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(Journal of Leukocyte Biology. 2001;70:737-744.)
© 2001 by Society for Leukocyte Biology

Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni

João A. Pedras-Vasconcelos, Laura Rosa Brunet and Edward J. Pearce

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY

Correspondence: Dr. Edward J. Pearce, Department of Microbiology and Immunology, C5-165 VMC, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell responses of interleukin (IL)-4-/- and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4+ cells isolated from the spleens of infected IL-4-/- mice. Surprisingly, these cells produced significantly less interferon (IFN)-{gamma} and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8+ cells isolated from infected IL-4-/- mice also produced less IFN-{gamma} than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4-/- mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8+ cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.

Key Words: helminth parasite • Th1/Th2 • proliferation • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-4 is considered a crucial cytokine for the development of helper T cell (Th) 2 responses and indirectly to be crucial for the negative regulation of Th1 responses [1 , 2 ]. The acute phase of murine Schistosoma mansoni infection is characterized by the development of a strong Th2 response, with the production of high levels of IL-4, IL-5, IL-10, and IL-13 in response to schistosome egg antigens (SEAs) [3 4 5 ]. These type-2 cytokines are produced by both CD4+ Th2 cells [4 ] and non-T cells [5 , 6 ], and they are induced primarily by parasite egg antigen [7 ]. Th2 responses play a significant role in the classical liver pathology of schistosomiasis by producing IL-13, which promotes hepatic fibrosis [6 , 7 ]. However, the Th2 response also plays an essential host-protective role during infection by inhibiting the excessive production of proinflammatory mediators, which if uncontrolled prove lethal [8 , 9 ]. Death in infected IL-4-/- mice is preceded by a severe cachexia that is in part mediated by reactive nitrogen intermediates [10 ]. CD4+ cells appear also to be involved in the fatal outcome of infection, because depletion of this subset with anti-CD4 monoclonal antibody (mAb) slows weight loss and prolongs survival [8 ]. SEA-specific spleen cell responses in infected IL-4-/- mice are characterized by significantly increased interferon (IFN)-{gamma} production and decreased type-2 cytokine production, suggesting that the CD4+ T cell response defaults in the Th1 direction [8 , 11 ]. This Th1 response occurs independently of IL-12 [12 ].

Here we report the results of studies in which we examined in detail the nature of the T cell response during acute schistosomiasis in IL-4-/- mice, to ascertain whether the lethal outcome of infection is due to the development of a pathologic Th1 response, as has been seen in other acute parasitic infections [13 14 15 16 ]. In addition, we analyzed CD8+ cell function in the IL-4-/- mice. This focus on CD8+ as well as CD4+ cells is based on findings that CD8+ cells from schistosome-infected mice are primed for IFN-{gamma} production and that IL-4 can promote production of IFN-{gamma} by these cells [17 ]. Moreover, recent evidence indicates that infection leads to the development of a schistosome antigen-specific CD8+ cell response [18 ], so it is of interest to ascertain whether changes in the function of these cells can be correlated with disease severity during infection. Our results indicate that in addition to the predicted absence of type-2 cytokines, acutely infected IL-4-/- mice suffer from an impairment in production of T cell-derived IFN-{gamma} after anti-CD3 stimulation. This defect was observed when splenocytes or isolated CD4+ or CD8+ cells were examined. For unseparated spleen cell populations and CD4+ cells, there is a concomitant deficiency in proliferation as detected by both [3H]thymidine incorporation and cell cycle analysis. The lack of a strong CD4+ Th1 response in these animals is consistent with results from other laboratories [19 , 20 ], which suggest that the absence of IL-4 alone is not sufficient for CD4+ cells to default strongly in the type-1 direction in vivo. The defect in CD8+ cell responses in the absence of IL-4 raises the possibility that IL-4 is more important than hitherto credited for the development of strong CD8+ cell responses during some infections.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and experimental infections
Six- to eight-week-old C57BL/6 (B6) mice were purchased from Taconic Farms (Germantown, NY). IL-4-/- B6 mice were bred at Cornell University (Ithaca, NY) [8 ]. Age-matched female mice were each infected by percutaneous exposure to 60–85 cercariae of S. mansoni (NMRI strain) [21 ]. Mice were sacrificed by asphyxiation with CO2.

