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Departments of Microbiology and Immunology and Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298;
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; and
Department of Medical Biophysics and Immunology, Ontario Cancer Institute, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2 and IFN-
, but not Fas or Fas ligand. The increased
susceptibility of CD44 KO mice to hepatitis correlated with the
observation that T cells from CD44 KO mice were more resistant to
activation-induced cell death when compared with the CD44 WT mice.
Together, these data demonstrate that activated T cells use CD44 to
undergo apoptosis, and dysregulation in this pathway could lead to
increased pathogenesis in a number of diseases, including
hepatitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise mechanism(s) by which T cells and macrophages induce
hepatitis is not known. Because a massive release of macrophage and T
cell-derived cytokines, including IL-2, IL-1, IL-6, TNF-, IFN-,
and GM-CSF, occurs with different kinetics in response to Con A, a role
has been envisaged for these cytokines in the development of the
hepatic lesions (5, 6). Nonetheless, the precise role of
cytokines in the pathogenesis of this immunoinflammatory condition
remains to be defined.
CD44 is an acidic, sulfated integral membrane glycoprotein ranging in molecular mass from 80 kDa up to 200 kDa (7, 8). Hyaluronic acid (HA)4 has been shown to be an important ligand for CD44 (8). CD44 is expressed by various lymphoid and nonlymphoid tissues (8, 9). The CD44 molecule has been demonstrated to participate in lymphocyte adhesion to the matrix, lymph node homing, and lymphopoeisis (8, 10). Studies from our laboratory have shown that on activation, T cells express increased levels of CD44, which can act as a signaling molecule in effector functions such as induction of cytotoxicity (11, 12). Similarly, mAbs against CD44 were shown to induce B cell growth and differentiation (13). Other studies have shown that mAbs directed against CD44 molecules can either up-regulate (14) or down-regulate (15) anti-CD3 and anti-CD2 mAb-induced proliferation of T cells. Recent studies from our laboratory also demonstrated that IL-2-induced vascular leak and damage to the endothelial cells is mediated by CD44 (16). Together, the above studies demonstrated that CD44 expressed on activated lymphocytes can interact with HA expressed on target cells and participate in cell injury leading to autoimmune reactions. In the current study, we used CD44 knockout (KO) mice to study Con A-induced hepatitis, speculating that such mice would be resistant to development of hepatitis. In contrast, we observed that CD44 KO mice exhibited enhanced pathogenesis in Con A-induced hepatitis. The enhanced hepatitis seen in these mice primarily resulted from inability of Con A-activated CD44-deficient T cells to undergo apoptosis. The current study demonstrates that CD44 plays an important regulatory role in the pathogenesis of Con A-induced hepatitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult female C57BL/6 (CD44) wild-type (WT) mice were purchased from the National Institutes of Health (Bethesda, MD). CD44 KO mice on C57BL/6 background were kindly provided by Amgen Institute (Toronto, Ontario) and bred in our animal facilities and screened for the CD44 mutation. These mice were back-crossed for 12 generations onto the C57BL/6 background and were susceptible to syngeneic tumors of C57BL/6 origin. The phenotype of these mice has been described elsewhere (16, 17).
Con A-induced hepatitis and its evaluation
To induce hepatitis, female CD44 WT and CD44 KO mice weighing 20–23 g were challenged with Con A (12 mg/kg body weight i.v. in 100 µl of saline). Control mice received 100 µl saline (i.v.). Plasma from individual mice was separated from blood obtained through the orbital plexus under anesthesia with IsoFlo (Abbott Laboratories, North Chicago, IL) at various time intervals after Con A injection. Plasma aspartate aminotransferase activity (AST) levels were measured with a commercial kit (Sigma, St. Louis, MO).
For histopathological studies, the harvested livers 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 morphologic changes, as described (16).
Isolation of lymphocytes infiltrating the liver
The isolation of liver-infiltrating mononuclear cells (MNC) was conducted as described by others (18, 19). Briefly, livers obtained 16 h after Con A injection from CD44 WT and CD44 KO mice were pressed through a 200-gauge stainless steel mesh (18) and suspended in RPMI 1640 medium supplemented with 5% FCS. After one washing with the medium, the cells were resuspended in 30 ml of medium, and infiltrating lymphocytes were separated from parenchymal hepatocytes and Kupffer cells by Histopaque density (1.09) gradient centrifugation. The cell suspension (35 ml) was overlaid on 15 ml of the Ficoll-Isopaque in a 50-ml conical plastic tube. Centrifugation was performed at 2500 x g for 30 min at room temperature. After centrifugation, 10 ml of the interface was aspirated, mixed with 20 ml of the medium in a 50-ml conical tube, and the cells were washed twice. The liver MNC were studied for the expression of cell surface markers. Also, the liver MNC were studied for their ability to undergo apoptosis with annexin V/propidium iodide (PI) assay as described below.
