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The Journal of Immunology, 2001, 166: 5726-5732.
Copyright © 2001 by The American Association of Immunologists

A Role for CD44 in the Production of IFN-{gamma} and Immunopathology During Infection with Toxoplasma gondii1

Sarah L. Blass*, Ellen Puré{dagger},{ddagger} and Christopher A. Hunter2,*

* Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; {dagger} Wistar Institute, Philadelphia, PA 19104; and {ddagger} The Ludwig Institute for Cancer Research, New York, NY 10058


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4+ T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4+ T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-{gamma}. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-{gamma}, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4+ T cells from infected mice resulted in the production of high levels of IFN-{gamma}, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-{gamma} production by CD4+ T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD44 is a glycoprotein found on the surface of many cell types, including lymphocytes and epithelial cells, and expression levels vary depending upon the origin and activation status of the cell (1). To date, CD44 is best recognized as an adhesion molecule that participates in lymphocyte adhesion to inflamed endothelium and tumor metastasis (1), but is not required for immune surveillance by naive lymphocytes (2). Although many cells express high levels of CD44, only cells with activated CD44 have the capacity to bind to CD44’s principal ligand, hyaluronan (HA)3 (3). TNF-{alpha} and signaling through the TCR/CD3 complex have been shown to be involved in the activation of CD44, whereas IL-4 down-regulates CD44 activation (4, 5, 6). HA is a component of the extracellular matrix and is ubiquitously expressed in a high m.w. form (HMW-HA) (1). At sites of inflammation, low m.w. HA (LMW-HA) accumulates via multiple mechanisms: 1) activated hyaluronidases, 2) oxidative degradation, as well as 3) de novo synthesis (7, 8, 9, 10, 11). It has been proposed that the deposition of LMW-HA at sites of inflammation directs effector/memory cell trafficking to these sites (1). However, CD44 has also been demonstrated to provide costimulatory signals for T cell activation (12, 13). For example, stimulation through CD44 has been reported to enhance T cell proliferation (14, 15, 16) and IL-2 production independently of CD28 (12, 13). In addition to T cells, stimulation through CD44 enhances macrophage production of proinflammatory mediators, including IL-12, IL-1{beta}, and TNF-{alpha}, as well as NK cell cytolytic activity (6, 17, 18).

A role for CD44 in the regulation of immune responses in vivo has been shown by studies in which anti-CD44 treatment inhibited the development of collagen-induced arthritis, IL-2-induced vascular leak syndrome, as well as optimal delayed-type hypersensitivity reactions (2, 19, 20). However, the function of CD44 during infection or infection-induced pathology has not been studied. Therefore, we chose to study the role of CyouD44 in the immune response to the intracellular pathogen, Toxoplasma gondii. Resistance to this parasite is dependent on the production of IL-12, which stimulates a strong Th1-typSde response characterized by the production of IFN-{gamma}, the major mediator of resistance (21, 22, 23). However, this protective response can also result in the development of severe immunopathology. For example, oral infection of C57BL/6 mice with large numbers of parasites results in a lethal CD4+ T cell-mediated, IFN-{gamma}-dependent inflammatory response in the ileum (24). Therefore, infection with T. gondii serves as a model system with which to dissect the role of CD44 in the generation of protective immunity as well as infection-induced immunopathology.

The studies presented in this work reveal that infection with T. gondii results in up-regulation and activation of CD44, and treatment of infected mice with anti-CD44 was associated with markedly reduced pathology and enhanced survival. In addition, in vitro studies demonstrate that stimulation through CD44 results in enhanced production of IFN-{gamma} by CD4+ T cells from infected mice, which is dependent on endogenous IL-12. These studies demonstrate that CD44 is not required for resistance to T. gondii, but has an important role in the regulation of IFN-{gamma} production that contributes to immunopathology during this infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and parasites