Antibodies
GK1.5 (anti-CD4), 3.155 (anti-CD8), RA3-3A1/6.1 (anti-B220), MAC1 (anti-CD11b), J11D.2 (anti-HSA), M5/14.15.2 (anti-Ia), and 357.1 (anti-CD28) hybridomas were purchased from the American Type Culture Collection (Rockville, MD). The hybridomas RB6-8C5 (anti-GR1/Ly6-G) and H35.152 (anti-CD8) were kindly provided by R. Coffman (DNAX Research Institute, Palo Alto, CA) and by P. Scott (University of Pennsylvania, Philadelphia), respectively. mAbs were purified from hybridoma supernatants (SNs) by affinity chromatography on protein G.

The following were purchased as fluorescein-isothiocyanate (FITC) conjugates from PharMingen (San Diego, CA): RM-6-5 mAb anti-mouse CD4, RA3-6B2 mAb anti-mouse B220 (CD45R), and R3-34 rat immunoglobulin G1 (control mAb). Phycoerythrin (PE)- and cychrome (Cy)-conjugated 53-6.7 mAb anti-mouse CD8 were also from PharMingen.

T cell purification
Spleens were aseptically removed form wild-type (WT) and IL-4-/- mice carrying 6- to 8-week-old schistosome infections and from uninfected age-matched controls, and single cell suspensions were prepared by pressing the spleens through Falcon 2350 cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ). The resulting cell populations were washed with Dulbecco’s modified Eagle’s medium containing 15 mM HEPES, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (all components from Sigma Chemical Co., St. Louis, MO). Erythrocytes were lysed with Tris-buffered ammonium chloride. Cells that excluded trypan blue were counted and resuspended at 107/mL in CTCM (Dulbecco’s modified Eagle’s medium, 30 mM HEPES, 100 U/mL of penicillin, 100 µg/mL of streptomycin, 5 x 10-5 M 2-mercaptoethanol, and 10% fetal calf serum (all from Sigma). To purify T cells by negative selection, splenocytes were washed once, resuspended in column wash buffer, loaded onto murine T Cell selection columns (R&D Systems, Minneapolis, MN), and incubated for 10 min at room temperature. T cells were eluted, counted, and pelleted. To purify CD4+ cells, T cells were resuspended at 107/mL in a monoclonal antibody (mAb) cocktail containing 1:7 dilutions of H35.152, RA3-3A1/6.1, M5/14.15.2, MAC1, J11D, PK136, and RB-6-8C5 hybridoma SNs and were incubated for 15–30 min at 4°C. Cells were pelleted and washed once in CTCM. Sheep anti-rat immunoglobulin G Dynabeads (Dynal Inc., Lake Success, NY) were added directly to the resuspended cell pellet in a ratio of three to four beads per cell and incubated at 4°C for 30 min on a rocking platform. Labeled cells were removed using a Dynal MPC-6; three rounds of Dynabead addition and magnetic separation were performed. By substituting GK1.5 hybridoma SN for H35.152, the preceding protocol was used to purify CD8+ cells. These negative-selection protocols resulted in 88–95% CD4+ or CD8+ T cells as determined by follow-up analysis with FITC-conjugated RM-6-5 mAb (anti-mouse CD4) and Cy-conjugated 53-6.7 mAb (anti-mouse CD8) using a FacScalibur flow cytometer (Becton Dickinson Instruments). Contamination by the opposite T cell population was always less than 1%.

Assays for cell function
For most experiments, purified T cells were resuspended at 5 x 105/mL in CTCM and cultured at 1 x 105/well for 72 h at 37°C in 5% CO2. Splenocytes were resuspended at 5 x 106/mL in CTCM and cultured at 1 x 106/well under the same conditions. Round-bottom 96-well microtiter plates (Falcon; Becton Dickinson Labware) were coated with 145-2C11 mAb anti-mouse CD3{varepsilon} [depending on the experiment, 0.01–0.5 µg/well in 20 µL of phosphate-buffered saline (PBS); PharMingen] for 2 h at 37°C, after which wells were washed with 100 µL of PBS (Sigma), and 200 µL of cell suspension were added per well. Individual experimental conditions were set up in triplicate, and each experiment was repeated at least once. T cells were stimulated with plate-bound (pb) anti-CD3 mAb, plus or minus recombinant mouse IL-4 (300 U/mL; Genzyme, Cambridge, MA), recombinant human IL-2 (100 U/mL; TSI Washington Labs, Rockville, MD), soluble anti-CD28 mAb (5 µg/mL) or medium alone, and incubated at 37°C in 5% CO2.