In vivo Con A-induced apoptosis in T cells
At different time points after Con A injection, spleen cells were harvested. T cells from spleen were purified with nylon wool. Next, T cells were studied for apoptosis by TUNEL (20) and annexin/PI assays as described (21). To study apoptosis by TUNEL assay, the cells were fixed with 4% p-formaldehyde for 30 min at room temperature. The T cells were washed with PBS, permeabilized on ice for 2 min, and incubated with FITC-dUTP for 1 h at 37°C. To study apoptosis by annexin/PI assay, the T cells were stained and analyzed as outlined by the manufacturer (Roche, Indianapolis, IN). Briefly, the T cells were washed with PBS and stained with annexin V and PI for 20 min at room temperature. The cells were washed twice with PBS, and the fluorescence of the cells was measured by flow cytometry as described previously (21). The analysis was performed by a Coulter Epics V flow cytometer (Coulter, Miami, FL). Cells (5000) were analyzed per sample.
Con A-induced apoptosis in vitro
Spleens were harvested from CD44 WT and CD44 KO mice. T cells were enriched by nylon wool separation. The T cells (5 x 105) were stimulated with Con A (5 µg/ml) for 8, 16, or 24 h, after which the T cells were harvested, washed in PBS, and analyzed for apoptosis by the TUNEL assay.
TCR-induced activation and apoptosis
TCR-induced apoptosis was studied as described by Radvanyi et al. (22). Spleen cells were isolated from CD44 WT and CD44 KO mice. The spleens were placed into a stomacher bag containing RPMI 1640 medium supplemented with 5% FCS and prepared into a single-cell suspension with a laboratory homogenizer (Stomacher; Tekmar, Cincinnati, OH). Contaminating RBCs were lysed by resuspending the pellet in 3 ml of RBC lysing buffer (Sigma), and the splenocytes were washed and resuspended in RPMI 1640 medium supplemented with 10% FCS. The splenocytes (5 x 105) were stimulated with anti-CD3 mAb (5 µg/ml) for 48 h in 24-well plates. Next, the cells were harvested, and viable cells were purified by density gradient centrifugation over Histopaque (Sigma) The cells were added to 96-well tissue culture plates (5 x 105 cells/well) that had been precoated overnight (4°C) with 50 µl of anti-CD3 mAbs (5 µg/ml in PBS) and cultured with 50 U/ml IL-2 for 48 h. The cells were harvested, washed, and analyzed for apoptosis by annexin/PI staining as described above.
T cell proliferative responsiveness to mitogens
Splenocytes (5 x 105) from CD44 WT and CD44 KO mice were cultured in RPMI 1640 supplemented with 10% FCS and stimulated with Con A (5 µg/ml) or anti-CD3 (5 µg/ml) for 48 h in 96-well tissue culture plates (13). During the final 8 h, the cells were pulsed with 2 µCi of [3H]thymidine. DNA synthesis was determined by [3H]thymidine incorporation with a liquid scintillation counter.
Analysis of cell surface markers
CD44 WT and CD44 KO mice were injected with Con A (12 mg/kg body weight i.v. in 100 µl of saline). Twenty-four hours later, spleen cells were harvested and the lymphocyte population was screened for various cell surface markers by flow cytometry. Briefly, 1 x 106 spleen cells were incubated with Fc receptor block (BD PharMingen, San Diego, CA) followed by culture with PE-conjugated anti-mouse CD3, anti-mouse CD8, anti-mouse CD4, anti-mouse Mac3 (macrophages), or anti-CD45/B220 mAbs (B cells; BD PharMingen) on ice for 30 min. The cells were washed with PBS three times and analyzed for fluorescence with a Coulter Epics V flow cytometer (11).
Detection of Fas, Fas ligand (FasL), TNF-, IFN-, and IL-2
expression by semiquantitative RT-PCR
RT-PCR was conducted to detect Fas, FasL, TNF-, IFN-, and
IL-2 gene expression in a similar way as described
previously (12). The primers used were as follows: Fas
primer, 5'-GCACAGAAGGGAAGGAGTAC-3' and 5'-GTCTTCAGCAATTCTCGGGA-3'; FasL
primer, 5'-GAGAAGGAAACCCTTTCCTG-3' and 5'-ATATTCCTGGTGCCCATGAT-3';
IFN- primer, 5'-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-3' and
5'-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3'; TNF- primer,
5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3' and
5'-GTATGAGATAGCAAATCTGGCTGACGGTGTGGG-3'; IL-2 primer,
5'-TTCAAGCTCCACTTCAAGCTCTACAGCGGAAG-3' and
5'-GACAGAAGGCTATCCATCTCCTCAGAAAGTCC-3'; and mouse 
-actin primer,
5'-ATCCTGACCCTGAACTACCCCATT-3'; and
5'-GCACTGTAGTTTCTCTTCGACACGA-3'. PCR amplification products
were visualized in 1.5% agarose gels after staining with ethidium
bromide. The density of the PCR products for various cytokines was
compared, with -actin used as an internal control. Briefly, the spot
density of each band in the gel was measured with the AlphaImager 2000
digital imaging system (Alpha Innotech, San Leandro, CA). The
percent cytokine expression was calculated as follows: (density of
cytokine product/density of actin product) x 100.