Female C57BL/6 and CBA/CaJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). For experiments, 6- to 8-wk-old mice were inoculated orally with 100 cysts or i.p. with 20 cysts of the ME49 strain of T. gondii, which had been prepared from the brains of chronically infected CBA/CaJ mice. At the time of sacrifice, the small intestines were removed from infected C57BL/6 mice that had been treated with either rat IgG or anti-CD44 (IM7, rat IgG2b) Ab, fixed in 10% neutral buffered Formalin (Sigma, St. Louis, MO), and embedded in paraffin. Organs were sectioned (5 µm) and stained with hematoxylin and eosin for visualization of pathological changes. Soluble Toxoplasma Ag (STAg) was prepared from in vitro cultured tachyzoites of the RH strain of T. gondii. Purified parasites were suspended in distilled water and repeatedly freeze-thawed. Following centrifugation (20 min, 800 x g; 15 min, 10,000 x g), the Ag-containing supernatant was stored at -70°C. STAg was titrated to determine the optimal concentration for induction of cytokines and was used in these studies at 10 µg/ml.

Reagents

Anti-CD3{epsilon} mAb (145-2C11), anti-CD44 mAbs (IM7, KM81, and IRAWB14), and anti-IL-12p40 (C17.8; obtained from G. Trinchieri, Wistar Institute, Philadelphia, PA) mAb were prepared from hybridoma supernatants. Rat Ig was obtained from Sigma. LMW-HA (ICN Pharmaceuticals, Costa Mesa, CA), and HMW-HA (Kabi Pharmacia, Uppsala, Sweden) were used at 10 µg/ml, unless stated otherwise. Production of cytokines was determined by sandwich ELISA, as previously described (25, 26). Cytokine concentrations were determined from the appropriate standard curves using recombinant cytokines (murine IL-12; Genetics Institute, Cambridge, MA).

Flow cytometric analysis

Splenocytes were stained directly after their isolation using the following primary mAb: PE-conjugated anti-F4/80 (IgG2b) and anti-B220 (RA3-6B2, IgG2a) (Caltag, San Francisco, CA); anti-CD4 (RM4-5, IgG2a), anti-CD8 (53-5.8, IgG1), biotinylated anti-CD62 ligand (CD62L; Mel-14, IgG2a), biotinylated anti-CD44 (IM7, IgG2b) (BD PharMingen, San Diego, CA), and FITC-HA (27, 28). Appropriate isotype control mAb were obtained from BD PharMingen or Caltag and included in each experiment. To block nonspecific binding, cells were incubated for 15 min on ice with 50 µg/ml rat IgG (Sigma) plus 50 µg/ml Fc Block (BD PharMingen) in FACS buffer (PBS, 0.2% BSA Fraction V (Sigma), 4 mM NaN3). Cells were then stained for 30 min on ice with primary mAb. After one wash, appropriate samples were incubated for 30 min with FITC- or PE-conjugated streptavidin (BD PharMingen) or FITC-conjugated HA. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and CellQuest software (BD Biosciences). Samples were gated on leukocytes based on forward and side scatter, and 10,000 events within the lymphocyte gate were acquired for each sample.

Analysis of T cell responses

Spleens were harvested and dissociated into single cell suspensions in complete RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 50 µM 2-ME, 0.1 mM nonessential amino acids, 10 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml fungizone (Life Technologies). Erythrocytes were depleted using 0.83% ammonium chloride, and cells were washed twice in complete RPMI 1640 before further analysis. Splenocytes were plated at 2 x 106 cells/ml in a final volume of 200 µl and incubated at 37°C in 5% CO2 for 48 h. Cultures were left untreated or were stimulated with 10 µg/ml STAg or 1 µg/ml anti-CD3 mAb. Where indicated, anti-CD44 mAb (IM7, 10 µg/ml) or anti-IL-12 mAb (C17.8, 10 µg/ml) were added to cultures, while control cultures were incubated with rat IgG (10 µg/ml). For analysis of purified CD4+ T cells, splenocytes from uninfected and infected animals were stained with anti-CD4 mAb and sorted on a Vantage Cell Sorter (Cancer Center, University of Pennsylvania, Philadelphia, PA). The purity of these cell preparations was 95–97%, and cells were used at a cell density of 5 x 104 cells/well. CD4+ T cells were cultured in anti-CD3-coated plates. Plates were coated with either 0.01 µg/ml or 0.1 µg/ml anti-CD3 in sterile 1x PBS for 2 h at 37°C. Plates were washed with excess sterile 1x PBS three times before cells were added.

