CD44-deficient mice exhibit enhanced hepatitis after concanavalin A injection: evidence for involvement of CD44 in activation-induced cell death.

Administration of Con A induces severe injury to hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) caused 100% mortality in C57BL/6 CD44-knockout (KO) mice, although it was not lethal in C57BL/6 CD44 wild-type (WT) mice. Administration of lower doses of Con A (12 mg/kg body weight) into CD44 WT mice induced hepatitis as evident from increased plasma aspartate aminotransferase levels accompanied by active infiltration of mononuclear cells and neutrophils, and significant induction of apoptosis in the liver. Interestingly, CD44 KO mice injected with similar doses of Con A exhibited more severe acute suppurative hepatitis. Transfer of spleen cells from Con A-injected CD44 KO mice into CD44 WT mice induced higher levels of hepatitis when compared with transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44 KO mice was accompanied by increased production of cytokines such as TNF-alpha, IL-2 and IFN-gamma, 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.

Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus.

In an attempt to delineate the immunological alterations that may occur following treatment with estrogen, groups of C57BL/6 mice were treated with 75mg/kg body weight of beta-estradiol-17-valerate (E2) or the vehicle. The thymus from these mice were harvested on days 1, 4 and 7 following treatment. The thymocytes from E2-treated mice when cultured in vitro for 24h, showed increased levels of apoptosis when compared to controls. The apoptosis was demonstrable by both TUNEL assay and AnnexinV/propidium iodide (PI) staining. Also, thymic atrophy and increased apoptosis of thymocytes when cultured in vitro were seen when lower doses of E2 (5mg/kg) were administered. The thymus from E2-treated mice on days 4 and 7 also showed a decrease in the percentage of CD4(+)CD8(+) (DP) T cells and an increase in the percentage of CD4(-)CD8(-) (DN), CD4(+) and CD8(+) T cells. However, the total cellularity of all T cell subsets in the thymus was decreased following E2 treatment. Earlier studies from our laboratory and elsewhere have demonstrated that in thymocytes undergoing apoptosis, there is increased expression of surface markers including CD3, alphabetaTCR and CD44 with a simultaneous decrease in the expression of J11d. Similar changes were observed in thymocytes from mice on days 4 and 7 following E2 treatment. These data therefore confirmed that the thymocytes were indeed undergoing apoptosis following E2 treatment. Together, our studies suggest for the first time that estrogen may induce thymic atrophy by triggering apoptosis.

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.

Structural characterization and in vivo immunosuppressive activity of neuroblastoma GD2.

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, GD2. GD2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of GD2 in vivo were then determined in an established murine model. GD2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus GD2-treated mice; p < 0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2 x 10(6) cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as GD2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.

Immunotoxicity of AZT: inhibitory effect on thymocyte differentiation and peripheral T cell responsiveness to gp120 of human immunodeficiency virus.

3'-Azido-2',3'-dideoxythymidine (AZT) is extensively used in the treatment of human immunodeficiency virus (HIV) infection. Currently, it is debated whether AZT should be offered to symptomless HIV-infected individuals in the hope of delaying or even preventing progression to acquired immunodeficiency syndrome (AIDS). However, before the chronic use of AZT, it is essential to establish whether this drug alters the differentiation and functions of T cells particularly because HIV infects CD4+ T cells, as well as investigate whether AZT would alter the T cell response to HIV-encoded antigens. In the current study, therefore, we investigated the effect of administration of AZT into mice for 7-14 days on T cell differentiation in the thymus and the ability of T cells to respond to HIV antigens in the periphery. Our data demonstrated that AZT, when administered orally at 500 mg/kg body wt or higher concentrations for 14 days, caused marked thymic involution with significant decrease in the percentage of CD4+CD8+ T cells and increase in the percentage of CD4-CD8-, CD4+, and CD8+ T cells. AZT treatment did not affect the cellularity of the spleen or the ratios of T cells in the periphery. Also, splenic T cells from AZT-treated mice did not demonstrate marked decrease in their ability to respond to mitogens in vitro. However, when AZT-treated mice were immunized with foreign antigens, such as gp120 of HIV or conalbumin, the T cells demonstrated significant decrease in their ability to respond to these antigens. When AZT was added to cultures, it was found to inhibit the proliferation of T cells to mitogens as well as the differentiation of cytotoxic T lymphocytes (CTL). Interestingly, addition of exogenous IL-2 to CTL cultures reconstituted the decreased CTL response. Furthermore, administration of IL-2 into AZT-treated mice could also reconstitute the decrease in thymic cellularity induced by AZT. These data indicate that AZT, when present at the time of T cell differentiation or responsiveness to antigens in vivo, can mediate significant inhibition of such functions thereby suggesting that AZT may affect the immune response to HIV antigens.