Detection of cytokines in the serum by ELISA
CD44 WT and CD44 KO mice were injected with Con A (12 mg/kg body
weight i.v.). At 8 or 24 h after injection, serum was separated
from blood collected from the orbital plexus and allowed to clot. The
serum was obtained by centrifugation at 2000 x g for
20 min. The serum levels of various cytokines (IL-2, IFN-, and TNF)
were determined by the methods described in the Quantikine M ELISA kits
(R&D Systems, Minneapolis, MN).
Detection of apoptosis in the liver by the TUNEL method
In situ apoptosis in liver was detected as described elsewhere (23). To this end, liver from control and Con A-injected mice were aseptically removed and fixed in 10% neutral formalin solution. Paraffin-embedded sections measuring 5 µm were adhered to slides. Deparaffinization was done by heating the sections at 60°C for 25 min. Rehydration was conducted by transferring the slides through the following solutions: twice in xylene for 5 min, twice in 95% ethanol for 5 min, twice in 70% ethanol for 5 min, and 10 min in distilled water. The tissues were treated with 20 µg/ml proteinase K (Sigma) in 10 mmol Tris-HCl, pH 8, for 30 min at 37°C and then washed with PBS twice for 10 min. Endogenous alkaline-phosphatase were inactivated by treating the slides with 10 mmol levamisole (Sigma) at room temperature for 1 h. The sections were washed with PBS for 10 min and covered with TdT-FITC-dUTP enzyme-labeling solution and then incubated at 37°C in a humidified incubator for 1.5 h. The slides were rinsed for 10 min in PBS and covered with alkaline phosphatase converter solution. After 1 h of incubation, the slides were washed twice with PBS for 10 min and purple substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) was added. Dark purple color was visible in 15–25 min. The slides were washed and counterstained with eosin, and the cover-slip was placed on mounting medium. The nuclear staining was evaluated under a light microscope. Similar slides also were also stained with hematoxylin-eosin to detect lymphocyte infiltration and to evaluate tissue structure.
Adoptive cell transfer
Groups of five female CD44 WT and CD44 KO mice were challenged with Con A (12 mg/kg body weight i.v. in 100 µl of saline). Control mice received 100 µl of saline. Twenty-four hours later, the spleen cells were harvested and washed with PBS. The RBC were lysed, and the spleen cells were washed three times with PBS before adoptive transfer. Spleen cells (1 x 108 cells by i.v. route) from CD44 WT or CD44 KO mice were adoptively transferred into CD44 WT or CD44 KO mice. Plasma from individual mice was collected 24 h after the adoptive transfer. Plasma AST levels were measured with a commercial kit as described above.
Statistical analysis
The statistical comparison between experimental and control groups was conducted by Student’s t test, and p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the nature of hepatocellular damage seen after
administration of Con A, CD44 WT and CD44 KO mice were initially
injected with 20 mg/kg body weight of Con A i.v. in 100 µl of saline.
At this dose, all CD44 KO mice died within a few hours, whereas CD44 WT
mice survived. To further address the mechanism by which Con A triggers
increased toxicity in CD44 KO mice, we used a lower nonlethal dose (12
mg/kg) in all subsequent experiments. To investigate the extent of
hepatitis, plasma AST levels were measured at different time points
after Con A injection. As shown in Fig. 1
, in both CD44 WT and CD44 KO mice,
there was a significant increase in the plasma AST level, thereby
indicating that hepatitis was induced after Con A injection. Increased
AST levels were seen as early as 6 h after Con A injection,
reaching a peak at 12 h and declining thereafter. At 48 h,
the plasma AST reached normal levels. Although, CD44 WT and CD44 KO
mice exhibited similar kinetics, CD44 KO mice exhibited higher plasma
AST levels, particularly at 12 and 24 h after Con A injection.
These data indicated that CD44 KO mice may develop more severe acute
suppurative hepatitis than the CD44 WT mice (Fig. 1). Similar results
also were obtained by histological studies and in situ apoptosis
staining (Fig. 2). Hematoxylin-eosin
staining of liver section was conducted 48 h after Con A challenge
because of significant liver damage seen at this time point. Damage to
hepatocytes and presence of mixed inflammatory infiltrate consisting of
lymphocytes, macrophages, and neutrophils was observed in both CD44 WT
and CD44 KO mice. However, in CD44 KO mice, the liver damage was more
severe than WT mice because of the larger areas of liver destruction.