Cell SNs were collected at 24 and 72 h after the initiation of the cultures and stored at -20°C until used for cytokine determinations. IL-2 in 24-h culture SN was measured by enzyme-linked immunosorbent assay (ELISA), using mAb JES6-1A12 and biotinylated-mAb JES6-5H4 (PharMingen) as capture and secondary antibodies, respectively. IL-4, IL-5, IL-10, and IFN-{gamma} levels in 72-h SN were determined by cytokine-specific two-site ELISAs, as described previously [17 ]. Each SN was assayed over a series of dilutions.

Thymidine incorporation was used as an indicator of cell proliferation. Splenocyte aliquots were added to 96-well round-bottom plates at 2.5 x 105 cells per well and cultured for 5 days with either medium alone or pb mAb anti-CD3. Twelve to 18 h before the end of the culture, [3H]thymidine (Amersham Corp., Arlington Heights, IL) was added (1 µCi/well). On day 5, cells were harvested, and thymidine incorporation into DNA was determined by liquid scintillation counting; the proliferation index is the ratio of anti-CD3-induced thymidine incorporation to the incorporation in medium alone.

For some experiments, cellular DNA content of cultured cells was analyzed using the flow cytometry method of Nicoletti et al. [22 ]. Briefly, after the removal of 72-h-culture SNs, purified T cells or unfractionated splenocytes were resuspended gently in 100 µL of a 20-µg/mL solution of propidium iodide (PI; Sigma) in hypotonic lysis buffer (0.1% sodium citrate and 0.1% Triton X-100; Sigma). The suspension was incubated overnight at 4°C in the dark and analyzed with a FacScalibur flow cytometer to determine the PI fluorescence of individual nuclei. Contents of replicate wells were pooled, data on 10,000 nuclei were collected, and the cell cycle stages of the nuclei were determined by looking at the intensity of the FL-2 area on a linear scale. In some experiments, this same technique was performed on fresh splenocyte suspensions using 1 x 106 initial cells and 0.5 mL of PI-containing hypotonic lysis buffer.

Apoptosis analysis
Apoptosis-associated changes in the composition and permeability of plasma membranes were monitored using the R&D apoptosis detection kit (which uses Annexin V-FITC and PI to discriminate between viable, apoptotic, and necrotic cells) as per the manufacturer’s instructions. This system was used in combination with 7-amino-actinomycin D staining (7AAD; Calbiochem-Behring, La Jolla, CA) by adapting the method of Philpott et al. [23 ], which flow cytometrically distinguishes viable, apoptotic, and necrotic cells by the increasing binding of 7AAD measured as fluorescence intensity. This triple-staining technique allows the identification of early apoptotic cells that have lost membrane asymmetry (Annexin-V+ [24 ]) and have altered membrane permeability (7AADdim [23 ]) but are still viable (PI-). Briefly 0.5–1 x 106 splenocytes from infected or normal mice (WT and IL-4-/-) were washed twice in PBS and resuspended in 100 µL of Annexin binding buffer (HEPES-buffered saline solution supplemented with 25 mM CaCl2). The cell suspension was stained with Annexin V-FITC (1 µg/mL final), PI (5 µg/mL final), and 7AAD (20 µg/mL final) for 20 min at room temperature in the dark. Volume was brought to 0.5 mL with Annexin binding buffer, and cells were immediately analyzed by three-color flow cytometry using a FacScalibur instrument. Cells were considered apoptotic if they were intact as measured by forward- and side-scatter and if they stained Annexin-FITC+ PI- 7AADdim. To examine apoptosis in specific T cell populations, splenocytes were first stained for CD4 or CD8 surface markers using PE-labeled antibodies and then stained for apoptosis (without PI) and analyzed by three-color flow cytometry.

