Role of CD44 in activation-induced cell death: CD44-deficient mice exhibit enhanced T cell response to conventional and superantigens

Robert J. McKallip1, Yoon Do1, Michael T. Fisher1, John L. Robertson2, Prakash S. Nagarkatti1 and Mitzi Nagarkatti1

1 Departments of Microbiology and Immunology and Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298. USA 2 Department of Biomedical Sciences and Pathobiology, Virginia–Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Correspondence to: M. Nagarkatti, Department of Microbiology and Immunology and Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Box 980678, Richmond, VA 23298-0678, USA. E-mail address: mnagark{at}hsc.vcu.edu
Transmitting editor: P. W. Kincade


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cells upon activation are known to up-regulate CD44 expression. However, the precise function of CD44 on activated T cells is not clear. In this report, we demonstrate that signaling through CD44 plays an important role in activation-induced cell death (AICD). CD44 knockout (KO) mice had an elevated in vivo primary and in vitro secondary response to challenge with conalbumin, anti-CD3 mAb and staphylococcal enterotoxin A (SEA), which correlated with reduced AICD when compared to CD44 wild-type mice. In addition, CD44 KO mice exhibited increased delayed-type hypersensitivity response to dinitrofluorobenzene. In a model examining in vitro AICD, splenocytes from CD44 KO mice showed resistance to TCR-mediated apoptosis when compared to splenocytes from CD44 wild-type mice. In addition, signaling through CD44 led to increased apoptosis in TCR-activated but not resting T cells from CD44 wild-type mice without affecting Fas expression. Injection of SEA into mice deficient in CD44 and Fas (CD44 KO/lpr) led to an increased primary response when compared to mice that expressed CD44 but not Fas (CD44 WT/lpr), suggesting that the enhanced response to SEA was dependent on CD44 but not Fas expression. Administration of anti-CD44 mAb into CD44 wild-type mice caused a significant decrease in antigen-specific T cell response. Together, these data implicate CD44 as an important regulator of AICD in T cells. Furthermore, targeting CD44 in vivo may constitute a novel approach to induce apoptosis in activated T cells, and therefore to treat autoimmune diseases, allograft rejection and graft versus host disease.

Keywords: apoptosis, cell surface molecules, transgenic/knockout


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In addition to lymphocyte proliferation and differentiation, cell death by apoptosis plays an important role in the development and maintenance of the immune system. The first example of regulation of the immune system by cell death was described in the education and maturation of immature T cells in the thymus (14). Upon entering the thymus immature T cells go through an intricate process by which self-reactive cells are eliminated by apoptosis, thereby reducing the potential development of autoimmune disorders. Once in the periphery, lymphocytes are regulated by another process, known as activation-induced cell death (AICD) (57). AICD plays a vital role in maintaining immune homeostasis and is characterized by the induction of apoptosis. AICD mainly occurs in hyperactivated T cells or after strong re-stimulation of activated lymphocytes, leading to their clearance. This specific regulation is believed to be critical for preventing the development of autoimmune disorders. The exact mechanism by which AICD is initiated remains unclear, but is believed to take place by signaling through cell surface molecules located on activated lymphocytes. The interaction between Fas and Fas ligand is one signaling pathway believed to play a major role in the induction of apoptosis (811). In addition to Fas and Fas ligand, a number of other molecules have been shown to influence the induction of apoptosis and AICD. One molecule that has received recent attention is CD44.

CD44 is a widely distributed surface glycoprotein expressed by a number of lymphoid and non-lymphoid cells (12,13). The principal ligand of CD44 is believed to be hyaluronic acid. The interaction between CD44 and its ligand plays an important role in a number of immunological processes, such as lymphocyte migration, extravasation, activation and cytolytic activity (1419). In addition, recent evidence suggests that CD44 may also be involved in the induction of apoptosis; however, the exact role that CD44 plays in apoptosis remains unclear. A number of reports suggest that the signaling through CD44 can protect a cell from apoptosis. For example, ligation of CD44 can lead to resistance to drug-induced apoptosis in various tumor cells (2022). In contrast, up-regulation and signaling through CD44 can also lead to enhanced apoptosis. For example, ligation of CD44 can lead to apoptosis in fibroblast, neutrophils as well as certain lymphocyte populations (2325). Recent evidence from our laboratory demonstrated that CD44 knockout (KO) mice show increased susceptibility to concanavalin A (Con A)-induced hepatitis, which directly correlated with decreased levels of lymphocyte apoptosis in these mice (26). These apparent discrepancies as to the role of CD44 in the induction of apoptosis can possibly be explained by the fact that CD44 exists as multiple isoforms. The isoforms are expressed during different stages of activation and evidence suggests that the individual CD44 isoforms may play specific roles in the regulation of the immune response (2730). For example, it has been shown that specific isoforms are only expressed on activated lymphocytes and antibodies directed against specific CD44 isoforms are able to prevent the development of trinitrobenzene sulfonic acid-induced colitis (31).

