The selective ablation of interleukin 2-producing cells isolated from transgenic mice.

To better understand the requirement for interleukin 2 (IL-2) in specific immune responses, we have established the use of cell ablation to selectively eliminate T cells that produce IL-2. To accomplish this we have generated transgenic mice that express the herpes simplex virus 1-thymidine kinase (HSV-TK) gene under the transcriptional control of the murine IL-2 promoter that renders IL-2-producing cells sensitive to the cytotoxic effects of the antiviral drug ganciclovir (GANC). HSV-TK activity was specifically expressed in activated T cells from transgenic mice. When CD4 T cells from transgenic mice were stimulated with the superantigen staphylococcal enterotoxin A (SEA) in the presence of GANC, proliferation and IL-2 production were almost completely inhibited and the activated CD4+V beta 3+ T cell population, eliminated. Proliferation was not restored by adding IL-2, showing that most proliferating cells are not bystander cells. In contrast, the proliferative response to concanavalin A (Con A) was only partially inhibited by treatment of CD4 T cells with GANC, although the efficiency of eliminating IL-2-producing cells was shown to be comparable with that achieved using SEA. This suggests that a portion of the proliferative response to Con A occurs via an alternative pathway not requiring IL-2 synthesis and release.

GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells.

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.

Dystrophin-deficient myocardium is vulnerable to pressure overload in vivo.

OBJECTIVE: Dystrophin provides mechanical reinforcement to the membranes of myocytes. Dystrophin abnormalities are known to cause cardiomyopathy and skeletal muscle disorders; however, the pathogenesis of these abnormalities remains unclear. Dystrophin-deficient skeletal muscle is vulnerable to stresses such as stretch and hypo-osmotic shock. We investigated whether the myocardium of dystrophin-deficient (mdx) mice shows increased vulnerability to acute pressure overload in vivo. METHODS AND RESULTS: Abdominal aortic banding was performed in 12-week-old mdx and control mice. The aortic pressure was measured by cannulation of the right carotid artery at the time of sacrifice. Systolic pressures in mdx mice at 0, 1, 2, 7 and 14 days after aortic banding were 100 +/- 11, 119 +/- 7, 123 +/- 4, 134 +/- 11 and 130 +/- 10 mmHg, respectively. Microscopic analysis revealed focal lesions in the left ventricular wall in banded mdx mice. These lesions consisted of damaged myocytes and inflammatory cells, and also of fibrosis at a late stage. Similar lesions were not observed in non-banded or banded control mice. The proportion of areas of lesions to total left ventricular area increased over time: 1.0 +/- 0.6% in mdx mice without aortic banding (sham, n = 6), and 1.7+/-1.4% 1 day (n = 6, vs. sham, NS), 2.6 +/- 1.9% 2 days (n = 7, vs. sham, P < 0.05), 6.3+ /- 6.5% 7 days (n = 13, vs. sham, P < 0.05) and 9.9 +/- 8.3% 14 days after aortic banding (n=15, vs. sham, P < 0.01). Furthermore, linear regression analysis revealed a significant correlation between percentage of lesion area and systolic pressure in mdx mice (P < 0.05). CONCLUSION: Dystrophin-deficient myocardium is more vulnerable than normal myocardium to pressure overload in vivo. This result has two clinical implications: (1) the patients with dystrophynopathy, such as the Duchenne and the Becker types of muscular dystrophy and X-linked type of dilated cardiomyopathy, who develop arterial hypertension should be treated aggressively, and (2) they should avoid stresses that elevate blood pressure.

[Involvement of Fas and Fas ligand in case of human fulminant hepatitis].

Fas antigen has been found to be a cell surface molecule which can induce apoptosis. Since it has been reported that administration of anti-Fas monoclonal antibody into mouse peritoneal cavity causes fulminant hepatitis (FH) in vivo, the Fas-Fas ligand system is thought to play important roles in massive hepatocytes death in FH. In murine system, there are several reports which showed Fas-Fas ligand system involved in FH. In human, the expression of Fas antigen was markedly increased on the cell surface of the hepatocytes and Fas ligand was expressed on the hepatic infiltrated lymphocytes. It is suggested that the Fas-Fas ligand system also play important roles in human FH.

Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells.