In contrast, the degree and the nature of inflammation was similar in
CD44 WT and CD44 KO mice. Next, the liver sections were examined for
apoptosis with TUNEL assay. Because apoptosis occurs at earlier time
points, it was studied at 16 h after Con A injection. The dark
purple area demonstrated nuclei positive for DNA fragmentation (Fig. 2). It was noted that CD44 KO mice had a higher number of nuclei
positive for DNA fragmentation when compared with CD44 WT mice (Fig. 2). Together, the above data demonstrated that CD44 KO mice exhibited
increased susceptibility to Con A-induced hepatitis when compared with
the CD44 WT mice.
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, IFN-, IL-2, Fas, and FasL mRNA in the
liver after Con A treatment
Increased mRNA expression of cytokines such as TNF-, IFN-,
and IL-2 have been considered to play a key role in the development of
Con A-induced hepatitis (24). To this end, total liver RNA
was isolated from CD44 WT and CD44 KO mice 2 and 8 h after
treatment of mice with Con A. mRNA was reverse transcribed and
amplified by PCR with primers specific for TNF-, IFN-, IL-2, Fas,
and FasL. Mouse -actin was used as a housekeeping gene in the
experiment. In these experiments, CD44 KO mice were found to exhibit
higher levels of mRNA for TNF-, IFN-, and IL-2 when compared with
CD44 WT mice (Fig. 3). However, mRNA of
Fas and FasL expression in CD44 WT and CD44 KO mice was very similar.
To quantitatively measure the mRNA expression in liver tissue, a
semiquantitative PCR was conducted with the AlphaImager
2000 digital imaging system. At 2 h (Fig. 4A) and 8 h (Fig. 4B) after Con A injection, the percentage of various
cytokine mRNA when compared with -actin levels was measured. The
data shown in Fig. 4 suggested that CD44 KO mice had increased levels
of TNF-, IFN-, and IL-2 mRNA expression but not Fas and FasL when
compared with the CD44 WT mice.
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, and TNF- in
response to in vivo Con A treatment
To further corroborate the increased levels of TNF-, IFN-,
and IL-2 in CD44 KO mice at the protein level, serum samples from Con
A-injected mice were collected 8 and 24 h after Con A
administration. The sera were tested for the presence of cytokines by
ELISA. As seen from Fig. 5, the IFN-
and IL-2 levels were similar in both the CD44 WT and CD44 KO mice
8 h after Con A injection. However, at 24 h, the CD44 KO mice
exhibited significantly higher levels of IFN- and IL-2 when compared
with the CD44 WT mice. The TNF- levels also were significantly
higher in CD44 KO mice at 8 h after Con A injection. However, at
24 h, both the CD44 WT and CD44 KO mice exhibited undetectable
levels of TNF. This may result from use of the TNF produced in
hepatitis induction as suggested by other studies (25, 26). These data confirmed the transcriptional analysis of
cytokine production and suggested that CD44 KO mice exhibited more
cytokine production than CD44 WT mice in response to Con A.
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To rule out the possibility that the increased susceptibility of
CD44 KO mice to hepatitis was caused by differential activation of T
cell subsets when compared with the CD44 WT mice, lymphocyte
subpopulations in spleen and liver infiltration were determined by flow
cytometry (Fig. 6). At different time
points after Con A treatment, spleen cells or liver infiltrating MNC
from CD44 WT and CD44KO mice were harvested and the lymphocyte
subpopulations were screened by flow cytometry (Fig. 6, A–E). There was statistically no significant
difference in the proportions of T cells (CD4+,
CD8+, CD3+), B cells, and
macrophages in the spleen when analyzed 0–36 h after Con A injection
between CD44 WT and CD44 KO mice. Similar results also were obtained
when cells infiltrating the liver were screened (Fig. 6F).
These data suggested that the increased hepatitis seen in CD44 KO mice
was not caused by altered presence of lymphocyte/macrophage
subpopulations.
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Recent studies from our laboratory have demonstrated that
CD44-deficient T cells are more resistant to activation-induced cell
death (AICD; unpublished data). Therefore, we tested the hypothesis
that CD44 KO mice are more susceptible to Con A-induced hepatitis
because the Con A-activated T cells fail to undergo apoptosis, thereby
continuing to elicit hepatitis. To this end, CD44 WT and CD44 KO mice
were injected with Con A, and 8–48 h later, T cells purified from the
spleen were tested for apoptosis by TUNEL assay. In both groups of
mice, early signs of apoptosis were detected at 8 h after Con A
injection (Fig. 7A). The
apoptosis induction peaked at 24 h and declined by 48 h.