Statistics
Statistical analysis was by Student’s t-test. P values of <=0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The absence of IL-4 during infection led to diminished splenic enlargement which correlated with decreased lymphocyte proliferation and increased apoptosis. One of the most striking features of schistosomiasis in WT animals is the splenomegaly that develops after the onset of egg production. This splenomegaly was found to be far less prominent in infected IL-4-/- mice. Spleens from infected IL-4-/- mice weighed less (data not shown) and contained fewer nucleated cells than those from infected WT animals (Fig. 1A ). Flow-cytometric analysis of splenocyte suspensions from infected WT and IL-4-/- mice revealed that the percentage of CD8+ cells significantly decreased in the absence of IL-4 (Fig. 1B) . In contrast, the percentages of B220+ cells and of CD4+ cells remained unaffected by the absence of IL-4 (Fig. 1B) , whereas the percentage of NK1.1+ cells was significantly elevated in the infected IL-4-/- versus WT mice. However, because splenomegaly in infected IL-4-/- mice was less prominent, the total cell numbers for all splenic cell types were reduced. These differences intensified as infected IL-4-/- mice neared death (data not shown). No differences were seen in splenic populations between uninfected WT and IL-4-/- animals (Fig 1B) .



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  		Figure 1. Reduced splenomegaly during infection in the absence of IL-4. (A) Numbers of nucleated cells in individual spleens from uninfected (NORMAL) or 7-week-infected WT or IL-4-/- (KO) mice. Results are means ± sd of five spleens from one experiment and are representative of three experiments. S. mansoni-infected WT mice have significantly more nucleated cells in their spleens than similarly infected IL-4-/- mice (*, P<0.005). "106" = 106. (B) Percent of spleen composed of CD4+, CD8+, or B220+ cells as determined by flow cytometry, in erythrocyte-free single-cell suspensions made from two to three pooled spleens from uninfected (NORMAL) WT or IL-4-/- (KO) mice or 6.5- to 7.5-week-infected WT or IL-4-/- animals. In the spleens of infected IL-4-/- mice, the percentage of CD8+ cells was significantly decreased whereas that of NK1.1+ cells was significantly increased (*, P<0.05 in both cases) compared with the spleens of infected WT mice. Results are means ± se of 5–13 experiments.

 
The reduced splenomegaly observed in infected IL-4-/- mice compared with WT mice could be explained by increased rates of spleen cell apoptosis, decreases in splenocyte proliferation, or a combination of both. To measure apoptosis in the spleens of these animals, we examined by flow cytometry the binding of FITC-labeled Annexin V, in combination with the DNA-binding dyes 7-AAD and PI. This triple-staining technique identifies early apoptotic cells that have lost membrane asymmetry (Annexin V+) and have altered membrane permeability (7AADdim) but are still viable (PI-). Flow-cytometric analysis performed immediately ex vivo revealed a consistently greater percentage of splenocytes in the early stages of apoptosis in infected IL-4-/- mice compared with infected WT animals (Fig. 2A ). In addition to slightly increased levels of apoptosis, spleen cells from infected IL-4-/- mice also proliferated less well than WT cells after culture with anti-CD3 mAb (Fig. 2B) . The increased percentage of apoptotic cells correlated well with the decreased splenocyte proliferation index for cultures from infected IL-4-/- mice. This trend was also observed for isolated splenic CD4+ cells but not for CD8+ cells (Fig. 2A 2B) . No differences in proliferation or percentage of apoptotic cells were found between cultures from uninfected WT and IL-4-/- mice (data not shown). These results indicated that differences in T cell proliferation and death contribute to the observed differences in splenic hyperplasia in infected IL-4-/- and WT animals.



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  		Figure 2. Increased splenocyte apoptosis (A) and decreased splenocyte proliferation (B, C) in acutely infected IL-4-/- versus WT mice. (A) The percentages of apoptotic cells (Annexin-FITC+ PI- 7-AADdim) in unfractionated splenocyte populations (Spl) from 7-week-infected WT or IL-4-/- (KO) animals were determined by flow cytometry; to determine the percentages of apoptotic CD4+ or CD8+ cells, PI was replaced by specific PE-labeled mAbs. Results are means ± se of three experiments. Compared with WT splenocytes, a higher percentage of IL-4-/- splenocytes was apoptotic immediately ex vivo (*, P<0.05). (B) Unfractionated splenocyte populations (Spl) or purified CD4+ or CD8+ cells were cultured in the presence of mAb anti-CD3 for 5 days, and proliferation was measured by [3H]thymidine uptake. Results are means ± sd of triplicate wells and are representative of three experiments. IL-4-/- (KO) splenocytes and purified CD4+ cells had significantly lower proliferative responses than did corresponding WT cells (*, P<0.01). (C) Cell cycle analysis of the nuclei from splenocytes (Spl) or purified CD4+ or CD8+ cells, after 72 h of culture with anti-CD3. Cells were lysed under hypotonic conditions, the DNA was stained with PI, and the percentages of apoptotic ("A"), resting ("G0/G1"), or dividing ("S+G2/M") nuclei were determined by flow cytometry. Significantly fewer (*, P<0.05) splenocytes or CD4+ cells proliferated in the IL-4-/- (KO) cultures than in cultures of WT cells. Data are means ± se from five experiments.