Naive CD4+ T cells initially transcribe both IL-4 and IFN-gamma when stimulated with either the mitogen Con A or Ag in the presence of IL-4 or IL-12 and, therefore, appear uncommitted as to the pathway of differentiation they will follow. However, when stimulated either with Con A or with Ag in the presence of IL-4, CD4+ T cells become primed to follow the Th2 differentiation pathway, and we show now that by 48 h of culture in this environment these cells extinguish IFN-gamma gene transcription. Likewise, priming in the presence of IL-12 leads to the development of Th1 cells, which switch off the expression of the IL-4 gene. To clarify the Th1 differentiation pathway, we performed ablation studies using IL-4 thymidine kinase transgenic mice. When the antiviral drug ganciclovir was added 1 day after primary stimulation in the presence of IL-12, IFN-gamma- and IL-4-producing cells were ablated. In contrast, when ganciclovir was added 2 days after primary stimulation, IL-4-producing cells, but not IFN-gamma-producing cells, were ablated. Thus, our studies show that by 48 h after activation, Th1 or Th2 cells have already become polarized to the differentiation pathway that they will follow. As the differentiation toward Th1 and Th2 effector cells proceeds, substantial amounts of IFN-gamma and IL-4 mRNA accumulate, while the mRNAs of the corresponding lineage (i.e., IFN-gamma in the case of Th2 cells, and IL-4 in the case of Th1 cells) diminish to undetectable levels. IL-4R is up-regulated during T cell differentiation by a mechanism mediated mainly by IL-4. The fact that IL-12 priming does not suppress IL-4-dependent IL-4R up-regulation shows that both IL-4 mRNA and cytokine are produced by IL-12-primed naive CD4+ T cells during differentiation into Th1 cells. Naive CD4+ T cells, therefore, begin as uncommitted cells which express both Th1 and Th2 cytokines that rapidly extinguish the expression of the inappropriate cytokine as the commitment toward the effector lineages is made.

Regulation of CD4+ T cell differentiation.

Naive T cells can be induced to differentiate from an uncommitted precursor to T helper 1 (Th1) and Th2 cells. During this differentiation, genes for transcription factors are activated, and transcription factors such as AP-1 accumulate. To study this activation, we have developed reporter transgenic mice for a number of factors, including AP-1. Naive T cells require two signals to activate AP-1. However, upon becoming effector cells, activation through diacylglycerol analogues is sufficient. Rested effector cells lose accumulated AP-1, and the induction of AP-1 synthesis requires both Ca2+ diacylglycerol signals, but not co-stimulation.

Hepatitis B virus-DNA transfected myeloma cell-specific cytotoxic T cells in chronic hepatitis B patients.

To study the mechanisms of hepatitis B virus (HBV)-induced chronic hepatitis (B-CH), we took chronic hepatitis B patients' peripheral blood lymphocytes (PBL) and examined their cytotoxic activities against human myeloma cells (ARH77) transfected by HBV-DNA. Two different transfected cells, one expressing HBV envelope antigens (S6) and the other expressing HBV core antigens (C4), were prepared and used as targets in the in vitro cytotoxic test. We found that PBL of B-CH patients had specific cytotoxic activity against these target cells (S6, 22.0 +/- 4.8%; C4, 21.6 +/- 4.8%), whereas no remarkable cytotoxic activity was observed in non-B chronic hepatitis patients as well as asymptomatic chronic HBV carriers. These specific cytotoxic activities were inhibited with anti-CD3 antibody, hence these killer cells belonged to T cells (cytotoxic T cells; CTL). The requirement of HLA class 1 antigens to exert these CTL activities was demonstrated by the absence of CTL activity with PBL obtained from HLA-nonidentical B-CH patients and by the inhibition of their activities with anti-HLA class 1 antibody. Thus, our results indicate that, at least two different CTL, one recognizing envelop antigen and the other recognizing core antigen, exist in chronic hepatitis B patients.

The relationship of IL-4- and IFN gamma-producing T cells studied by lineage ablation of IL-4-producing cells.

Subsets of CD4 T cells are defined by the cytokines that they produce; these cytokines determine the effector function of these cells. Cloned CD4 T cells fall into two subsets, producing either interferon-gamma (IFN gamma) or interleukin-4 (IL-4) in combination with other cytokines, and are called Th1 and Th2 cells, respectively. The lineage relationship between naive T cells and effector Th1- and Th2-type cells is unclear. We generated transgenic mice in which IL-4-producing cells express herpes simplex virus 1 thymidine kinase and are eliminated by ganciclovir (GANC). Activation of transgenic T cells in the presence of GANC eliminates IL-4 and IFN gamma production, showing that IL-4- and IFN gamma-producing cells express or have expressed IL-4. These results show that effector cells producing either IL-4 or IFN gamma have a common precursor, which expresses the IL-4 gene.

Effect of alpha-interferon on hepatitis B virus-specific cytotoxic T cells.