Statistical analyses

Two-tailed unpaired Student’s t test was performed using the INSTAT software (GraphPad, San Diego, CA), and p < 0.05 was considered significant. The Fisher’s exact test was used to determine statistical significance of survival data, and p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of CD44 expression and activation status following infection with T. gondii

To characterize the role of CD44 during infection, C57BL/6 mice were infected i.p. or orally and FACS was used to assess levels of CD44 expression and activation by splenocytes during the acute phase of infection. At day 7 after infection, CD4+ and CD8+ T cells, as well as B cells, NK cells, and macrophages, from infected mice all expressed increased levels of CD44 compared with uninfected mice. Fig. 1Go, A and B, shows a representative FACS plot for CD4+ T cells, and similar results were seen in other cell populations. This increased expression of CD44 correlated with decreased expression of CD62L (Fig. 1Go, C and D), consistent with an activated phenotype of CD4+ T cells from infected mice. Furthermore, to assess the activation status of CD44, cells were stained with FITC-HA. Whereas CD4+ T cells from uninfected mice failed to bind FITC-HA, a small percentage of CD4+ T cells from infected mice did bind FITC-HA. Of all the cell populations analyzed, only CD4+ T cells (Fig. 1GoF) and F4/80+ macrophages (data not shown) expressed activated CD44. Further characterization revealed that the population of HA-binding CD4+ T cells expressed low levels of CD62L (Fig. 1GoF). To determine whether HA binding was CD44 mediated, the KM81 anti-CD44 Ab was used. This Ab binds to the HA binding site of CD44 and is used to show specficity of the CD44/HA interaction (29). The addition of KM81 to splenocytes from infected mice inhibited FITC-HA binding (Fig. 1Go, F and H), indicating that the HA binding was a function of CD44 activation status. Another CD44 Ab, IRAWB14, induces conformational changes in CD44, thereby inducing CD44-HA binding (27). In a typical experiment, the addition of IRAWB14 to cells induced 53% of CD4+ spleen cells from infected mice to bind HA vs 26% of CD4+ spleen cells from uninfected mice (data not shown). Together, these data indicate that infection results in the emergence of a subpopulation of CD4+ cells, perhaps Ag specific, that express an effector/memory phenotype (CD62Llow), which bind HA.



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  		FIGURE 1. Up-regulation and activation of CD44 on CD4+ T cells day 7 after infection with T. gondii. Splenocytes from C57BL/6 mice infected orally with T. gondii (100 cysts ME49) were prepared for FACS analysis of CD44 and CD62L expression and CD44 activation, as described in Materials and Methods. Analysis was performed on CD4+ splenocytes, and the data shown are from a representative experiment of three performed with three mice per experimental group (broken lines represent isotype control). A and B, CD44 expression; C and D, CD62L expression; E–H, CD44 activation. Binding of FITC-HA was inhibited by 30-min preincubation with mAb KM81, which inhibits the binding of activated CD44 to HA.

