamma} were significantly lower in BL/6 mice than in CBA mice (p < 0.05); the levels of IL-5 and IL-10 were not significantly different between both strains. B, Pooled MLN cells from 5 BL/6 and 5 CBA mice were cultured in the presence of 20 µg/ml SEA for 24 h to measure IL-2 and for 48 h to measure IFN-{gamma}, IL-5, and IL-10. Cytokines in the culture supernatants were measured by ELISA, and levels are expressed as means of triplicate determinations ± SEM. IL-2 and IFN-{gamma} levels are significantly lower in BL/6 mice than in CBA mice (p < 0.01); the levels of IL-5 and IL-10 were not significantly different between both strains. Background cytokine levels from unstimulated cultures were subtracted.

 
Enhanced apoptosis of cytokine-producing CD4+ granuloma T cells in BL/6 mice

The marked difference in overall cytokine production between the BL/6 and CBA mice was further examined at the single cell level by flow cytometry, for the purpose of assessing how the environment affected the viability of individual lesional cytokine-producing CD4+ T cells. These experiments were conducted under conditions in which granuloma cells were stimulated with SEA for 24–36 h, further incubated for 5 h with PMA/ionomycin in the presence of monensin as a Golgi traffic inhibitor, and triple stained for 7-AAD, CD4, and the various cytokines. The data in Table II show that there was a significantly greater percentage of apoptotic Th1- and Th2-type cytokine-producing CD4+ T cells in the granulomas from BL/6 mice. Interestingly, the greatest difference was observed in the case of IL-2-producing CD4+ T cells, as 77% of these were apoptotic in the BL/6 mice, as opposed to only 37% in the CBA strain.


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  		Table II. Percentage of live and apoptotic cytokine-producing CD4+ T cells in hepatic granulomas from 8-wk infected BL/6 and CBA micea

 
Exogenous IL-2 prevents apoptosis of CD4+ T cells from BL/6 mice in vitro

The low levels of secreted cytokines and the decreased viability of cytokine-producing cells in BL/6 mice are of singular relevance in the case of IL-2, as IL-2 and the related cytokine IL-15 are the main growth factors for T cells (44). To ascertain whether there was a direct linkage between the high proportion of apoptotic CD4+ T cells and the reduced levels of IL-2, we added rmIL-2 to cultures of sorted, live granuloma lymphoid cells from 8-wk-infected BL/6 mice, and determined their level of apoptosis after 48 h of culture. As shown in Fig. 6, rmIL-2 significantly reduced the number of apoptotic CD4+ T cells by over 43%; this was readily reversed in the presence of sufficient neutralizing anti-IL-2 mAb. Importantly, following the addition of IL-2 to the cultures, the number of cells was comparable to those in the unstimulated cultures, suggesting that lymphocyte proliferation was not a significant factor in the reduced apoptosis. Attempts at rescuing apoptotic BL/6 CD4+ T cells in vitro with the use of cytokine-rich supernatants from SEA-stimulated CBA cells (see Fig. 5) were unsuccessful, probably because the final concentration of growth factors, or their stability in solution, was not adequate to exert functional effects (data not shown).



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  		FIGURE 6. rIL-2 added in vitro inhibits apoptosis of granuloma CD4+ T cells from 8-wk-infected BL/6 mice. Sorted, nonapoptotic granuloma cells from 8-wk-infected BL/6 were cultured in the presence or absence of the indicated amounts of rmIL-2 and neutralizing anti-IL-2 mAb for 48 h. Cells were stained with anti-CD3 and anti-CD4 mAb and subjected to the TUNEL assay, as described in Materials and Methods. Results are means ± SD of duplicate determinations in one experiment, which is representative of three with similar results. The percentage of CD3+CD4+ T cells undergoing apoptosis significantly decreases in the presence of 50 U/ml and 100 U/ml of rmIL-2 (both p < 0.05).