Interestingly, at 16 and 24 h after Con A injection, the apoptosis
induction in T cells was markedly higher in CD44 WT mice when compared
with the CD44 KO mice (Fig. 7A). A representative
experiment conducted 24 h after Con A injection has been
depicted in Fig. 7B that indicated clearly that CD44 KO mice
had a significantly lower percentage of apoptotic cells (27.8%) when
compared with CD44 WT mice (60.2%).
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C, when T cells from Con A-injected mice were analyzed at
24 h, the WT mice had a significantly higher proportion of
annexin+ and
annexin+/PI+ cells when
compared with the CD44 KO mice. Furthermore, when purified liver MNC
were analyzed 16 h after Con A injection, the cells from the CD44
KO mice exhibited decreased levels of apoptosis when compared with the
CD44 WT mice (Fig. 8).
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It was possible that T cells from CD44 KO mice were more resistant
to Con A-induced apoptosis because they exhibited decreased
responsiveness to Con A stimulation. To address this, spleen cells from
CD44 WT and CD44 KO mice were stimulated with Con A or anti-CD3
mAbs, and T cell proliferation was measured. As shown in Fig. 9, there was no significant difference in
the ability of T cells from CD44 KO mice to proliferate when compared
with T cells from CD44 WT mice.
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To further corroborate that T cells from CD44 KO mice were more
resistant to apoptosis, T cells from CD44 WT and CD44 KO mice were
cultured in vitro with Con A and the ability of cells to undergo AICD
was studied. The results demonstrated that at 8, 16, and 24 h
after incubation with Con A, more apoptosis was seen in CD44 WT when
compared with CD44 KO mice (Fig. 10).
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B, the CD44 WT T cells
exhibited an increased percentage of apoptotic cells when compared with
T cells from CD44 KO mice. This difference did not result from altered
expression of CD3 because T cells from CD44 KO and CD44 WT mice
exhibited similar levels of CD3 (Fig. 11A). These data
suggested that T cells from CD44 KO mice were more resistant to
TCR-based apoptosis when compared with T cells from CD44 WT
mice.
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It was not clear from the previous experiments whether CD44
expression on immune cells or hepatic cells was playing a critical role
in hepatitis induction. To investigate this, adoptive transfer
experiments were conducted. Twenty-four hours after Con A treatment,
purified T cells from CD44 WT or CD44 KO mice were adoptively
transferred into normal CD44 WT mice (1x108
cells/mouse i.v.). AST levels were measured 24 h after the
adoptive transfer. Transfer of Con A-activated T cells from CD44 WT
mice into normal CD44 WT mice triggered increased AST levels, thereby
indicating that Con A-activated T cells were capable of inducing
hepatitis. Interestingly, similar transfer of Con A-activated T cells
from CD44 KO mice into normal CD44 WT mice induced higher levels of AST
when compared with the WT
WT group (Fig. 12). Transfer of Con A-activated CD44
WT cells into CD44 KO mice induced similar AST levels as the WTWT
group (data not shown). Together, these data demonstrated that Con
A-activated CD44-deficient T cells were able to induce increased levels
of AST when compared with Con A-activated CD44+ T
cells. Also, when Con A-activated CD44+ T cells
were transferred into CD44 WT or CD44 KO mice, the levels of AST were
similar, thereby suggesting that the CD44 expression in the liver did
not influence the hepatitis induction. Thus, CD44 expression on T cells
rather than hepatocytes played a crucial role in Con A-induced
hepatitis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-, which
may contribute to more severe liver damage, as seen in CD44 KO mice
when compared with the CD44 WT mice. Furthermore, adoptive transfer
experiments corroborated this observation by demonstrating that
transfer of activated CD44-deficient spleen cells into WT mice could
induce higher levels of AST when compared with transfer of Con
A-activated CD44 WT spleen cells into CD44 WT mice. These data together
demonstrated that CD44 is actively involved in the Con A-induced
hepatitis.
Con A is a potent T cell mitogen with tropism for the liver. In mice,
Con A induces an acute hepatitis that is a model of T
lymphocyte-mediated liver injury (1). Previous studies
have shown that Con A-induced hepatitis requires IL-2, IFN-, and
TNF- (25, 27). In addition, Fas-FasL interaction
(26) and perforin-granzyme system (28) also
were shown to play an important role in Con A-induced hepatitis. In
this study, we observed that the increased hepatitis seen in CD44 KO
mice correlated with increased production of cytokines such as TNF-,
IL-2, and IFN-, but not Fas or FasL. This increased cytokine
production in CD44 KO mice may result from the fact that Con
A-activated T cells from CD44 KO mice were more resistant to apoptosis
when compared with similar cells from CD44 WT mice. In the current
study, it was noted that the livers from Con A-injected mice exhibited
marked induction of apoptosis. These data are consistent with the
previous studies that FasL is involved in Con A-induced hepatitis
(27). In addition, TNF- also has been shown to induce
hepatotoxicity after Con A injection (26).