 
CD44 is required for the development of infection-induced immunopathology

Oral infection of C57BL/6 mice with T. gondii results in the development of severe inflammation of the small intestine, associated with death of the host (24). To determine whether CD44 played a role in the development of the immunopathology induced by T. gondii, anti-CD44 mAb was administered in vivo on days 5 and 7 following oral infection. Treatment of infected mice with anti-CD44 resulted in survival of 75% (9/12) of mice, whereas only 7% (1/14) of C57BL/6 mice treated with rat Ig survived until day 15 after infection (Fig. 2GoA). The enhanced survival of mice treated with anti-CD44 was significant at day 8 and all time points thereafter, as determined by the Fisher’s exact test (all p values < 0.05). Analysis of splenocytes and intraepithelial lymphocytes from infected mice treated with anti-CD44 did not reveal any loss of T cells (data not shown); thus, the enhanced survival of anti-CD44-treated mice is not a consequence of CD4+ T cell depletion. These results are supported by those of Brocke et al. (30), which showed that in vivo {alpha}-CD44 treatment reduces inflammation in the CNS, without depleting CD4+ T cells in the periphery. In contrast to oral infection, anti-CD44 treatment before or following i.p. infection of C57BL/6 mice with T. gondii had no effect on survival (data not shown), indicating that CD44 is not required for resistance to T. gondii. Because anti-CD44 treatment enhanced survival of orally infected mice in comparison with control-treated mice, small intestines were harvested for histological analysis. Gross examination revealed that small intestines from rat Ig-treated animals were enlarged, bloody, and inflamed, whereas the small intestines from anti-CD44-treated animals were normal in appearance. Histological analysis of the small intestines revealed that infected C57BL/6 mice treated with rat Ig had large, severe inflammatory infiltrates as well as gross areas of necrosis (Fig. 2GoB). In contrast, infected mice that were treated with anti-CD44 Ab had greatly reduced levels of inflammation and necrosis compared with rat Ig-treated animals (Fig. 2GoC). Together, these results indicate that treatment of infected mice with anti-CD44 antagonizes the development of the inflammatory response in the gut and this is associated with increased survival.



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  		FIGURE 2. CD44 is required for the development of infection-induced immunopathology. A, C57BL/6 mice were infected orally with T. gondii (100 cysts of ME49). Anti-CD44 mAb and rat Ig were administered (200 µg) i.p. on days 5 and 7 after infection. In vivo anti-CD44 mAb treatment resulted in survival of 75% (9 of 12) of mice, whereas 93% (13 of 14) of mice treated with rat Ig died between days 7 and 11 after infection. The data presented are pooled from three experiments, each containing four to five mice per experimental group. B and C, Comparison of immunopathology in the small intestine. C57BL/6 mice were infected orally with T. gondii (100 cysts of ME49). Anti-CD44 mAb (C) and rat Ig (B) were administered (200 µg) i.p. days 5 and 7 after infection. Small intestines were collected at day 10 after infection with T. gondii, fixed in 10% formalin, and stained with hematoxylin and eosin. In vivo treatment with anti-CD44 mAb resulted in reduced inflammation and tissue necrosis in the small intestine in comparison with rat Ig-treated mice, which has been previously described during per oral infection with T. gondii (24 ). Similar results were seen in three different experiments with five mice per experimental group.