 
Exogenous administration of IL-2 significantly reduces CD4+ T cell apoptosis in vivo and exacerbates the egg-induced immunopathology in infected BL/6 mice

To ascertain whether IL-2 plays a consequential role in regulating CD4+ T cell apoptosis in vivo, we injected increasing amounts of rIL-2 i.p. to BL/6 mice during the fifth to eighth week of infection, which is the time of egg granuloma formation and growth. At 8 wk postinfection, the mice were sacrificed and CD4+ T cell apoptosis was measured ex vivo by the TUNEL assay. As shown in Fig. 7A, the proportion of apoptotic CD4+ T cells was dramatically reduced in both the granulomas as well as the MLN from the IL-2-treated mice. Most importantly, this drop in CD4+ T cell apoptosis correlated with a significant increase in granuloma size (Fig. 7B). These findings demonstrate that IL-2, per se, can prevent CD4+ T cell apoptosis, which, in turn, results in the exacerbation of granulomatous inflammation.



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  		FIGURE 7. rIL-2 treatment in vivo inhibits granuloma CD4+ T cell apoptosis and exacerbates the hepatic egg-induced immunopathology. BL/6 mice were injected daily i.p. with progressively increasing doses of human rIL-2 (10,000–100,000 IU/mouse) diluted in PBS for over a period of 3 wk, starting at 5 wk postinfection. A, At 8 wk postinfection, granuloma and MLN cells from IL-2-treated and untreated mice were stained ex vivo with anti-CD3 and anti-CD4 mAb, and subjected to the TUNEL assay, as described in Materials and Methods. The percentage of CD3+CD4+ T cells undergoing apoptosis is markedly decreased in the IL-2-treated mice. Results are means ± SD of duplicate determinations in one experiment, which is representative of two with similar results. B, The extent of granuloma formation in the same mouse groups was evaluated, as described in Fig. 1. Granulomas in the IL-2-treated BL/6 mice were significantly larger than those in the untreated mice (p < 0.001). Results shown, expressed in mean surface units ± SEM, are from a representative experiment of two performed. Seventy to ninety granulomas from five mice per group were evaluated in this experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined CD4+ T cell apoptosis as a possible mechanism underlying the significant difference in immune response and hepatic granulomatous inflammation between the low pathology BL/6 and the high pathology CBA strains of mice during infection with S. mansoni. Our results clearly demonstrated significantly more CD4+ T cell apoptosis in granulomas and MLN from infected BL/6 than in those from CBA mice. These findings indicate that the magnitude of CD4+ T cell-mediated immunopathology correlates inversely with the extent of CD4+ T cell apoptosis. The independence of antigenic stimulation together with a relatively lower expression of Fas, as well as the high affinity receptor for IL-2 (CD25) and the activation marker CD69, suggested that the observed apoptosis in the BL/6 mice was not a manifestation of AICD. In fact, the most likely effector mechanism of AICD in T cells, involving Fas/FasL, was formally excluded by demonstrating the persistence of intact levels of CD4+ T cell apoptosis and immunopathology in infected Fas- or FasL-deficient mice. Another possible pathway of AICD involving TNF (19) was considered unlikely, because TNF was undetectable in granuloma cell supernatants from BL/6 mice (45).

The role of CD8+ T cells as inducers of CD4+ T cell apoptosis was also examined in detail, as these cells were significantly increased in the granulomas from BL/6 mice and, above all, because they have been frequently implicated in the immunomodulation of the immunopathology in schistosomiasis (9, 10, 11). However, the CD4+ T cell apoptosis was similar in intact and CD8+ T cell-depleted granuloma cell cultures, as well as in granulomas obtained from infected WT or CD8-deficient BL/6 mice. These findings indicate that a perforin/granzyme effector mechanism involving CD8+ T cells does not regulate the CD4+ T cell apoptosis either in vitro or in vivo; CD8+ T cells also fail to influence the level of the hepatic egg-induced immunopathology, as previously reported (6).