To study the mechanism of the effects of alpha-interferon (alpha-IFN) on chronic hepatitis B, we examined its effect on hepatitis B virus (HBV)-specific cytotoxic T cells (CTL). Using two different HBV-DNA transfected human myeloma cell lines, one expressing hepatitis B core antigen (HBcAg; C4) and the other expressing hepatitis B surface antigen (HBsAg; S6) as targets in cytotoxic tests in vitro, peripheral blood mononuclear cells obtained from chronic hepatitis B patients who were treated with alpha-IFN were examined for their cytotoxic activity against these transfectants. During the treatment with alpha-IFN, in association with a decline of serum alanine amino transferase levels, CTL activities were significantly reduced. An inhibition study in vitro revealed that alpha-IFN did not directly inhibit these CTL activities, indicating that alpha-IFN may inhibit the induction of CTL, and thereby may be related to the reduction of hepatocyte injury.

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure.

OBJECTIVE: The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients' serum. METHODS: On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. RESULTS: By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. CONCLUSIONS: The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

A conditionally active form of STAT6 can mimic certain effects of IL-4.

Binding of IL-4 to its cognate receptor leads to the activation of a number of signaling pathways within the cell. Activation of the transcription factor STAT6 by JAK family protein tyrosine kinases has been shown to be essential for the full response of cells to IL-4. To elucidate the role of STAT6 in IL-4 signaling, we have constructed and expressed in cells a conditionally active form of the protein (STAT6:ER*) by fusing STAT6 to a modified form of the hormone-binding domain of the estrogen receptor. Activation of STAT6:ER* by 4-hydroxytamoxifen leads to specific activation of STAT6-regulated gene expression including the activation of a STAT6 reporter construct and induction of CD23 in B cell lines. Interestingly, in contrast to native STAT6, activation of STAT6:ER* occurs in the absence of detectable tyrosine phosphorylation of the fusion protein. This type of conditional system will be helpful in dissecting the mechanisms and specificity of transcriptional regulation by the STAT family of transcription factors.

Effect of B7.1-transfected human colon cancer cells on the induction of autologous tumour-specific cytotoxic T cells.

BACKGROUND: The induction of tumour-specific immunity is important for advanced cancer therapy. There are many molecules, including costimulatory molecules, that have been identified as the activator for tumour-specific T cells. METHODS: To induce autologous tumour-specific cytotoxic T lymphocytes (CTL) more effectively, we studied whether the expression of the B7 gene may render human colon cancer cells able to stimulate autologous peripheral blood mononuclear cells (PBMC) to become tumour-specific cytotoxic T cells. After the establishment of a B7.1 gene transfected tumour cell line, Cw2/B7.1, we first examined its stimulatory effect on autologous PBMC and subsequently, its effect on the induction of parental cell (Cw2)-specific CTL. RESULTS: The results showed that Cw2/B7.1 had a more potent stimulatory effect on PBMC for the induction of both proliferation and cytotoxicity than Cw2. By adding a low dose of interleukin-2, Cw2/B7.1-activated killer cell activity was significantly increased. The specificity of Cw2/B7.1-activated killer cells was demonstrated by the absence of their cytotoxicity to either human lymphocyte antigen (HLA)-A33 identical (ORF) or HLA-non-identical (MT) allogenic colon cancer cell lines. Furthermore, such Cw2-specific cytotoxic activity was significantly reduced by the deletion of CD8+ cells but not CD4+ cells, indicating that these killer cells were mainly CD8+ T cells. CONCLUSIONS: Thus, our results demonstrate that, by using B7.1 gene-transfected tumour cell lines, we effectively induced autologous tumour-specific CTL. These results will provide us with new tools for adoptive immunotherapy for colon cancer patients.

Repeated thermal therapy upregulates arterial endothelial nitric oxide synthase expression in Syrian golden hamsters.

It has been previously reported that sauna therapy, a thermal therapy, improves the hemodynamics and clinical symptoms in patients with chronic heart failure and also improves endothelial function, which is impaired in such patients. The present study investigated whether the improvements observed with sauna therapy are through modulation of arterial endothelial nitric oxide synthase (eNOS) expression. Eight male Syrian golden hamsters underwent sauna therapy, using an experimental far infrared-ray dry sauna system, at 39 degrees C for 15 min followed by 30 degrees C for 20 min daily for 4 weeks. Control group hamsters were placed in the sauna system switched off at room temperature of 24 degrees C for 35 min. Immunohistochemistry found greater amounts of the immunoreactive products of eNOS in the endothelial cells of the aorta and carotid, femoral and coronary arteries in the sauna group than in the control group. Western blot analysis also revealed that 4-week sauna therapy significantly increased eNOS expression in aortas by 50% in 4 series of independent experiments with an identical protocol (p<0.01). In reverse transcription polymerase chain reaction assay, the eNOS mRNA in aortas was greater in the sauna group than in controls, with a peak at 1-week of sauna therapy (approximately 40-fold increase). In conclusion, repeated thermal therapy upregulates eNOS expression in arterial endothelium.

Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster.

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.