In the current study we tested the hypothesis that CD44 plays an important role in the down-regulation/homeostasis of the immune response by facilitating AICD. To test this hypothesis, we used the CD44 KO mouse and demonstrated that absence of the CD44 molecule on activated lymphocytes leads to an unregulated immune response characterized by reduced induction of apoptosis and that signaling of activated lymphocytes through CD44 leads to increased induction of apoptosis.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C57BL/6 mice (6–8 weeks old) referred to as CD44 wild-type and C57BL/6 lpr/lpr mice referred to as CD44 WT/lpr were purchased from the National Institute of Health (Bethesda, MD) and Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice deficient in CD44 (CD44 KO) were generated at Amgen Institute (Toronto, Canada) and bred in the animal facility at Virginia Commonwealth University. The phenotype of these mice has been described previously (32,33). Mice deficient in CD44 and Fas on C57BL/6 background were generated by crossbreeding CD44 KO mice with lpr/lpr mice and referred to as CD44 KO/lpr mice. The phenotype of these mice has been recently described by us (manuscript submitted).

Evaluation of the primary immune response in vivo
CD44 wild-type and CD44 KO mice were injected s.c. into their rear footpads with conalbumin (100 µg/footpad) mixed 1:1 with complete Freund’s adjuvant (CFA), anti-CD3 mAb (145-2C11) (25 µg/footpad) mixed 1:1 with CFA or staphylococcal enterotoxin A (SEA) (10 µg/footpad) as described (34). Between 3 and 7 days following the injection, the popliteal lymph nodes draining the injection site were harvested and placed into a stomacher bag containing 10 ml complete RPMI (10% FCS). The lymph nodes were prepared into a single-cell suspension using a stomacher laboratory blender and washed 3 times in complete RPMI. The lymph node cellularity was determined by Trypan blue exclusion.

Use of anti-CD44 mAb to block the response to conalbumin in vivo
CD44 wild-type mice were injected s.c. into their rear footpads with conalbumin (100 µg/footpad) mixed 1:1 with CFA as described above along with 500 µg of anti-CD44 mAb (9F3) or isotype control mAb (R3-34) by the i.p. route (3538). In separate experiments CD44 wild-type mice were treated with 200 µg of anti-CD44 (IM-7) or isotype control mAb (A95-1). Seven days following the injection the popliteal lymph nodes draining the injection site were harvested, weighed and placed into a stomacher bag containing 10 ml complete RPMI (10% FCS). The lymph nodes were prepared into a single-cell suspension using a stomacher laboratory blender and washed 3 times in complete RPMI. The lymph node cellularity was determined by Trypan blue exclusion.

Assessments of the proliferative response in vitro
Lymph node cells or splenocytes (5 x 105) from CD44 wild-type or CD44 KO mice were cultured in 96-well flat-bottomed plates in the presence or absence of 25 µg/ml conalbumin, 2 µg anti-CD3 mAb (145-2C11) or 2 µg SEA for 24–96 h. The cells were pulsed with 2 µCi [3H]thymidine during the final 6 h of culture and harvested using a cell harvester (Skatron, Sterling, VA). The incorporated labeled DNA was determined using a ß-scintillation counter.

Detection of apoptosis after conalbumin stimulation
Apoptotic events were quantified at the single-cell level using the TUNEL method. Popliteal lymph node cells were cultured for 24–96 h in RPMI alone or re-stimulated as described above. The cells were harvested and washed twice with PBS. To analyze apoptosis by TUNEL, cells were fixed on ice with 4% parafomaldehyde for 30 min. The cells were washed with PBS and permeabilized for 2 min. Finally, the cells were incubated for 1 h at 37°C with FITC–dUTP and TdT. The cells were washed and analyzed using a flow cytometer.

In vivo delayed-type hypersensitivy (DTH) response
CD44 wild-type and CD44 KO mice were sensitized on their abdomens with 0.5% dinitroflourobenzene (DNFB) in 4:1 acetone:olive oil (20 µl). Five days later, the mice were challenged with 20 µl of 0.2% DNFB in 4:1 acetone:olive oil (stimulated) on their right ear and 4:1 acetone:olive oil on their left ear (negative control). The DTH response was measured 24, 48, 72 and 96 h later by measuring the increase in thickness of the stimulated ear compared to the negative control. In addition, mice not receiving the sensitization were included as additional negative controls.

For histopathological studies, the ears were fixed in 10% buffered formalin and embedded in paraffin. Five-micrometer sections were affixed to slides, deparaffinized and stained with hematoxylin & eosin to assess morphological changes.

Assessments of CD44-induced apoptosis
Splenocytes (5 x 105) from CD44 KO and CD44 wild-type mice were cultured in 96-well plates that were precoated with 50 µl of 5 µg/ml anti-CD44 mAb (IM7) or isotype control (A95-1). The cells were stimulated with soluble anti-CD3 mAb (145-2C11) for 24–48 h, and harvested and washed twice with PBS. The presence of apoptotic cells was determined using the TUNEL method as described above.

TCR-induced apoptosis assay
Splenocytes (5 x 106) from CD44 KO and CD44 wild-type mice were suspended in 10% RPMI and cultured in 24-well plates in the presence of 2 µg/ml anti-CD3 mAb (145-2C11) for 48 h. The cells were then washed twice and cultured for an additional 24 h in the presence of 50 U/ml rIL-2. The cells were harvested, washed twice and cultured for 24–48 h in 96-well flat-bottomed plates that were either uncoated or precoated with anti-CD3 mAb (5 µg/ml in 50 µl/ml PBS overnight). The proliferative response was measured by pulsing with 2 µCi [3H]thymidine during the final 6 h of culture. Apoptosis was determined by the TUNEL method, as described above.