 
The increased expression of apoptotic markers ex vivo could be an indicator or result of defective proliferative responses in the spleens of infected IL-4-/- mice, because cells that are more prone to apoptose would likely have undergone activation-induced cell death on restimulation and thus would have been unable to incorporate [3H]thymidine. To better understand the relationship between these two events, we used hypotonic lysis and PI staining [20 ] to examine the cell cycle stage of the nuclei at the end of the culture period. This approach revealed that in infected IL-4-/- mice compared with infected WT mice, the percentages of dividing nuclei (those in S or G2/M phases) were significantly lower in anti-CD3-stimulated splenocyte and CD4+ cell cultures but not in CD8+ cell cultures (Fig. 2C) . No significant differences were observed in the cell cycle profiles for normal IL-4-/- versus WT splenocytes (data not shown). These results suggest that the defect during infection probably lies in the initiation and/or maintenance of cell division, more than in an increased propensity for apoptosis.

Failure of spleen cells from infected IL-4-/- mice to make high levels of IFN-{gamma} after anti-CD3 stimulation
Previous work from our laboratory has shown that splenocyte cultures from acutely infected IL-4-/- animals produce primarily IFN-{gamma} and little IL-5 or IL-10 in culture with SEA [8 ]. Conversely, under comparable conditions, cultures from infected WT animals produce little IFN-{gamma} but high levels of IL-4, -5, and -10 [8 ]. This IFN-{gamma} production led us to hypothesize that CD4+ Th cell responses in S. mansoni-infected IL-4-/- mice default in the Th1 direction. Because we have been interested in the cytokines produced specifically by T cells, we used the polyclonal T cell stimulus mAb anti-CD3 to examine cytokine production by splenocytes from 7-week-infected WT and IL-4-/- animals or from the respective uninfected controls. Splenocytes from uninfected WT and IL-4-/- mice produced little IL-5 and IL-10 and similar levels of IFN-{gamma} and IL-2, and they proliferated to a similar extent (data not shown). Splenocytes from infected WT animals produced high levels of IL-5, IL-10, and IFN-{gamma} (Fig. 3 ). We have shown that this mixed cytokine profile is due to the development of strong concurrent CD4+ Th2 and type-1 CD8+ responses during the acute phase of schistosomiasis in WT mice [17 ]. Contrary to what we observed after SEA stimulation, spleen cells from infected IL-4-/- mice made little IFN-{gamma} in response to anti-CD3. Indeed, of the cytokines measured, only IL-2 was produced in quantity by these cells.



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  		Figure 3. Splenocytes from acutely infected IL-4-/- mice failed to make high levels of IFN-{gamma} after stimulation with anti-CD3. Unfractionated splenocyte populations from either infected WT or IL-4-/- (KO) mice were cultured for 72 h with mAb anti-CD3, and the levels of IFN-{gamma}, IL-2, IL-5, and IL-10 in the SNs were measured. Results are the means ± sd of triplicate wells and are representative of three experiments. All differences between WT and KO cells were highly (P<0.001) significant, except as indicated by the asterisk, where the ; P value was < 0.05.

 
Little production of IFN-{gamma} in vitro by CD4+ cells from infected IL-4-/- mice
To directly address whether the CD4+ cells in infected IL-4-/- animals are strongly Th1-like, we examined the cytokines produced by purified splenic CD4+ cells after T cell receptor (TCR) cross-linking. CD4+ cells from uninfected WT or IL-4-/- animals produced little measurable IL-5, IL-10, or IFN-{gamma} but produced comparable levels of IL-2 (data not shown). Splenic CD4+ cells from infected WT mice were strongly Th2-like, producing high levels of IL-5 and IL-10, low levels of IFN-{gamma}, and some IL-2 (Fig. 4 ). In contrast, CD4+ cells from infected IL-4-/- mice produced only IL-2 in quantity (Fig. 4) . These results mirror those obtained using similarly stimulated whole-splenocyte cultures.