The strikingly low production of IL-2 by granuloma and MLN cells from BL/6 suggested, instead, that the apoptosis of the CD4+ T cells could be due to growth factor deprivation, as IL-2 represents the most powerful growth factor for T cells (44, 46). This hypothesis was tested and indeed substantiated by demonstrating that exogenous IL-2 significantly reversed the apoptosis both in vitro and in vivo. This reversion was far greater than that seen in the in vitro response to the related cytokine IL-15; IL-4 and IFN-{gamma} had no such effect (data not shown). Most importantly, the decrease in apoptosis mediated by IL-2 also correlated with an exacerbation of granulomatous inflammation in vivo. This observation suggests that the treatment with IL-2 resulted in an increased survival of CD4+ T cells capable of mediating disease. Interestingly, exogenous administration of IL-2 (47) or of anti-IL-2 mAb (48) has been previously reported to respectively exacerbate or ameliorate granuloma formation in the high pathology CBA and C3H mice.

T cell apoptosis has been demonstrated to play a role in the control of pathology associated with a variety of parasitic diseases (22, 23, 24, 25, 26, 27, 28). In schistosomiasis, T cell apoptosis in egg granulomas from schistosome-infected BL/6 mice was first described by Rumbley et al. (49); these authors later postulated that the apoptosis was precipitated by SEA and was mediated by Fas, although an exceedingly high amount of Ag was required to achieve a relatively modest in vitro effect (29). Lundy and Boros (30) similarly reported Fas-mediated splenic CD4+ T cell apoptosis precipitated by FasL-expressing CD5+ B-1a cells, which, in turn, were induced by IL-10 and IL-4 produced by the same CD4+ T cells stimulated with SEA; however, this mechanism of apoptosis could not be demonstrated in granuloma CD4+ T cells. The latter study was performed in high pathology CBA mice, so, even if operative in vivo, the apoptosis appears to be of limited host-protective value to this strain. Finally, to explain the gradual shift of the antischistosome immune response toward the Th2 type, Estaquier et al. (31) suggested that the Th1-type cytokine-producing cells are deleted by AICD in a process involving IL-10. Taken together, these studies leave open the possibility for the existence of more than one means to induce T cell apoptosis, and also suggest that different strains of mice may develop different forms of apoptosis. Of particular importance is a recent study by Carneiro-Santos et al. (32) in human schistosomiasis demonstrating greater T cell apoptosis in patients with mild intestinal schistosomiasis than in those with the severe hepatosplenic form of disease. This study lends relevance to T cell apoptosis as a plausible means to regulating disease severity in vivo.

A question that remains unanswered is what role CD4+ T cell apoptosis plays in setting the level of morbidity associated with the schistosome infection in any given patient or mouse strain. From our findings and those of others, it does appear that active or passive apoptosis can be a useful means to silence some pathogenic CD4+ T cells; however, it is unlikely to be the only mechanism to achieve this effect. Rather, the level of immunopathology in each case seems to be a product of factors influencing the availability, state of activation, and ultimate function of the egg Ag-specific CD4+ T cells. Thus, effective control of pathogenic CD4+ T cells could be attained through deletion by cell death, such as demonstrated in this work, in combination with peripheral T cell unresponsiveness by virtue of anergy and/or active suppression. The design and implementation of strategies to induce and maintain specific CD4+ T cell down-regulation will be of particular importance in those individuals suffering from, or prone to, severe disease.


   Acknowledgments
 
We thank Drs. Ann Marshak-Rothstein, Shyr-Te Ju, and Luk Van Parijs for helpful suggestions and/or critically reading the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI-18919 and AI-48736. Back

2 L.I.R. and G.A.M. contributed equally to this work. Back

3 Current address: Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155, piso 13. (1121) Buenos Aires, Argentina. Back

4 Address correspondence and reprint requests to Dr. Miguel J. Stadecker, Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111. E-mail address: miguel.stadecker{at}tufts.edu Back

5 Abbreviations used in this paper: SEA, schistosome egg Ag; 7-AAD, 7-amino actinomycin D; AD, actinomycin D; AICD, activation-induced cell death; {beta}2m, {beta}2-microglobulin; FasL, Fas ligand; MLN, mesenteric lymph node; PFA, paraformaldehyde; rmIL, mouse rIL; SAP, saponin; WT, wild type. Back

Received for publication March 10, 2003. Accepted for publication June 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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