CD44 is a family of cell surface glycoproteins with proposed functions in extracellular matrix binding, cell migration, lymphopoeisis, and lymphocyte homing (8). Gantner et al. (25) proposed that in Con A-induced hepatitis, liver-infiltrating T lymphocytes are recruited from the spleen and migrate to the liver where either activated macrophages or T cells may directly cause hepatocyte death. In the current study, we screened different subsets of lymphocytes in spleen and liver. However, we did not find any significant changes in CD44 KO mice when compared with CD44 WT mice. These data excluded the possibility that increased hepatitis seen in CD44 KO mice was caused by the presence of activated lymphocyte subsets. Previous studies have shown that CD44 is involved in the migration of lymphocytes to organs, including the liver (8). However, in an earlier study with CD44 KO mice, we observed in IL-2-induced vascular leak syndrome that the lymphocyte infiltration in lungs and liver was similar to CD44 WT mice (16). A histological study in the current investigation also showed that similar levels of infiltration of cells were seen in the livers of Con A-injected CD44 WT and CD44 KO mice. These data suggested that increased hepatitis seen in CD44 KO mice was not caused by altered migration of lymphocytes to the liver. When liver MNC from Con A-injected mice were screened for apoptosis, cells from CD44 KO mice were found to be more resistant to apoptosis when compared with the cells from CD44 WT mice. It is possible that such resistant cells may contribute toward increased hepatitis as seen in CD44 KO mice.
T cells on activation undergo apoptosis, a process termed as AICD (29, 30). The role of AICD in Con A-induced hepatitis has not been studied previously. In the Con A-induced hepatitis model, how T cells undergo apoptosis on Con A activation may be crucial for mice to recover from the disease. CD44 has been shown to be up-regulated after T cell activation after Con A injection, thereby suggesting that CD44 could be a good candidate for inducing apoptosis. Indeed, we found that CD44-deficient T cells showed increased resistance to apoptosis both in vivo and in vitro, which could lead to prolonged survival of Con A-activated T cells in CD44 KO mice when compared with CD44 WT mice. These data also indicated that CD44 expressed on activated T cells may play a crucial role in AICD. The mechanism by which CD44 participates in AICD is not clear. We have shown previously that T cells on activation express increased levels of CD44 (11, 12). HA has been shown to serve as an important ligand for CD44 (8). Thus, interaction between CD44 and HA on activated T cells may trigger apoptosis. Therefore, T cells deficient in CD44 may fail to undergo apoptosis and continue to produce cytokines, thereby causing enhanced hepatitis. To this end, we recently have generated mice that are deficient in CD44 and Fas. We found that such mice develop more severe lymphoproliferative disease than mice that are CD44+ but Fas-deficient (unpublished data). Furthermore, we have observed that T cells from CD44- Fas- mice are more resistant to AICD than CD44+Fas- mice. In the current study, we also noted that T cells from CD44 KO mice were more resistant to AICD when stimulated with anti-CD3 mAbs. Together, these studies suggested that CD44 may play an important role in AICD.
CD44 is expressed on a wide range of lymphoid and nonlymphoid cells (8). Thus, it was not clear whether CD44 expression in the liver tissue or on lymphocytes was playing a role in Con A-induced hepatitis. To this end, we conducted the adoptive transfer experiment and found that Con A-activated T cells were capable of inducing hepatitis in both WT and CD44 KO mice. In fact, T cells from Con A-injected CD44 KO mice, when transferred into CD44 WT mice, induced increased plasma AST levels when compared with similar cells transferred from CD44 WT mice into CD44 WT mice. It should be noted that the plasma AST levels in adoptive transfer experiments were not as high as the AST levels seen in mice injected directly with Con A. This may be because the transferred cells may not home well. In fact, in a recent report, the authors injected the cells into the spleen to induce significant degree of hepatitis (4). Other investigators have suggested that in addition to the T cells from the spleen (1), NK1+ T cells may play a role in Con A-induced hepatitis in the liver (31). Consistent with the current study, Con A-induced hepatitis has been associated with the infiltration of lymphocytes, monocytes, and neutrophils (32). Although the role of T lymphocytes and macrophages in the pathogenesis of Con A-induced hepatitis has been clearly established (1), the exact role played by neutrophils is not clear. It is possible that neutrophils may accumulate because of the plethora of cytokines produced by Con A-activated T cells.
In summary, the current study demonstrates for the first time that activated T cells may use CD44 to undergo apoptosis. Thus, dysregulation in the expression of CD44 may lead to increased survival of activated T cells, which in turn may trigger enhanced autoimmune reactions.