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  		Figure 4. CD4+ cells from infected IL-4-/- mice produced little IFN-{gamma} after stimulation with anti-CD3. Purified CD4+ cells (90% CD4+, <1% CD8+) from either infected WT or IL-4-/- (KO) mice were cultured for 72 h with mAb anti-CD3, and the levels of IFN-{gamma}, IL-2, IL-5, and IL-10 in the SNs were measured by ELISA. Results are the means ± sd of triplicate wells and are representative of three experiments. All differences between WT and KO cells were highly (P<0.001) significant, except as indicated by the asterisk, where the P value was <0.05.

 
In an attempt to enhance IFN-{gamma} production by CD4+ cells from infected IL-4-/- mice, we added exogenous help in the form of recombinant IL-2 or soluble anti-CD28 mAb. For IL-4-/- CD4+ cells, recombinant IL-4 was also added. Each of these additional stimuli increased IFN-{gamma} production, with CD28 ligation being most effective in this regard, pushing IFN-{gamma} levels close to those of equivalently stimulated CD4+ cells from WT mice (Fig. 5 ).



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  		Figure 5. IFN-{gamma} production by CD4+ cells from infected IL-4-/- mice was dependent on costimulation through CD28. Purified CD4+ cells (90% CD4+, <1% CD8+) from either infected WT or IL-4-/- (KO) mice were cultured for 72 h with mAb anti-CD3 and in the presence or absence of IL-2 (100 U/mL), soluble anti-CD28 mAb (5 µg/mL), or IL-4 (300 U/mL), after which the levels of IFN-{gamma} in the SNs were measured. Results are means ± sd of triplicate wells and are representative of three experiments. *, P < 0.05. ND, not done.

 
Impairment of CD8+ cell responses in the absence of IL-4
From previous studies we know that much of the IFN-{gamma} produced when splenocytes from schistosome-infected mice are stimulated with mAb anti-CD3 is CD8+ cell derived [4 ]. Hence, we asked whether the low level of IFN-{gamma} produced by spleen cells from infected IL-4-/- mice reflects a dysfunctional CD8+ cell population. CD8+ cells from infected WT mice were found to produce significantly more IFN-{gamma} than do CD8+ cells from infected IL-4-/- animals (Fig. 6 ). This impaired IFN-{gamma} production was not due to decreased autocrine help from IL-2 because CD8+ cells from IL-4-/- mice produced IL-2 at levels comparable with those produced by WT CD8+ cells (Fig. 6) . Moreover, the difference in IFN-{gamma} levels was not due to a difference in the activation threshold of CD8+ cells from infected WT and IL-4-/- mice, because CD8+ cells from WT mice made significantly more IFN-{gamma} regardless of the concentration of mAb anti-CD3 used for stimulation. Attempts to enhance IFN-{gamma} production using exogenous IL-2, IL-4, and soluble anti-CD28 mAb, although partially successful, did not bring the production of this cytokine to WT levels (Fig. 7 ). These results suggest that S. mansoni-infected IL-4-/- mice have impaired CD8+ IFN-{gamma} responses during the acute phase of disease.



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  		Figure 6. Impaired IFN-{gamma} production but normal IL-2 production by CD8+ cells from infected IL-4-/- mice. CD8+ cells were purified to >93% (<1% CD4+ cells) by negative selection, from either infected WT or IL-4-/- (KO) mice, and cultured for 72 h with mAb anti-CD3 at the concentrations shown. Levels of IFN-{gamma} and IL-2 in the SNs were measured by ELISA. Results are means ± sd of triplicate wells and are representative of two experiments. *, P < 0.05.