Detection of cell surface markers
Lymph node cells were analyzed for the expression of various cell surface markers using flow cytometric analysis. Antibodies used included phycoerythrin- or FITC-conjugated anti-Fas (Jo2), anti-TCR (H57-597) and anti-CD44 mAb (IM7) (PharMingen, San Diego, CA). Cells (1 x 106) were stained with 1 µg primary mAb for 30 min on ice and washed twice with PBS. The fluorescence was determined using a flow cytometer (Beckman Coulter, Fullerton, CA).

Detection of Fas expression using RT-PCR
RT-PCR was conducted to detect Fas as described previously. The primers for Fas were 5'-GCACAGAAGGGAAGGAGTAC-3' and 5'-GTCTTCAGCAATTCTCGGGA-3' and mouse ß-actin primer 5'-ATCCTGACCCTGAACTACCCCATT-3' and 5'-GCACTGTAGTTTCTTCTT CGAC. PCR products were visualized in 1.0% agarose gels after staining with ethidium bromide. The density of the PCR products for Fas was compared to ß-actin which was used as an internal control.

Statistical analysis
Groups of five mice were used in most experiments, and the statistical analysis between experimental and control groups was conducted by Student’s t-test and P < 0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD44 KO mice demonstrate increased lymph node cellularity and resistance to apoptosis following antigenic challenge
Using the well-developed in vivo popliteal lymph node assay, we compared the response of CD44 KO and CD44 wild-type mice to antigenic challenge with conalbumin. Mice were injected s.c. into their rear footpads with conalbumin and 7 days later, the increase in cellularity of the draining lymph node, when compared to unstimulated controls, was determined (Fig. 1A). The results from this experiment showed that the CD44 KO mice exhibited an increased response to conalbumin when compared to CD44 wild-type mice (Fig. 1A). The total cellularity of the unstimulated node was 0.72 ± 0.60 x 106/node. Upon injection with conalbumin, the cellularity increased to 8.81 ± 0.60 x 106/node in CD44 wild-type mice. In comparison, the CD44 KO mice exhibited a cellularity of 15.12 ± 1.0 x 106/node, resulting in a 72% increase over the control response.



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Fig. 1. CD44 KO mice demonstrate an increased popliteal lymph node cellularity and reduced apoptosis after in vivo challenge with conalbumin. The immune response of CD44 KO and CD44 wild-type mice was assessed by determining the increase in lymph node cell number after injection of conalbumin s.c. into their rear footpads (A). Seven days following the conalbumin injection, the draining lymph node cellularity was determined by Trypan blue dye exclusion. The data represent the mean cellularity per lymph node ± SEM using groups of five mice. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice. (B) A representative experiment on detection of apoptosis in lymph node cells after in vivo injection with conalbumin. Lymph node cells from mice injected with conalbumin were harvested as described above and analyzed for apoptosis, using the TUNEL method, following 24 h of culture in tissue culture medium in vitro.

 
Previous studies from our laboratory have demonstrated that AICD is difficult to detect in the lymph nodes following antigenic challenge due to rapid clearance of apoptotic cells by phagocytic cells. However, when such antigen-activated T cells were cultured in vitro with medium, increased levels of apoptosis were detected when compared to naive T cells similarly cultured (39). Therefore, to study AICD, CD44 wild-type and CD44 KO mice were injected with conalbumin s.c. in vivo as described above and lymphocytes isolated from the draining lymph nodes were analyzed for apoptosis either directly or following culture in tissue culture medium. The lymph node cells from conalbumin-immunized mice failed to exhibit apoptosis when tested directly (data not shown). However, upon culture, lymph node cells from CD44 wild-type mice showed significant apoptosis (Fig. 1B). In contrast, the lymph node cells isolated from conalbumin-immunized CD44 KO mice similarly cultured were resistant to apoptosis (Fig. 1B), thereby suggesting that the increased cellularity found in the CD44 KO mice may be related to decreased induction of apoptosis following antigenic challenge.

Lymph node cells from CD44 KO mice exhibit an elevated proliferative response and resistance to apoptosis following secondary stimulation in vitro
To study AICD, the response of conalbumin-sensitized lymph node cells from CD44 KO and CD44 wild-type mice to re-stimulation with the recall antigen in vitro was examined. CD44 KO and CD44 wild-type mice were injected with conalbumin as described above. After 5 days, the draining lymph node cells were harvested and prepared into a single-cell suspension. The sensitized cells were stimulated with conalbumin or cultured in medium alone. At various time points, the proliferative response was determined by [3H]thymidine uptake. As shown in Fig. 2(A), sensitized lymphocytes from CD44 KO mice exhibited a higher response at 48 h when compared to the CD44 wild-type mice. However, at 96 h, the cells from the CD44 wild-type mice showed a dramatic reduction in cell proliferation indicative of AICD, while the cells from CD44 KO mice continued to proliferate at a high level.



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Fig. 2. Lymph node cells from CD44 KO mice exhibit an elevated secondary immune response in vitro and reduced apoptosis. Lymph node cells from CD44 wild-type or CD44 KO mice were immunized with conalbumin as described in Fig. 1. Seven days later, the draining lymph node cells were re-stimulated with conalbumin for 48 or 96 h. (A) The proliferative response was determined by pulsing the cells for the final 8 h with [3H]thymidine followed by ß-scintillation counting. The data represent the mean ± SEM of triplicate wells. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice. (B) A representative experiment in which lymph node cells from conalbumin-immunized CD44 wild-type and CD44 KO mice were re-stimulated with conalbumin in vitro for 48–96 h. The level of apoptosis was determined using the TUNEL method followed by flow cytometric analysis. The percentage of apoptotic cells and the mean fluorescence intensity (MFI) has been depicted in each histogram.

