17ß-Estradiol (E-2) administration to male (NZB × SWR)F1 mice results in increased Id(LN)F1-reactive memory T-lymphocytes and accelerated glomerulonephritis.

While it has been shown that estradiol treatment accelerates the onset of lupus nephritis with autoantibody production and kidney damage in both male and female lupus-prone mice, the specific mechanism(s) involved are unknown. Our previous work has shown that alterations in Id(LN)F(1)-reactive T cells and Id(LN)F(1)+ antibodies correlated closely with the onset of autoimmune nephritis in female F(1) progeny of SWR and NZB (SNF(1)) mice, supporting a critical role for the Id(LN)F(1) idiotype in the development of disease. Since male SNF(1) mice normally do not develop nephritis, we tested whether administration of 17ß-estradiol (E-2) to male SNF(1) mice would increase Id(LN)F(1) IgG levels and autoreactive T cells, and further, induce nephritis. We found that E-2-treated male SNF(1) mice developed nephritis with the same time course and mean survival as normal female SNF(1) mice. Moreover, it appeared that the mechanism involved increased serum Id(LN)F(1)(+)IgG and its deposition in kidney glomeruli, preceded by a striking twofold increase in T-lymphocytes expressing the memory phenotype (CD44(+)CD45RB(lo)) predominantly in the Id(LN)F(1)-reactive T-cell population. In addition, we noted that cells with this phenotype were increased in the nephritic kidneys of treated mice, suggesting a direct involvement of those cells in the renal pathology. E-2 treatment also induced increased numbers of pathogenic Id(LN)F(1)+ antibody-producing B cells and elevated presentation of pathogenic Id(LN)F(1)+ peptide. Taken together, these results suggest a mechanism of E-2-induced acceleration of autoimmune disease in lupus-prone mice may involve expansion of autoreactive idiotypic T and B-cell populations.

Terminal deoxynucleotidyl transferase is found in prothymocytes.

Terminal deoxynucleotidyl transferase is an enzyme which has the unique property of polymerizing polydeoxynucleotides onto a primer in the absence of a template (1,2). This enzyme is found both in the thymus and the bone marrow of birds, rodents, and humans (3-7). Whether the marrow cells that contain terminal transferase are related to thymocytes, or are on a separate pathway of differentiation, is not yet known (7,8). To determine the lineage of the murine bone marrow cells that have terminal transferase, we have investigated whether these cells have the antigen Thy-1 induced on the cells by treatment with thymopoietin (9). Thymopoietin is known to induce a set of characteristic T-cell markers including the Thy-1 alloantigen on the surface of a subpopulation of bone marrow cells committed to T-cell differentiation (prothymocytes) (10). Destruction of Thy- 1-positive cells after exposure to thymopoietin allows elimination of a substantial fraction of those bone marrow cells that can repopulate an irradiated thymus (11). We find that such an elimination after induction with the thymic polypeptide removes a substantial amount of terminal transferase from the bone marrow cell population, suggesting that at least one-half of the marrow cells bearing this enzyme are related to those found in the thymus.

Expression of murine gamma fetal antigen in adult hematopoietic tissue and during induced differentiation of Friend erythroleukemia cells.

Quantitative analysis of extracts of various normal adult CD-1 mouse tissues indicated that the serologically defined murine gamma fetal antigen (gamma-FA) was expressed at high levels in hematopoietic tissue in general and in bone marrow (BM) in particular. Metabolic labeling of isolated BM cells indicated that the BM was a site of gamma-FA synthesis in the adult animal. The size(s) of the antigen immunoprecipitated from labeled BM cells (35 and 27 kilodaltons) with anti-gamma-FA serum correlated well with molecular weight estimates of fibrosarcoma-fetal mouse-associated gamma-FA, as determined by molecular sieve chromatography. For ascertainment of the relationship between hematopoietic cell differentiation and gamma-FA content, a multiparameter flow cytometric approach was used to evaluate gamma-FA levels in Friend erythroleukemia (FL) cells as a function of growth state (blast or dimethyl sulfoxide-differentiated) and cell-cycle compartment. Differentiated G1-arrested FL cells (G1D) possessed significantly lower gamma-FA-associated immunofluorescence as compared to control cells in the G0-G1 substate. Remaining S- and G2 + M-phase cells in differentiated populations demonstrated an even greater reduction in gamma-FA content relative to control cells in the corresponding cell-cycle phases. The available data support the tentative classification of gamma-FA as a murine differentiation antigen.