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Acknowledgments |
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Footnotes |
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2 D.C. and R.J.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Mitzi Nagarkatti, Department of Microbiology and Immunology and Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Box 980678, Richmond, VA 23298-0678.
4 Abbreviations used in this paper: HA, hyaluronic acid; KO, knockout; WT, wild type; AST, aspartate aminotransferase activity; MNC, mononuclear cells; PI, propidium iodide; FasL, Fas ligand; AICD, activation-induced cell death.
Received for publication July 7, 2000. Accepted for publication February 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J. Exp. Med. 191:105. and TNF- production by liver-associated T cells and acute liver injury in rats administered concanavalin A. Immunol. Cell Biol. 76:542.[Medline]
-/- mice, but not in TNF--/- mice: role for IFN- in activating apoptosis of hepatocytes. J. Immunol. 159:1418.[Abstract]
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B. Ruffell and P. Johnson Hyaluronan Induces Cell Death in Activated T Cells through CD44 J. Immunol., November 15, 2008; 181(10): 7044 - 7054. [Abstract] [Full Text] [PDF]
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 V. L. Hegde, S. Hegde, B. F. Cravatt, L. J. Hofseth, M. Nagarkatti, and P. S. Nagarkatti Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids: Involvement of Regulatory T Cells Mol. Pharmacol., July 1, 2008; 74(1): 20 - 33. [Abstract] [Full Text] [PDF]
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M. Ramaswamy, C. Dumont, A. C. Cruz, J. R. Muppidi, T. S. Gomez, D. D. Billadeau, V. L. J. Tybulewicz, and R. M. Siegel Cutting Edge: Rac GTPases Sensitize Activated T Cells to Die via Fas J. Immunol., November 15, 2007; 179(10): 6384 - 6388. [Abstract] [Full Text] [PDF]
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 W. Jia, V. L. Hegde, N. P. Singh, D. Sisco, S. Grant, M. Nagarkatti, and P. S. Nagarkatti {Delta}9-Tetrahydrocannabinol-Induced Apoptosis in Jurkat Leukemia T Cells Is Regulated by Translocation of Bad to Mitochondria Mol. Cancer Res., August 1, 2006; 4(8): 549 - 562. [Abstract] [Full Text] [PDF]
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 L. Zhuo, A. Kanamori, R. Kannagi, N. Itano, J. Wu, M. Hamaguchi, N. Ishiguro, and K. Kimata SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum J. Biol. Chem., July 21, 2006; 281(29): 20303 - 20314. [Abstract] [Full Text] [PDF]
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J. Larkin, G. J. Renukaradhya, V. Sriram, W. Du, J. Gervay-Hague, and R. R. Brutkiewicz CD44 Differentially Activates Mouse NK T Cells and Conventional T Cells J. Immunol., July 1, 2006; 177(1): 268 - 279. [Abstract] [Full Text] [PDF]
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M. N. Ajuebor, A. I. Aspinall, F. Zhou, T. Le, Y. Yang, S. J. Urbanski, S. Sidobre, M. Kronenberg, C. M. Hogaboam, and M. G. Swain Lack of Chemokine Receptor CCR5 Promotes Murine Fulminant Liver Failure by Preventing the Apoptosis of Activated CD1d-Restricted NKT Cells J. Immunol., June 15, 2005; 174(12): 8027 - 8037. [Abstract] [Full Text] [PDF]
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 S. Hayer, G. Steiner, B. Gortz, E. Reiter, M. Tohidast-Akrad, M. Amling, O. Hoffmann, K. Redlich, J. Zwerina, K. Skriner, et al. CD44 is a determinant of inflammatory bone loss J. Exp. Med., March 21, 2005; 201(6): 903 - 914. [Abstract] [Full Text] [PDF]
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 R. S. Hauptschein, K. E. Sloan, C. Torella, R. Moezzifard, M. Giel-Moloney, C. Zehetmeier, C. Unger, L. L. Ilag, and D. G. Jay Functional Proteomic Screen Identifies a Modulating Role for CD44 in Death Receptor-Mediated Apoptosis Cancer Res., March 1, 2005; 65(5): 1887 - 1896. [Abstract] [Full Text] [PDF]
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 R. J. McKallip, M. Fisher, U. Gunthert, A. K. Szakal, P. S. Nagarkatti, and M. Nagarkatti Role of CD44 and Its v7 Isoform in Staphylococcal Enterotoxin B-Induced Toxic Shock: CD44 Deficiency on Hepatic Mononuclear Cells Leads to Reduced Activation-Induced Apoptosis That Results in Increased Liver Damage Infect. Immun., January 1, 2005; 73(1): 50 - 61. [Abstract] [Full Text] [PDF]