 
It was possible that the prolonged proliferative response seen in CD44 KO mice at 96 h was due to a decrease or delay in AICD. Therefore we measured the induction of apoptosis in CD44 KO and CD44 wild-type after re-stimulation of sensitized lymphocytes with conalbumin in vitro. These data demonstrated that at 96 h, CD44 wild-type mice exhibited increased levels of apoptosis (33.7%) when compared to cells from CD44 KO mice which exhibited 9.5% apoptosis (Fig. 2B). In contrast, at 48 h, the levels of apoptosis were similar in both the groups of mice. Together, these data suggested that the observed enhanced immune response to conalbumin seen in CD44 KO mice may be due to increased resistance to AICD.

CD44 KO mice show an elevated primary response to anti-CD3 mAb and SEA in vivo
The observation that CD44 KO mice exhibit increased lymph node cellularity was further supported by experiments using immunization with anti-CD3 mAb and SEA. CD44 KO and wild-type mice were injected s.c. into their rear footpads with either anti-CD3 mAb or SEA. Three days following the injection, the draining lymph nodes were removed and the viable cell numbers per lymph node were determined by Trypan blue dye exclusion (Fig. 3A). These data demonstrated that the draining lymph node cellularity was significantly elevated in CD44 KO mice when compared to that of CD44 wild-type mice following immunization with anti-CD3 mAb or SEA (Fig. 3A).



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Fig. 3. CD44 KO mice demonstrate increased primary and secondary response to anti-CD3 mAb and SEA. (A) The primary immune response of CD44 KO and CD44 wild-type mice was assessed by determining the increase in lymph node cell number after in vivo injection into their rear footpads with anti-CD3 mAb or SEA. Three days following the injection, the draining lymph node cellularity was determined by Trypan blue dye exclusion. The data represents the mean cellularity per lymph node ± SEM, using groups of five mice. (B) The secondary immune response to recall antigens was compared in CD44 wild-type and CD44 KO mice. Lymph node cells from CD44 wild-type or CD44 KO mice previously immunized with anti-CD3 mAb or SEA as described above were re-stimulated with the specific antigen for 72 or 96 h. The proliferative response was determined by pulsing the cells for the final 8 h with [3H]thymidine followed by ß-scintillation counting. The data represent the mean ± SEM of triplicate wells. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice.

 
Lymph node cells from CD44 KO mice demonstrate elevated secondary proliferative response to anti-CD3 mAb and SEA in vitro
The lymph node cells from CD44 wild-type and CD44 KO mice sensitized with anti-CD3 mAb or SEA in vivo as described above were re-stimulated with the specific antigen, and the proliferative response was determined at 72 and 96 h (Fig. 3B). The results showed that the CD44 KO mice exhibited a dramatically increased and prolonged secondary immune response in vitro. Together these experiments suggested that the absence of CD44 expression leads to a prolonged and increased proliferative response of sensitized lymphocytes to stimulation with recall antigen.

Increased DTH response in CD44 KO mice
The DTH assay is a classic experiment used to measure the secondary cellular response in vivo. Therefore we chose the DTH model to determine whether CD44 KO mice had an altered secondary cellular response in vivo. To this end, CD44 wild-type and CD44 KO mice were sensitized with DNFB and 5 days later challenged with DNFB on the right ear. The DTH response was studied 24–96 h later. As seen from Fig. 4(A), CD44 KO mice had a significantly elevated DTH response when compared to CD44 wild-type mice. Such an increase in DTH response in CD44 KO mice was present at 24, 48, 72 and 96 h after rechallenge with DNFB. These results suggested that the absence of CD44 leads to an increased DTH response in vivo. Hematoxylin & eosin staining of ear sections was performed 24 and 48 h following the rechallenge with DNFB. Figure 4(B) shows representative ear sections from CD44 wild-type and CD44 KO mice 48 h following rechallenge with DNFB. The ear section from the CD44 wild-type mice exposed to DNFB exhibited a mild diffuse inflammatory response with predominant infiltration of macrophages and lymphocytes when compared to the controls. The ear section from CD44 KO mice rechallenged with DNFB showed a slightly elevated diffuse inflammatory response when compared to the CD44 wild-type mice. Similar results were seen in ear sections taken 24 h following rechallenge with DNFB (data not shown). These data suggested that CD44 deficiency does not prevent the migration of mononuclear cells to the sites of inflammation and are consistent with our previous studies involving Con A- or IL-2-induced inflammation (26,32).



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Fig. 4. Increased DTH response in CD44 KO mice to DNFB. CD44 wild-type and CD44 KO mice were sensitized on their abdomens with 0.5% DNFB. Five days later, the mice were challenged with 0.2% DNFB on their right ear. (A) The DTH response was measured 24, 48, 72 and 96 h later by measuring increase in thickness of the sensitized ear when compared to the negative control. The data represent the mean ± SEM of groups from six mice. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice. (B) Hematoxylin & eosin staining of ear sections studied 48 h after challenge with 0.2% DNFB (magnification x200).