 


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  		Figure 7. IFN-{gamma} production by CD8+ cells from infected IL-4-/- mice failed to reach WT levels when exogenous help was provided. Purified CD8+ cells from either infected WT or IL-4-/- mice were cultured for 72 h with mAb anti-CD3 (0.5 µg/mL), and in the presence or absence of IL-2 (100 U/mL), IL-4 (300 U/mL), or soluble anti-CD28 mAb (5 µg/mL), and the levels of IFN-{gamma} in the SNs were measured. Results are means ± sd of triplicate wells and are representative of three experiments. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In WT mice infected with S. mansoni, responding CD4+ T cells are strongly Th2-like during the acute stage of the disease. We expected that in the absence of IL-4, the infection-induced CD4+ T cell response would default in the type-1 direction. However, our data show that, along with the anticipated failure to produce high levels of type-2 cytokines, T cells from acutely infected IL-4-/- mice were less capable that cells from infected WT animals of producing IFN-{gamma} after TCR ligation. This defect was observed when we examined unfractionated splenocytes, purified CD4+ cells, and purified CD8+ cells. For both the unfractionated splenocytes and purified CD4+ cells, we noted a concomitant deficiency in proliferation as detected by [3H]thymidine incorporation and cell cycle analysis.

Although we have not measured cell proliferation in vivo, the decreased spleen hyperplasia observed in infected IL-4-/- compared with WT mice (Fig. 1) suggests that the impaired proliferative responses detected in vitro likely mirror the in vivo situation in infected IL-4-/- animals. The slightly increased percentage of apoptotic splenic cells (Fig. 2) could be a reflection of an in vivo cell cycle block [25 , 26 ]. A combination of impaired proliferative responses and slightly increased rates of apoptosis (Fig. 2) could result in the substantial differences in spleen size and cell numbers that are observed between infected IL-4-/- and WT mice.

It is generally thought that Th1 response suppression during schistosomiasis results from endogenous IL-10 production [27 , 28 ]. However, NO and IFN-{gamma} produced during infection in the early phase of the egg-induced immune response could also play an autoregulatory role in Th1 response modulation, although their contributions may not be as obvious as that of IL-10. IFN-{gamma} can regulate in vivo proliferation and apoptosis of CD4+ Th1-like cells during Mycobacterium bovis BCG infection [29 ], and NO has been implicated in various infections as an inhibitor of Th1 responses [30 , 31 ]. NO itself seems not to affect the generation of type-2 responses and in some cases might instead favor them [30 31 32 ]. Possibly then, diminished T cell responses in infected IL-4-/- mice could reflect extensive autoregulation by IFN-{gamma} and/or NO. It is interesting that WT mice treated with aminoguanidine (an inducible nitric oxide synthase inhibitor) from day 35 of infection suffer exacerbated morbidity and the course of fatal disease in infected IL-4 mice is accelerated by the same treatment [33 ]; both results are consistent with NO playing a protective autoregulatory role. Additionally, we have recently shown that NO produced in culture, when splenic cells from infected IL-4-/- mice are activated with anti-CD3, plays a role in suppressing CD4+ and CD8+ cell proliferation (E. A. Patton, A. C. LaFlamme, J. Pedras-Vasconcelos, and E. J. Pearce, unpublished results). The finding that isolated CD4+ cells from infected IL-4-/- mice are less capable that those from infected WT mice of proliferating in response to anti-CD3 (Fig. 2) suggests either that they themselves are making NO or that additional factors, probably related to suboptimal costimulation through CD28 (Fig. 5) , are also participating. We assume that the low levels of antigen-driven IFN-{gamma} produced by spleen cells from infected IL-4-/- mice are greater than those produced by splenocytes from infected WT mice [8 ] due to the combination of appropriate costimulation and the absence of IL-10 in the cultures. Alternatively, other non-T cell subsets could be contributing IFN-{gamma} in this setting, with natural killer cells, splenic percentages of which increase in infected IL-4-/- mice (Fig. 1) , being a possible source.

Recent in vitro studies by several groups [34 , 35 ] indicate that epigenetic factors control the differentiation of naive CD4+ T cells into strong IFN-{gamma} or IL-4 producers. These factors require cell division to manifest themselves because differentiation of CD4+ cells into strong cytokine producers has been found to be blocked by cell cycle inhibitors [34 ]. The same cell cycle inhibitors did not appear to affect the generation of strong IL-2 producers, which might account for the discrepancy between IL-2 levels and cell division in cultures of cells from infected IL-4-/- mice; it is possible for the Th cells to produce IL-2 while producing little IFN-{gamma} due to a cell cycle block. On a similar note, in one study, production of IL-4 has been suggested to be required for the production of high levels of IFN-{gamma} [35 ], possibly by enhancing the proliferation of individual IFN-{gamma}-producing cells. Additionally, when the lineage relationship between naive CD4+ cells and Th1 and Th2 cells was assessed, it was found that both effector types necessarily express IL-4 early after activation [36 ]. One interpretation of this observation is that IL-4 can play an important role in the early clonal proliferation of all CD4+ cells.