Relative subunit composition of the ferritin synthesized by selected human lymphomyeloid cell populations.

Biosynthesis of ferritin subunits by cell sets isolated from normal human peripheral blood, spleens of Hodgkin's disease patients, and tumor cell lines were investigated. Normal mature hematopoietic cells made a ferritin with more H (21K) than L (19K) subunits. The reverse was found for a promyelocytic tumor cell line and tumor cell lines derived from other tissues. Two dimensional electrophoresis indicated H has a lower pI than L. Therefore relative proportions of the two subunits contribute to the electrophoretically distinct forms of the isoferritins. In response to increasing concentrations of iron in vitro, a selected monocyte population synthesized more H than L; L biosynthesis however increased more than H. Some possible regulatory implications of these observations are discussed.

Induction of lymphocyte differentiation by epidermal cultures.

Human and murine lymphoid cell populations were induced to express terminal deoxynucleotidyl transferase, a marker of early lymphoid differentiation, by exposure to allogeneic or syngeneic epidermal cells. Control growth medium, fibroblasts, or a mammary epithelial cell line did not induce this marker. These findings suggest that epidermal cells can induce lymphoid cell differentiation in vitro.

Role of estrogen receptor alpha in hematopoietic stem cell development and B lymphocyte maturation in the male mouse.

Although estrogens and estrogen receptors (ERs) are known to function in the male brain and reproductive tract, few studies have evaluated their involvement in the male hematopoietic and immune systems. This study was undertaken to determine the role of ERalpha in hematopoietic progenitor and B lymphocyte maturation. ERalpha knockout (ER-/-), wild-type (ER+/+), and radiation chimeric (ERalpha positive or negative in either nonhematopoietic or hematopoietic elements, or both) male mice were used to determine target tissues. ER-/- and ER+/+ animals showed similar hematopoietic progenitor profiles, but the ER-/- animals had fewer cells in all bone marrow B lymphocyte subpopulations. Animals receiving a pharmacological dose (5 mg/kg BW) of 17beta-estradiol (E2) with both elements, ER+/+, had decreased early hematopoietic progenitors and a shift toward a mature B cell subpopulation, whereas animals with both elements, ER-/-, showed changes only in early hematopoietic progenitors. Hematopoietic element ER+/+ animals exhibited greater E2-induced hematopoietic progenitor and B lymphocyte alterations than those having only nonhematopoietic ERalpha. These data indicate that 1) ERalpha is not necessary for regulating male mouse normal hematopoietic progenitor cell proportions, but is involved in B cell regulation; and 2) ERalpha in hematopoietic elements is predominantly responsible for mediating E2-induced hematopoietic and B cell changes.

HIV-1 infection abolishes CD4 biosynthesis but not CD4 mRNA.