 
The levels of CD3 and TCR are unaltered in CD44 KO splenocytes.
Next, we examined whether the increased immune response seen in the CD44 KO mice was directly related to altered expression of CD3 and/or TCR. Naive splenocytes from wild-type and CD44 KO mice were analyzed for the expression of cell surface CD3 and TCR (Fig. 5). The results showed that the percentage of CD3+ or {alpha}ßTCR+ T cells as well as the density of expression of these molecules was similar in both groups of mice. Together, these results suggested that T cells from CD44 KO mice express similar levels of CD3–TCR.



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Fig. 5. CD44 KO and CD44 wild-type splenocytes express similar levels of CD3 and {alpha}ßTCR. Splenocytes from CD44 wild-type and CD44 KO mice were stained for cell surface expression of CD3 and {alpha}ßTCR. The level of expression of the cell surface markers was determined by flow cytometric analysis.

 
Resistance of lymphocytes from CD44 KO mice to apoptosis is not due to differences in the expression of CD69 or Fas
It was possible that the reduced levels of apoptosis seen in antigen-activated lymphocytes from CD44 KO mice may result from altered expression of cell surface markers involved in apoptosis, such as Fas, and activation markers, such as CD69. Therefore, we examined the levels of expression of these molecules along with CD44 on splenocytes from CD44 wild-type and CD44 KO mice before and after stimulation with anti-CD3 mAb. Activation of splenocytes from CD44 wild-type mice led to a significant increase in the expression of CD69 and Fas which was almost identical to that seen from CD44 KO mice (Fig. 6). In addition, expression of Fas on anti-CD3 mAb-activated CD44 wild-type and CD44 KO splenocytes was also similar (Fig. 6). Furthermore, activation of CD44 wild-type splenocytes led to significant up-regulation of CD44, suggesting a possible role of CD44 in activated lymphocytes.



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Fig. 6. Effect of activation through CD3 on the expression of CD69, Fas and CD44 on splenocytes. Splenocytes from CD44 wild-type and CD44 KO mice were stimulated with anti-CD3 mAb (shaded histogram) or cultured in medium alone (empty histogram with dark borders) for 48 h. Next, the cells were stained for the expression of CD69, Fas and CD44. The percentage of positive cells and mean fluorescence intensity (MFI) was determined by flow cytometric analysis.

 
CD44 KO splenocytes are resistant to TCR-induced apoptosis (AICD) in vitro
Using a recently developed model for determining AICD in vitro (40), we further tested whether lymphocytes from CD44 KO mice were resistant to TCR-induced apoptosis. Briefly, splenocytes from CD44 KO and CD44 wild-type mice were stimulated for 48 h with anti-CD3 mAb in vitro. The activated cells were subsequently harvested and the viable cells were cultured for 24 h in medium containing 50 U/ml rIL-2. Finally, the cells were washed and cultured in plates precoated with anti-CD3 mAb. After 24 h, splenocytes from CD44 wild-type and CD44 KO mice not cultured on the anti-CD3 mAb-coated plates remained active as demonstrated by their high level of [3H]thymidine uptake (Fig. 7A). However, when activated splenocytes from CD44 wild-type mice were cultured on plates precoated with anti-CD3 mAb, the proliferative response was dramatically reduced, suggesting that the delivery of a second stimulus through the TCR on TCR-activated cells led to a decrease in the proliferative response. Interestingly, splenocytes from CD44 KO mice receiving the second stimulation through the TCR did not demonstrate a reduction in the proliferative response. Similar results were evident at 48 h, in which the CD44 KO splenocytes receiving the second stimulation continued to show an increased proliferative response when compared to the CD44 wild-type cells. Evaluation of the induction of apoptosis after 48 h revealed higher levels of apoptosis in the splenocytes from CD44 wild-type mice when compared to CD44 KO mice (Fig. 7B). Together, these results further confirmed that CD44 KO lymphocytes were more resistant to AICD.



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Fig. 7. Splenocytes from CD44 KO mice demonstrate an enhanced secondary proliferative response characterized by reduced induction of apoptosis in vitro. Splenocytes from CD44 wild-type and CD44 KO mice were stimulated in vitro for 48 h with anti-CD3 mAb, followed by a 24 culture with IL-2. The cells were then washed and cultured for 24–48 h in wells containing bound anti-CD3 mAb. The proliferative response (A) was determined by pulsing the cells during the final 8 h of culture with [3H]thymidine followed by ß-scintillation counting. The data represent the mean ± SEM of triplicate wells. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice. The cells were also analyzed for apoptosis (B) by TUNEL assay followed by flow cytometric analysis as described in Fig. 1.

 
Ligation of CD44 on activated splenocytes leads to increased apoptosis
In order to directly examine whether ligation of cell surface CD44 molecules triggers the induction of apoptosis, unstimulated and anti-CD3 mAb-stimulated splenocytes were incubated on plates that were precoated with anti-CD44 mAb or isotype control mAb. After 48 h, the induction of apoptosis was determined by TUNEL assay. The results showed that ligation of CD44 on cells cultured in medium induced similar levels of apoptosis (25.1%) as cells cultured with isotype control antibodies (25.6%) (Fig. 8A). However, when splenocytes were stimulated with anti-CD3 mAb and simultaneously exposed to anti-CD44 mAb, there was a dramatic increase in the induction of apoptosis (74.6%) when compared to the control antibodies (30.1%) (Fig. 8B). These results suggested that direct ligation of CD44 on TCR-activated lymphocytes leads to the induction of apoptosis.