 
Regulation of IFN-{gamma} production by CD44

Because administration of anti-CD44 mAb to infected mice inhibited the development of the CD4+ T cell-mediated IFN-{gamma}-dependent pathology in the gut (24), the role of CD44 in the production of IL-12 and IFN-{gamma} from infected mice was assessed. The systemic immune response of orally infected mice was monitored by analyzing serum levels of IL-12p40 and IFN-{gamma} (Fig. 3GoA). Mice infected with T. gondii and treated with anti-CD44 had significantly reduced serum levels of IFN-{gamma} at both 7 (*, p = 0.0027) and 10 days (p = 0.048; data not shown) after infection in comparison with rat Ig-treated mice, but there was no significant difference in the IL-12p40 levels between these experimental groups. IL-12p70 was not detected by ELISA or RIA. In addition, splenocytes from infected mice treated in vivo with anti-CD44 mAb and stimulated ex vivo with anti-CD3 mAb produced significantly reduced levels of IFN-{gamma} in comparison with rat Ig-treated mice (Fig. 3GoB; **, p = 0.0342). Interestingly, splenocytes from mice treated with anti-CD44 mAb and stimulated with STAg produced normal levels of IFN-{gamma}, but in vitro addition of anti-CD44 mAb resulted in a significant reduction in the production of IFN-{gamma} by splenocytes from infected mice treated in vivo with rat Ig (Fig. 3GoC; *, p = 0.0213) as well as CD44 (Fig. 3GoC; **, p = 0.0219). Splenocyte cultures from infected animals have high levels of parasite Ag, which stimulate IL-12 production. Therefore, we hypothesize that in vivo IM7 treatment had no effect on STAg-induced IFN-{gamma} production because the role of CD44 in IFN-{gamma} production is minimal in the presence of high levels of IL-12 and Ag (see Fig. 5Go). Furthermore, the addition of increasing concentrations of LMW-HA, a natural ligand for CD44, to splenocytes from infected C57BL/6 mice resulted in increased levels of IFN-{gamma} when cultured for 4 (data not shown) and 48 h (Fig. 4GoA). This effect was not observed with HMW-HA or splenocytes from uninfected mice that do not express activated CD44 (see Fig. 1Go). These results associate the protective effects of anti-CD44 treatment with a reduction in IFN-{gamma} levels and indicate a role for CD44 in the regulation of IFN-{gamma} production.



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  		FIGURE 3. Effect of in vivo treatment with anti-CD44 on the production of IFN-{gamma} and IL-12 by mice infected with T. gondii. C57BL/6 mice were infected orally with T. gondii (100 cysts of ME49), and anti-CD44 mAb and rat Ig were administered i.p. (200 µg) on days 5 and 7 after infection. IFN-{gamma} and IL-12p40 production were measured by ELISA, as described in Materials and Methods. A, Serum levels of IFN-{gamma} and IL-12 on day 7 after infection. Administration of anti-CD44 mAb treatment significantly reduced (*, p = 0.0027) serum levels of IFN-{gamma}, but did not affect serum levels of IL-12p40. The data presented are pooled from two experiments, each containing three to four mice per experimental group. B, Production of IFN-{gamma} in response to anti-CD3 mAb by splenocytes from infected animals. The addition of 10 µg/ml anti-CD44 mAb in vitro significantly reduced the production of IFN-{gamma} by splenocytes from infected mice treated in vitro with control Ab (*, p = 0.039). In addition, splenocytes from infected mice treated in vivo with anti-CD44 mAb had reduced IFN-{gamma} production in response to anti-CD3 mAb (1 µg/ml) in comparison with splenocytes from mice that received rat Ig (**, p = 0.0342). Results are presented as the means ± SE from three different experiments with three mice per group. Splenocytes from uninfected animals produce 1.8 ± 0.38 ng of IFN-{gamma} in response to anti-CD3 stimulation. C, In recall experiments using STAg (10 µg/ml), the addition of 10 µg/ml anti-CD44 mAb in vitro significantly reduced IFN-{gamma} production by splenocytes from both rat Ig (*, p = 0.0213) as well as anti-CD44 mAb-treated mice (**, p = 0.0219). Results are presented as the means ± SE from three different experiments with three mice per group. Splenocytes from uninfected animals produce 0.69 ± 0.22 ng of IFN-{gamma} in response to stimulation with STAg.

 


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  		FIGURE 5. LMW-HA directly enhances production of IFN-{gamma} by CD4+ T cells from mice infected with T. gondii. C57BL/6 mice were orally infected with T. gondii (100 cysts of ME49), and spleens were harvested 7 days after infection. Splenocytes were incubated with CD4-FITC and sorted on a high-speed FACSVantage SE to give a population of CD4+ T cells that were 97% pure. CD4+ T cells were used at a cell density of 5 x 104 cells/well and cultured with plate-bound anti-CD3 mAb, IL-12, and LMW-HA for 48 h at 37°C. IFN-{gamma} production was measured by ELISA, as described in Materials and Methods. CD4+ T cells from infected animals (closed symbols) produced increased levels of IFN-{gamma} when stimulated with suboptimal doses of plate-bound anti-CD3 mAb (0.01 µg/ml), IL-12 (0.01, 0.1, and 1 ng/ml), and LMW-HA (10 and 100 µg/ml) in comparison with CD4+ T cells from uninfected animals ({square}). Similar results were seen in three different experiments.