Although there is evidence to support the view that disease development in infected IL-4-/- mice is caused by the unregulated production of reactive nitrogen and oxygen intermediates [10 ], it is also possible that the diminished CD4+ cell response in these animals leads to altered regulation of granuloma formation and antibody production. Because granulomas and antibodies play important roles in protecting hepatic tissue from the toxic effects of molecules released from eggs [37 , 38 ], diminished Th cell function would be expected to increase the potential for hepatic damage during infection. In keeping with this view, one of the most notable consequences of inhibiting NO production in infected IL-4-/- mice is severe liver damage [33 ].

CD8+ cells also show defective IFN-{gamma} production in infected IL-4-/- mice (Fig. 6) , but unlike the CD4+ cells, levels of proliferation and IL-2 production by CD8+ cells from infected WT and IL-4-/- mice are similar. Nevertheless, there appears to be a high attrition rate of splenic CD8+ cells in infected IL-4-/- mice, with the already low percentage of CD8+ cells (Fig. 1) decreasing further as animals near death (J. Pedras-Vasconcelos and E. J. Pearce, unpublished results). This decrease is not seen with CD4+ cells, which suggests that CD4+ and CD8+ cells are affected differently by the absence of IL-4 during infection. Defects in IFN-{gamma} production have been observed during other chronic infections in IL-4-/- mice (i.e., mice infected by Toxoplasma gondii [39 ] and Candida albicans [40 ]). Considering the intracellular nature of both of these pathogens, the deficiency in IFN-{gamma} production in IL-4-/- mice could be due at least in part to problems with CD8+ cells. Thus, IL-4 might play a more important role in the generation of strong CD8+ cell responses during some types of infections than previously appreciated.

Previous work from our laboratory has shown that IL-4 can help CD8+ cells to produce IFN-{gamma} on stimulation with anti-CD3 [17 ]. Clearly IL-4 is unavailable in the infected IL-4-/- mice, which might result in suboptimal help for the induction of a CD8+ cell response. The lack of IL-4 during the ontogeny of an immune response might also increase the susceptibility of CD8+ cells to regulation by inflammatory mediators like tumor necrosis factor {alpha} (TNF-{alpha}) [41 ] and NO [42 ]. Recently, we showed that CD8+ cell proliferation was severely impaired when unfractionated spleen cells from infected IL-4-/- mice were activated by anti-CD3 and that CD8+ cell proliferation and IFN-{gamma} production could be increased by including an inorganic nitric oxide synthase inhibitor in the culture medium (Patton et al., unpublished data). Inhibition of CD8+ cell proliferation by NO in vivo might contribute to the increased attrition of CD8+ cells in IL-4-/- mice during the final stages of schistosomiasis. The fact that purified CD8+ cells from infected IL-4-/- mice proliferate normally (Fig. 2) supports the view that a mediator produced by another cell type is responsible for the inhibition of proliferation of this cell type when these cells remain in the splenic environment. The failure of isolated IL-4-/- CD8+ cells to produce WT levels of IFN-{gamma} indicates that non-IL-4 factors present in the splenic environment, in combination with a reduction in NO levels, can help CD8+ cells produce IFN-{gamma}.

In summary, the current study shows that S. mansoni-infected IL-4-/- B6 mice exhibit impaired splenic T cell responses. The initial expectation that, in the absence of IL-4, infection would lead to a strong type-1 response proved to be incorrect inasmuch as CD4+ cells from infected IL-4-/- mice, although qualitatively type 1, produced less IFN-{gamma} than WT CD4+ Th2 cells on anti-CD3 stimulation. Thus, in specific situations, IL-4 might be more important than hitherto credited for the development of strong type-1 immune responses.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant RO1-A132573 to E. J. P. Schistosome life stages were supplied through NIH-NIAID contract NOI-AI-55270. E. J. P. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology. The authors thank Drs. Eric Denkers, Dragana Jankovic, Anne LaFlamme, Sharon McGonigle, and Elisabeth Patton for helpful discussions.

Received April 1, 2001; revised August 10, 2001; accepted August 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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