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Fig. 8. Signaling through CD44 leads to increased apoptosis in anti-CD3 mAb-stimulated splenocytes. Splenocytes from CD44 wild-type mice were cultured with medium or anti-CD3 mAb and plated in wells coated with anti-CD44 mAb (5 µg/ml in 50 µl) or isotype control mAb for 48 h. The cells were harvested, washed and stained for apoptosis using the TUNEL method.

 
Ligation of CD44 on activated splenocytes does not affect Fas expression
In order to examine whether ligation of cell surface CD44 molecules triggers apoptosis by the induction of Fas expression, unstimulated and anti-CD3 mAb stimulated splenocytes were incubated on plates that were precoated with anti-CD44 mAb or isotype control mAb. After 6 h, the level of Fas mRNA transcripts was measured using RT-PCR (Fig. 9A) and after 24 h, the level of Fas protein was measured using flow cytometry (Fig. 9B). The results showed that stimulation with anti-CD3 mAb led to increased expression of Fas mRNA and protein. However, no increase in the expression of Fas mRNA or protein was observed after ligation of CD44 using mAb. Together these results suggested that apoptosis induced by ligation of CD44 was not directly due to up-regulation of Fas.



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Fig. 9. Signaling through CD44 in anti-CD3 mAb-stimulated splenocytes does not affect Fas expression. Splenocytes from CD44 wild-type mice were cultured with medium or anti-CD3 mAb and then plated in wells coated with anti-CD44 mAb or isotype control mAb. (A) At 6 h following stimulation total splenocyte RNA was isolated and mRNA was reverse transcribed, and amplified by PCR with primers specific for Fas and ß-actin. A photograph of ethidium bromide-stained amplicons is depicted. Lane 1 represents cells cultured with medium alone in wells coated with isotype control antibodies. Lane 2 represents cells cultured with medium alone in wells coated with anti-CD44 mAb (5 µg/ml in 50 µl). Lane 3 represents cells cultured with anti-CD3 mAb in wells coated with isotype control antibodies. Lane 4 represents cells cultured with anti-CD3 mAb plated in wells coated with anti-CD44 mAb. (B) The percentage of Fas+ cells and mean fluorescence intensity (MFI) was determined by flow cytometric analysis.

 
CD44 KO/lpr mice exhibit elevated primary immune response to SEA when compared to CD44 WT/lpr mice in vivo
To further address whether CD44-mediated apoptosis was independent of Fas, we compared the responsiveness of CD44 KO/lpr (CD44Fas) mice to that of CD44 WT/lpr (CD44+Fas) mice following SEA injection. To this end, CD44 WT/lpr and CD44 KO/lpr mice were injected with SEA as described in Fig. 3. Three days following the injection, the draining lymph nodes were removed and the viable cell number of the lymph nodes was determined by Trypan blue dye exclusion (Fig. 10). The results showed that the CD44 KO/lpr mice had a significantly enhanced primary response when compared to the CD44 WT/lpr mice, suggesting that the enhanced immune response seen in CD44-deficient mice was not dependent on the expression of Fas.



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Fig. 10. CD44 KO/lpr mice exhibit elevated primary immune response to SEA when compared to CD44 WT/lpr mice in vivo. The primary immune response of CD44 KO/lpr and CD44 WT/lpr mice was assessed by determining the increase in lymph node cell number after in vivo injection into their rear footpads with SEA. Three days following the injection, the draining lymph node cellularity was determined by Trypan blue dye exclusion. The data represents the mean cellularity per lymph node ± SEM in groups of five mice. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice.

 
Use of antibodies against CD44 to suppress the response to conalbumin in vivo
To examine the effect of ligation of CD44 on the response to a foreign antigen in vivo, CD44 wild-type mice were injected with conalbumin and 500 µg anti-CD44 mAb (9F3) or isotype control mAb. In a second set of experiments, CD44 wild-type mice were injected with conalbumin and a separate anti-CD44 mAb (IM7) or isotype control. Seven days later, the response to conalbumin was determined by examining the increase in draining lymph node weight and cellularity, and was compared to lymph nodes from unstimulated mice. As shown in Fig. 11, injection of anti-CD44 mAb, 9F3 (Fig. 11A) or IM7 (Fig. 11B), significantly inhibited the conalbumin-stimulated increase in draining lymph node weight and cellularity. We ruled out the possibility that anti-CD44 mAb-treatment led to complement-mediated killing by showing that the number of cells staining positive with the anti-CD44 mAb (IM7) in unstimulated mice was not altered following in vivo treatment the anti-CD44 mAb (9F3) (data not shown). In addition, in experiments using the anti-CD44 mAb IM7, the induction of complement-mediated lysis was unlikely due to the fact that the isotype of IM7 is IgG2a which is a poor mediator of complement-mediated lysis. It is possible that the antibodies against CD44 may cause shedding of CD44, as reported by Camp et al. (41). The above results suggested that ligation of CD44 can significantly inhibit the immune response to conalbumin in vivo.



