Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage.

Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.

Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects.

Epidermal Langerhans cells (LC) are associated anatomically with epidermal nerves, and a product of these nerves, calcitonin gene-related peptide (CGRP), inhibits the antigen-presenting capacity of LC and macrophages. As the CGRP receptor appears to be coupled to Gs alpha protein, which in turn activates adenylate cyclase, the ability of CGRP to induce cAMP in LC was examined and correlated with functional effects. LC were isolated from murine epidermal cells using antibodies on magnetic microspheres. Exposure to CGRP induced a significant increase in cAMP content, which could be inhibited by coculture with a truncated form of CGRP [CGRP-(8-37)] that is a specific competitive inhibitor of CGRP. Substance P and calcitonin failed to induce cAMP in LC. Although culture in CGRP reduced the ability of murine epidermal cells enriched for LC content to present pigeon cytochrome c to a responsive clone or to present antigen for elicitation of delayed-type hypersensitivity in immune mice, culture in forskolin had little or no effect on antigen presentation despite increased cAMP content of LC as much or more than that induced by CGRP. The effect of CGRP on antigen presentation in these systems could be blocked with CGRP-(8-37). CGRP inhibited the induction of B7-2 by lipopolysaccharide on peritoneal macrophages and a LC line, whereas calcitonin did not. CGRP induces specific accumulation of cAMP in LC and inhibits LC antigen-presenting function by a receptor-mediated event. However, the induction of cAMP by itself does not account for inhibition of antigen presentation. Suppression of the expression of B7-2 may be one mechanism by which CGRP inhibits antigen presentation.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Induction of mammary epithelial hyperplasias and mammary tumors in transgenic mice expressing a murine mammary tumor virus/activated c-src fusion gene.

Activation of the c-Src tyrosine kinase has been implicated as an important step in the induction of mammary tumors in both mice and humans. To directly assess the effect of mammary gland-specific expression of activated c-Src, we established transgenic mice that carry a constitutively activated form of c-src under transcriptional control of the murine mammary tumor virus long terminal repeat. Female mice derived from several independent transgenic lines lactate poorly as a consequence of an impairment in normal mammary epithelial development. In addition to this lactation defect, female mice frequently develop mammary epithelial hyperplasias, which occasionally progress to frank neoplasias. Taken together, these observations suggest that expression of activated c-Src in the mammary epithelium of transgenic mice is not sufficient for induction of mammary tumors.

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences.

B-lymphocyte-specific class switch recombination is known to occur between pairs of 2- to 10-kb switch regions located immediately upstream of the immunoglobulin constant heavy-chain genes. Others have shown that the recombination is temporally correlated with the induction of transcription at the targeted switch regions. To determine whether this temporal correlation is due to a mechanistic linkage, we have developed an extrachromosomal recombination assay that closely recapitulates DNA deletional class switch recombination. In this assay, the rate of recombination is measured between 24 and 48 hr posttransfection. We find that recombinants are generated in a switch sequence-dependent manner. Recombination occurs with a predominance within B-cell lines representative of the mature B-cell stage and within a subset of pre-B-cell lines. Transcription stimulates the switch sequence-dependent recombination. Importantly, transcription activates recombination only when directed in the physiologic orientation but has no effect when directed in the nonphysiologic orientation.

Aod1, the immunoregulatory locus controlling abrogation of tolerance in neonatal thymectomy-induced autoimmune ovarian dysgenesis, maps to mouse chromosome 16.

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.

Bclx regulates the survival of double-positive thymocytes.

The bclx gene has been shown to regulate programmed cell death in vitro. We now show that Bclx expression increases dramatically when T cells differentiate from CD4- CD8- (double negative) thymocytes to CD4+ CD8+ [double positive (DP)] thymocytes. In contrast single-positive (SP) thymocytes express negligible amounts of Bclx protein. This expression pattern contrasts with that of Bcl2, which is present in double-negative thymocytes, down-regulated in DP thymocytes, and reinduced upon maturation to SP thymocytes. Elimination of Bclx by gene targeting dramatically shortens the survival of DP thymocytes but not the survival of SP thymocytes or peripheral SP T cells. These data suggest that the induction of Bclx during thymic maturation plays a critical role in regulating the length of time DP thymocytes survive in the absence of selection.

Antitumor promotion by phenolic antioxidants: inhibition of AP-1 activity through induction of Fra expression.

Induction of phase 2 detoxification enzymes by phenolic antioxidants can account for prevention of tumor initiation but cannot explain why these compounds inhibit tumor promotion. Phase 2 genes are induced through an antioxidant response element (ARE). Although the ARE resembles an AP-1 binding site, we show that the major ARE binding and activating protein is not AP-1. Interestingly, AP-1 DNA binding activity was induced by the phenolic antioxidant tert-butylhydroquinone (BHQ), but the induction of AP-1 transcriptional activity by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by this compound. BHQ induced expression of c-jun, junB, fra-1, and fra-2, which encode AP-1 components, but was a poor inducer of c-fos and had no effect on fosB. Like c-Fos and FosB, the Fra proteins heterodimerize with Jun proteins to form stable AP-1 complexes. However, Fra-containing AP-1 complexes have low transactivation potential. Furthermore, Fra-1 repressed AP-1 activity induced by either TPA or expression of c-Jun and c-Fos. We therefore conclude that inhibitory AP-1 complexes composed of Jun-Fra heterodimers, induced by BHQ, antagonize the transcriptional effects of the tumor promoter TPA, which are mediated by Jun-Fos heterodimers. Since AP-1 is an important mediator of tumor promoter action, these findings may explain the anti-tumor-promoting activity of phenolic antioxidants.