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 A. Krettek, G. K. Sukhova, U. Schonbeck, and P. Libby Enhanced Expression of CD44 Variants in Human Atheroma and Abdominal Aortic Aneurysm: Possible Role for a Feedback Loop in Endothelial Cells Am. J. Pathol., November 1, 2004; 165(5): 1571 - 1581. [Abstract] [Full Text] [PDF]
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Y. Do, R. J. McKallip, M. Nagarkatti, and P. S. Nagarkatti Activation through Cannabinoid Receptors 1 and 2 on Dendritic Cells Triggers NF-{kappa}B-Dependent Apoptosis: Novel Role for Endogenous and Exogenous Cannabinoids in Immunoregulation J. Immunol., August 15, 2004; 173(4): 2373 - 2382. [Abstract] [Full Text] [PDF]
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 K. M.A. Rouschop, M. E. Sewnath, N. Claessen, J. J.T.H. Roelofs, I. Hoedemaeker, R. van der Neut, J. Aten, S. T. Pals, J. J. Weening, and S. Florquin CD44 Deficiency Increases Tubular Damage But Reduces Renal Fibrosis in Obstructive Nephropathy J. Am. Soc. Nephrol., March 1, 2004; 15(3): 674 - 686. [Abstract] [Full Text] [PDF]
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 Y. Do, A. Q. Rafi-Janajreh, R. J. Mckallip, P. S. Nagarkatti, and M. Nagarkatti Combined deficiency in CD44 and Fas leads to exacerbation of lymphoproliferative and autoimmune disease Int. Immunol., November 1, 2003; 15(11): 1327 - 1340. [Abstract] [Full Text] [PDF]
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R. J. McKallip, M. Fisher, Y. Do, A. K. Szakal, U. Gunthert, P. S. Nagarkatti, and M. Nagarkatti Targeted Deletion of CD44v7 Exon Leads to Decreased Endothelial Cell Injury but Not Tumor Cell Killing Mediated by Interleukin-2-activated Cytolytic Lymphocytes J. Biol. Chem., October 31, 2003; 278(44): 43818 - 43830. [Abstract] [Full Text] [PDF]
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X.-M. Xu, Y. Chen, J. Chen, S. Yang, F. Gao, C. B. Underhill, K. Creswell, and L. Zhang A Peptide with Three Hyaluronan Binding Motifs Inhibits Tumor Growth and Induces Apoptosis Cancer Res., September 15, 2003; 63(18): 5685 - 5690. [Abstract] [Full Text] [PDF]
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 C.-M. Hogerkorp, S. Bilke, T. Breslin, S. Ingvarsson, and C. A. K. Borrebaeck CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays Blood, March 15, 2003; 101(6): 2307 - 2313. [Abstract] [Full Text] [PDF]
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W. R. Waters, T. E. Rahner, M. V. Palmer, D. Cheng, B. J. Nonnecke, and D. L. Whipple Expression of L-Selectin (CD62L), CD44, and CD25 on Activated Bovine T Cells Infect. Immun., January 1, 2003; 71(1): 317 - 326. [Abstract] [Full Text] [PDF]
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G. Matsumoto, S. Tsunematsu, K.-i. Tsukinoki, Y. Ohmi, M. Iwamiya, A. Oliveira-dos-Santos, D. Tone, J. Shindo, and J. M. Penninger Essential Role of the Adhesion Receptor LFA-1 for T Cell-Dependent Fulminant Hepatitis J. Immunol., December 15, 2002; 169(12): 7087 - 7096. [Abstract] [Full Text] [PDF]
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R. A. O'Connor and E. Devaney Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae of Brugia pahangi Infect. Immun., November 1, 2002; 70(11): 5997 - 6004. [Abstract] [Full Text] [PDF]
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R. J. McKallip, Y. Do, M. T. Fisher, J. L. Robertson, P. S. Nagarkatti, and M. Nagarkatti Role of CD44 in activation-induced cell death: CD44-deficient mice exhibit enhanced T cell response to conventional and superantigens Int. Immunol., September 1, 2002; 14(9): 1015 - 1026. [Abstract] [Full Text] [PDF]
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R. J. McKallip, C. Lombard, M. Fisher, B. R. Martin, S. Ryu, S. Grant, P. S. Nagarkatti, and M. Nagarkatti Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease Blood, June 28, 2002; 100(2): 627 - 634. [Abstract] [Full Text] [PDF]
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M. I. Tammi, A. J. Day, and E. A. Turley Hyaluronan and Homeostasis: A Balancing Act J. Biol. Chem., February 8, 2002; 277(7): 4581 - 4584. [Full Text] [PDF]
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E. A. Turley, P. W. Noble, and L. Y. W. Bourguignon Signaling Properties of Hyaluronan Receptors J. Biol. Chem., February 8, 2002; 277(7): 4589 - 4592. [Full Text] [PDF]
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