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Fig. 11. Effects of administration of anti-CD44 mAb on the response to conalbumin in vivo. CD44 wild-type mice were injected s.c. into their rear footpads with PBS (unstim) or conalbumin (100 µg/footpad) mixed 1:1 with CFA along with (A) 500 µg of anti-CD44 mAb (9F3) or isotype control mAb (R3-34), or (B) 200 µg of anti-CD44 mAb (IM7) or isotype control mAb (A95-1). Seven days following the injection the popliteal lymph nodes draining the injection site were harvested. The response was measured by determining the increase in lymph node weight and cellularity. The data represents the mean ± SEM of groups of five mice. An asterisk indicates statistically significant difference when compared to the CD44 wild-type mice.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the current study, we demonstrated that CD44 plays an important role in AICD. CD44 KO mice exhibited an elevated primary and secondary response to conalbumin, SEA and anti-CD3 mAb in vivo. In addition, CD44 KO mice had an increased DTH immune response to DNFB in vivo. Further studies into the mechanism revealed that the increase in the proliferative response correlated with increased resistance to induction of apoptosis. We confirmed that CD44 expression played an important role in regulating AICD using an in vitro model of AICD and showing that a secondary stimulus of anti-CD3 mAb in CD44 KO splenocytes led to a significantly reduced level of apoptosis when compared to CD44 wild-type splenocytes. In addition, we demonstrated that stimulation with anti-CD3 mAb led to increased expression of CD44 and that ligation of CD44 on activated cells led to induction of apoptosis. Moreover, treatment with anti-CD44 mAb led to suppression of the response to conalbumin in vivo. Taken together, this study provides strong evidence that CD44 plays an important and direct role in AICD.

AICD is believed to play a major role in regulating the immune response by maintaining homeostasis. A hallmark feature of cells undergoing AICD is apoptosis. Numerous reports suggest that signaling through Fas plays an important role in the induction of apoptosis. In this study we showed that anti-CD3 mAb-induced activation led to a similar increase in Fas expression in both wild-type and CD44 KO splenocytes. However, induction of AICD by re-stimulation with anti-CD3 mAb was dramatically reduced in CD44 KO splenocytes. This observation combined with the results demonstrating increased apoptosis in anti-CD3 mAb-activated T cells following CD44 ligation strongly suggested a direct role of CD44 in AICD. Interestingly, we found that ligation of CD44 after stimulation for 24 or 48 h with anti-CD3 did not significantly increase in the level of apoptosis. Ligation of CD44 at the time of activation was necessary to significantly elevate levels of apoptosis. This suggests that in order to effectively induce apoptosis the ligation of CD44 must accompany the stimulatory signal or at least be present very early in the stimulation process. Although these results do not exclude the importance of Fas expression in AICD, they do suggest that additional molecules, such as CD44, may act in concert with or independent of Fas to achieve maximal cell death.

Initial studies from our laboratory, using in vitro assays to compare the proliferative response of splenocytes from CD44 wild-type and CD44 KO mice to stimulation with various polyclonal mitogens, failed to show a significant difference between the two groups (26). This finding led to the initial conclusion that CD44 deficiency does not alter the primary immune response to mitogens in vitro. However, injection of a polyclonal T cell mitogen such as Con A caused enhanced hepatitis in CD44 KO mice when compared to CD44 wild-type mice (26). These studies prompted us to undertake more detailed analysis of antigen-primed responsiveness in vivo and AICD to delineate the role of CD44. In the current study using in vivo immunization we found that CD44-deficient mice when compared to CD44 wild-type mice had a significant elevation in cell number in their draining lymph nodes following primary challenge with anti-CD3 mAb, SEA or conalbumin. The increased cellularity in the lymph nodes of the CD44 KO mice can be explained by at least two scenarios. First, the lack of CD44 may lead to an increased proliferative response and/or a decrease in AICD. Also, these events may be inter-related inasmuch as reduced AICD may lead to prolonged cell proliferation. We have noted that the proliferative response of naive splenocytes from CD44 wild-type and CD44 KO mice to stimulation with anti-CD3 mAb, Con A and SEA in vitro was similar (26) (data not shown), thereby suggesting that the increased cell numbers observed in the lymph nodes of immunized mice were not the result of an increased proliferative rate, but possibly due to increased resistance to apoptosis. Furthermore, the reason why T cells from CD44 KO mice when stimulated in culture with mitogens respond normally, whereas when challenged in vivo with antigens demonstrate enhanced response, can be explained by the fact that in vitro proliferative responses to mitogens are measured within 48 h at the peak DNA synthesis. Thereafter the response declines rapidly. This may not permit the detection of AICD. In contrast, following in vivo challenge with antigens, the prolonged primary response includes both the proliferative stage followed by a decline phase involving AICD (42). Thus, the T cells from CD44 KO mice, being more resistant to AICD, may continue to mount an enhanced immune response. Such cells may also account for the increased secondary response as seen with contact hypersensitivity following DNFB rechallenge.

CD44 has been shown to regulate a number of processes important for cell migration and trafficking including various cell–cell and cell–matrix interactions (14,15). We cannot rule out the possibility that the response to conalbumin in vivo might be due to differences in migration and/or retention of mononuclear cells. Previous studies have shown that blocking CD44 with mAb can inhibit lymphocyte migration into the peritoneal cavity, suggesting that CD44 plays an important role in lymphocyte migration (14). However, in the current study we demonstrated that the migration and infiltration of lymphocytes in CD44 KO mice were not altered in response to DNFB. This finding is consistent with our previous studies using IL-2-induced vascular leak or Con A-induced hepatitis models in which mononuclear cells from CD44 KO mice showed normal migration and homing (26,32). Whether this lack of a defect in lymphocyte migration seen in CD44 KO mice is due to a compensatory mechanism remains a possibility.