 


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  		FIGURE 4. LMW-HA-induced production of IFN-{gamma} is IL-12 dependent. C57BL/6 mice were infected orally with T. gondii (100 cysts of ME49), and spleens were harvested on day 7 after infection and cultured for 48 h. IFN-{gamma} and IL-12p40 production were measured by ELISA, as described in Materials and Methods. A, Splenocytes from infected mice produce IFN-{gamma} in a dose-dependent manner to LMW-HA ({circ}), but not HMW-HA ({square}). Splenocytes from uninfected mice did not produce IFN-{gamma} in response to either LMW- or HMW-HA (data not shown). Similar results were seen in three different experiments with three mice per group. B, The addition of 10 µg/ml anti-IL-12 mAb ({square}) in vitro inhibits LMW-HA-enhanced IFN-{gamma} production by splenocytes from infected mice. Similar results were obtained in three different experiments using three mice per group. C, Splenocytes from either uninfected ({circ}) or infected ({square}) mice did not produce IL-12p40 in response to LMW-HA (0–100 µg/ml). Similar results were obtained in an additional experiment using pooled splenocytes with three mice per experimental group. D, In recall experiments using STAg (10 µg/ml), the addition of 10 µg/ml anti-CD44 mAb in vitro had no effect on IL-12p40 production by splenocytes from both rat Ig as well as anti-CD44 mAb-treated mice. Results are presented as the means ± SE from three different experiments with three mice per group.

 
Previous studies have shown that LMW-HA can stimulate thioglycolate-elicited peritoneal macrophages to produce IL-12 (17), an important cofactor for the production of IFN-{gamma}. To determine whether the ability of LMW-HA to enhance the production of IFN-{gamma} was due to its ability to stimulate the production of IL-12, anti-IL-12 mAb was added in vitro to splenocytes stimulated with LMW-HA. Anti-IL-12 (10 µg/ml) Ab treatment ablated IFN-{gamma} production in response to increasing doses of LMW-HA (Fig. 4GoB). However, the presence of increasing doses of HA did not enhance production of IL-12 (Fig. 4GoC), and anti-CD44 Ab had no effect on STAg-induced production of IL-12 (Fig. 4GoD). One possible explanation for these results is that the IL-12 induced by LMW-HA was consumed by cells that make IFN-{gamma}. Therefore, levels of IL-12 mRNA were analyzed by RNase protection assay. These data demonstrated that LMW-HA does not stimulate increased levels of IL-12p40 mRNA (data not shown). Thus, it appears that under these experimental conditions, HA does not induce production of IL-12, but the ability of HA to enhance production of IFN-{gamma} is dependent on endogenous IL-12. This latter conclusion was supported by experiments in which splenocytes from infected IL-12p40 knockout mice required exogenous IL-12 for HA-induced production of IFN-{gamma} (data not shown).

LMW-HA enhances the production of IFN-{gamma} by purified CD4+ T cells

The studies described above demonstrate a role for CD44 in the regulation of IFN-{gamma} production by splenocytes that is dependent upon IL-12, but do not distinguish whether this is a direct or an indirect effect on T cells. Therefore, a series of experiments was designed to determine whether LMW-HA could directly enhance the production of IFN-{gamma} by purified CD4+ T cells from infected and uninfected mice as well as to determine the role of TCR-mediated stimulation and IL-12 in this process. LMW-HA alone had little effect on the production of IFN-{gamma}, but when added in combination with suboptimal doses of plate-bound anti-CD3 (0.01 µg/ml) and increasing concentrations of IL-12, resulted in the production of high levels of IFN-{gamma} by CD4+ T cells from infected animals, but not uninfected animals (Fig. 5Go). However, it should be noted that in the presence of high concentrations of plate-bound anti-CD3 (0.1 µg/ml) and IL-12 (1 ng/ml), high levels of IFN-{gamma} were produced and the addition of HA did not augment this response (data not shown). In fact, similar results using a T cell clone were recently described (31). Nevertheless, these results demonstrate that the addition of HA to activated CD4+ T cells enhances production of IFN-{gamma} in conjunction with TCR and IL-12 stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, there have been several reports demonstrating that CD44 is a signaling molecule in many cell types and is involved in the regulation of inflammatory responses (32, 33, 34, 35, 36), but nothing is known about its role during infection. The function of CD44 during inflammation has primarily been attributed to a role in the trafficking of leukocytes into local inflammatory sites (1). However, it is now appreciated that CD44 has many other roles in the immune system and has been shown to regulate cytotoxicity (17, 37, 38), enhance production of proinflammatory cytokines and chemokines (17, 37, 38), and provide costimulation for T cells (12, 13). In this study, we demonstrate that infection with T. gondii leads to the emergence of a population of CD62Llow CD4+ T cells, which express an activated form of CD44, and identify a novel role for CD44 in the regulation of IFN-{gamma} production. In addition, these studies address the function of CD44 during infection and demonstrate that although CD44 is not required for resistance to T. gondii, it is required for the development of infection-induced pathology. The studies of Liesenfeld and colleagues (24) demonstrated that the T. gondii-induced pathology in the small intestine is dependent on the presence of CD4+ T cells and IFN-{gamma}. Interestingly, the protective effect of anti-CD44 treatment in this model was associated with reduced production of IFN-{gamma}. Together with our finding that LMW-HA enhanced the production of IFN-{gamma} by CD4+ T cells, these results suggest that the direct inhibition of CD4+ T cell production of IFN-{gamma} is an important component of the antiinflammatory effect of anti-CD44. Although the studies presented in this work do not address whether blockade of CD44 affects the trafficking of T cells during this infection, previous studies have demonstrated that CD44 is required for lymphocyte trafficking into the skin during cutaneous delayed-type hypersensitivity reactions (2), and that anti-CD44 treatment of mice injected i.p. with staphylococcal enterotoxin B blocks the recruitment of Ag-specific V{beta}8+ T cells, but not macrophages and neutrophils, into the peritoneum (39). In the studies presented in this work, preliminary analysis of the effects of anti-CD44 treatment revealed no significant alteration in the numbers of intraepithelial lymphocytes. However, these data have to be interpreted with care, as the infection-induced pathology results in an almost complete loss of gut architecture, and we found it difficult to distinguish intraepithelial and lamina propria lymphocyte populations in this experimental system. However, our preliminary studies (S. L. Blass and C. A. Hunter, unpublished observations) indicate that administration of anti-CD44 following i.p. infection does not alter the cellular composition of the inflammatory cells in the peritoneum, and suggest a limited role for CD44 in the trafficking of inflammatory cells during toxoplasmosis.