The finding that CD44 KO mice have an elevated response to DNFB is contrary to a previous report which suggested that CD44 deficiency did not significantly alter the DTH response to DNFB (33). This apparent discrepancy between this study and that of ours might be explained by differences in experimental design. In their study, Schmits et al. (33) compared the DTH response in CD44+/– mice to the response in CD44–/– and found that there was no statistically significant difference between the two groups. In our study, we compared the DTH response of CD44–/– to CD44+/+ mice and found an elevated response in the KO mice. It was reported by Schmits et al. (33) that the CD44+/– mice express lower levels of CD44 than CD44+/+ mice. This alteration in the expression of CD44 may help to explain the discrepancies between the two studies. In addition, Schmits et al. reported that there was an increase in number and size of granuloma formed in response to Cryptosporidium parvum in CD44-deficient mice. These data support our findings suggesting an enhanced immune response in the CD44 KO mice. In a separate study, it was shown that treatment with anti-CD44 mAb leads to a suppressed DTH response, which is consistent with our findings of suppression of the response to conalbumin by anti-CD44 mAb (41).

In contrast to the current study and that of others (2325) demonstrating enhanced induction of apoptosis by signaling through CD44, some reports have shown that signaling through CD44 can enhance lymphocyte survival by suppressing the induction of apoptosis (2022,31). Such discrepancies may have resulted from the fact that in addition to the standard form of CD44, a number of different variant isoforms of CD44 can be expressed on lymphocytes. CD44 is encoded by 20 exons and by alternative splicing up to 10 variant exons can be inserted within the extracellular region, leading to the possible expression of a large number of CD44 isoforms. Therefore, the wide range of functions attributed to CD44 may result from the expression of different isoforms and that expression of some isoforms may lead to enhanced apoptosis while others may suppress apoptosis. Evidence for this possibility can be seen when comparing studies examining the role of CD44 in apoptosis. In a model of autoimmune reaction involving Con A-induced hepatitis, we observed that CD44 KO mice exhibited enhanced hepatitis when compared to the CD44 wild-type mice (24). In this study, CD44 deficiency led to increased resistance to Con A-induced apoptosis and such activated effector T cells were responsible for causing enhanced hepatitis. In contrast, a separate study showed that mice deficient in CD44 variant exon 7 were resistant to experimental colitis due to a higher rate of apoptosis at inflamed lesions (31). These data suggested that CD44v7 helped in the survival of effector lymphocytes. These studies are consistent with our observation that CD44 KO mice exhibited decreased vascular leak syndrome and endothelial cell injury following IL-2 therapy (23). Thus IL-2 treatment may up-regulate certain CD44 variant isoforms essential for survival of LAK cells which in turn cause endothelial cell injury. Together, these studies stress the need to further investigate the role played by CD44 variant isoforms in T cell activation and apoptosis.

Although we provide evidence which suggests that CD44 plays an important role in AICD, very little is known about the mechanism by which CD44 acts. Consistent with other reports, we demonstrated that CD44 expression is significantly increased on anti-CD3 mAb-activated splenocytes. Interestingly, we showed that only activated but not naïve cells were susceptible to anti-CD44 mAb-induced apoptosis, suggesting that the increased expression of CD44 may lead to increased binding of CD44 to its ligand resulting in the induction of apoptosis. Also, lymphocytes have been shown to acquire the ability to recognize hyaluronic acid, the primary ligand for CD44 upon activation (43). Lastly, activated T lymphocytes express unique isoforms of CD44 which may be involved in induction of apoptosis (44). In addition, some studies have characterized the molecular pathways triggered following signaling through CD44. For example, ligation of CD44 led to an increase in T lymphocyte intracellular Ca2+ (19), an increase in tyrosine phosphorylation of Zap-70 and enhanced kinase activity of p56lck (45). In addition, CD44 is associated with the tyrosine kinases Lck and Fyn found in lipid rafts thought to be important in TCR signaling (45). Therefore, it is possible that binding of CD44 on activated cells directly signals the cells to undergo AICD. Another possible mechanism by which increased expression of CD44 may act to mediate AICD is by enhancing the signaling by the TCR (25), leading to more efficient induction of apoptosis. Finally, ligation of CD44 may directly lead to the up-regulation of various molecules directly involved in apoptosis signaling. For example, it was shown that cross-linking or ligation of CD44 by HA resulted in the rapid up-regulation of Fas on rheumatoid synovial cells (46). In the current study, however, we failed to detect any increase in the expression of Fas on activated T cells following ligation of CD44. Furthermore, our experiments using CD44 WT/lpr and CD44 KO/lpr mice demonstrated that the increased resistance to AICD seen in CD44-deficient T cells was independent of Fas expression.

In the current study, we demonstrated that ligation of CD44 in vitro on activated but not naive T cells led to the induction of apoptosis. Furthermore, administration of anti-CD44 mAb into CD44 wild-type mice caused a significant decrease in the antigen-specific response. These data suggested that targeting CD44 may constitute a novel and effective approach to induce apoptosis in activated T cells in vivo. Such an approach may be useful in the treatment of autoimmune diseases, allograft rejection and graft versus host diseases involving activated T cells.


    Acknowledgements
 
We would like to thank Dr Tak W. Mak for providing CD44 KO mice. This work was supported in part by grants from National Institute of Health (HL058641, ES09098 and HL10455).


    Abbreviations
 
AICD—activation-induced cell death

CFA—complete Freund’s adjuvant

Con A—concanavalin A

DNFB—dinitrofluorobenzene

DTH—delayed-type hypersensitivity

KO—knockout

SEA—staphylococcal enterotoxin A


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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