The finding that infection with T. gondii leads to the emergence of a population of T cells that express activated CD44 raises several questions about the function of these cells and how CD44 activation is regulated during infection. Presumably, the T cells that express activated CD44 are specific for Ags of T. gondii, but it remains unclear whether these cells are an effector population or have some other role in the immune response to T. gondii. Although the events that lead to the activation of CD44 following infection are uncertain, it is known that TNF-{alpha}, which is produced following infection with T. gondii (40, 41), as well as stimulation through the TCR, results in activation of CD44 (39). Nevertheless, it is clear that infection with T. gondii leads to the priming of CD4+ T cells to produce high levels of IFN-{gamma} when stimulated with LMW-HA in combination with signaling through the TCR and IL-12R. The molecular basis for this effect is not clear, but signaling through CD44 results in the activation of NF-{kappa}B (34, 35), a phosphatidylinositol 3-kinase-dependent pathway that is modulated by protein kinase C (36), as well as tyrosine kinases (42). Several of these pathways have been linked to the production of IFN-{gamma} (43, 44, 45), and other stimuli such as CD28, IL-18, IL-1, and TNF-{alpha}, which can enhance IL-12-mediated production of IFN-{gamma} by T cells (46, 47, 48, 49), also activate NF-{kappa}B. Thus, the ability of CD44 to signal through NF-{kappa}B (34) may provide a mechanism whereby CD44 engagement directly enhances the production of IFN-{gamma}. Regardless, several questions remain about the interactions between the three stimuli (TCR, IL-12, and CD44) that lead to the production of high levels of IFN-{gamma}. It has been well documented that signaling through the TCR leads to the up-regulation and activation of CD44 (4, 39, 50), and IL-12 can also increase surface expression of several activation markers (CD25, ICAM, VCAM) on T cells (51, 52). Whether stimulation of T cells with IL-12 directly regulates the expression and activation status of CD44, or whether stimulation through CD44 leads to increased expression of the IL-12R, are unknown and are the subject of ongoing studies in these laboratories.

The finding that infection with T. gondii leads to the activation of CD44 by CD4+ T cells, that LMW-HA enhances the production of IFN-{gamma} (these studies), and that the Th2 cytokine IL-4 down-regulates activated CD44 (6) raises the question of whether activated CD44 is preferentially expressed by Th1 cells. In addition, because CD44 is involved in trafficking of T cells, it may have a similar role to the P-selectin/P-selectin glycoprotein ligand 1 adhesion molecules, which are involved in the specific recruitment of Th1 cells (53). However, because a Th1-type response is required for resistance to T. gondii (22, 23, 54) and because blockade of CD44 did not result in increased susceptibility to infection, it appears that CD44 is not essential for the development of Th1-type responses. Moreover, preliminary work in these laboratories indicates that CD44 knockout mice infected orally or i.p. survive this infection (our unpublished observations). Therefore, we propose that although CD44 has a role in the production of IFN-{gamma}, it is not essential for the development of Th1-type responses, but may be important in the regulation of their function during inflammation. Further studies are required to determine whether CD44 has different roles in the regulation of Th1- and Th2-type responses. Nevertheless, the studies presented in this work have identified a novel role for CD44 in the regulation of the production of IFN-{gamma}, a cytokine implicated in the pathogenesis of many T cell-mediated inflammatory conditions. The development of immune therapies that block T cell activation and production of cytokines, for example the use of antagonists of the B7/CD28 and CD40/CD40L interactions, has been shown to be useful in various models of autoimmunity and transplantation (55, 56, 57, 58). The finding that blockade of CD44 can antagonize infection-induced, IFN-{gamma}-dependent immune pathology identifies CD44 as a target for the treatment of inflammatory conditions such as arthritis, inflammatory bowel disease, and other autoimmune diseases in which CD44 has been implicated (38, 59, 60, 61, 62).


   Acknowledgments
 
We thank Dr. Jay Farrell and Dr. Phillip Scott for their insightful comments and support during these studies and the preparation of this manuscript. In addition, we also thank J. Moore and C. Pletcher at the University of Pennsylvania Cancer Center Flow Cytometry and Cell Sorting Resource for excellent technical advice and support.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 42334-01 and AI 45813-01 and Center Grant P30 DK50306. S.L.B. is supported by National Institutes of Health Training Grant 5T32 EY07131. Back

2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, School of Veterinary Medicine, 226 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: HA, hyaluronan; HMW-HA, high m.w. HA; LMW-HA, low m.w. HA; STAg, soluble Toxoplasma Ag; CD62L, CD62 ligand. Back

Received for publication September 22, 2000. Accepted for publication February 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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