In order to improve understanding of how HIV-1 infection down-modulates cell surface membrane expression of CD4, we have measured several parameters of CD4 expression in the human tumor T-cell lines CEM and MOLT-4 at different times after infection. Three independent HIV-1 isolates were used including one that encodes a truncated nef protein and another that appeared to be noncytolytic against CEM. The level of CD4 mRNA, the rate of biosynthesis of CD4 protein, and the percentage of CD4-positive cells were measured. With each viral isolate it was found that infection led to a specific and almost complete inhibition of CD4 protein biosynthesis. This substantially exceeded, at every time point after infection, a concomitant reduction in CD4 mRNA. Hence an inhibition of translation probably accounts for much of the decline in the rate of CD4 biosynthesis. This implicates a novel selective translational inhibition of host gene expression by HIV-1 as a factor in the disappearance of surface membrane CD4 from infected cultures.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced thymic atrophy and lymphocyte stem cell alterations by mechanisms independent of the estrogen receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has both agonist and antagonist effects on estrogen-mediated activities and estrogen receptor (ER) levels in epithelial tissues following exposure. We previously demonstrated that TCDD alters bone marrow lymphocyte stem cells, including prothymocytes, as measured by functional assays and alterations in the lymphocyte stem cell-specific markers terminal deoxynucleotidyl transferase (TdT) and recombinase activating gene-1 (RAG-1). We have also shown that 17 beta-estradiol valerate (E2V) affects lymphocyte stem cells by reducing TdT and RAG-1 mRNA. It has been suggested that the effect of TCDD on these lymphocyte stem cells may be mediated directly or indirectly through estrogenic action and/or the ER. Studies were designed to evaluate whether endogenous estrogens or the ER mediate TCDD-elicited bone marrow alterations and thymic atrophy. Ovariectomy did not alter the sensitivity of mice to TCDD-induced thymic atrophy or to a reduction in TdT biosynthesis in bone marrow cells compared with either intact or sham-operated mice. The pure estrogen antagonist ICI 164,384 blocked E2V-induced uterine hypertrophy, thymic atrophy and reductions in lymphocyte stem cell markers. However, the antiestrogen failed to protect against TCDD-elicited thymic atrophy or bone marrow alterations in intact animals. The results are consistent with the hypothesis that the effects of TCDD on the thymus and/or bone marrow are mediated by mechanisms independent of estrogens or the ER.

Effect of 3'-methoxy-4'-nitroflavone on benzo[a]pyrene toxicity. Aryl hydrocarbon receptor-dependent and -independent mechanisms.

This laboratory has studied a number of flavone derivatives for aryl hydrocarbon receptor (AhR) agonist and antagonist potential using cell-free and cell culture systems. The current report extends these investigations by testing the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) for in vivo activity. Wild-type C57Bl/6 male mice were treated with solvent, benzo[a]pyrene (B[a]P; 150 mg/kg), or concurrently with B[a]P and 3'M4'NF (60 mg/kg; delivered as a split dose). Since B[a]P is bioactivated to genotoxic metabolites by AhR-regulated enzymes, we measured B[a]P-induced chromosomal damage in peripheral blood (i.e. micronuclei) to characterize the antagonistic potential of 3'M4'NF in vivo. The influence of AhR signal transduction was investigated further by challenging wild-type and Ahr null allele mice with B[a]P with and without a 3'M4'NF co-treatment. The micronucleus data obtained from these experiments indicated that 3'M4'NF can attenuate the genotoxicity of B[a]P significantly. Since 3'M4'NF also protected Ahr null allele mice from B[a]P-induced genetic damage, it was apparent that AhR-independent mechanisms contribute to the effects observed. However, as opposed to the protective effects observed with the micronucleus endpoint, histological observations and lethality data indicated that some B[a]P effects are enhanced by 3'M4'NF. Potentiated B[a]P toxicity may be explained by inhibition of basal and induced CYP1A1/2 activities. Both in vitro and in vivo data presented herein support this hypothesis.

Polycistronic effects of catabolite repression on the lac operon.

The catabolite repression caused by glucose and glucose-6-phosphate has been studied for both beta-galactosidase and thiogalactoside transacetylase, the products of the operator proximal and distal cistrons of the lac operon, respectively. We find that both cistrons are affected coordinately by this form of repression. We also find that a single alteration at the lac promoter region is sufficient to abolish sensitivity to repression of both cistrons. From this, we conclude that there is only one target site for catabolite repression in the lac operon.

Influence of aromatic hydrocarbon receptor-mediated events on the genotoxicity of cigarette smoke condensate.

The role of aromatic hydrocarbon receptor (AhR)-mediated events on the genotoxicity of mainstream cigarette smoke condensate was investigated. In vitro studies with mouse hepatoma cells stably transfected with a DRE-dependent luciferase reporter indicate that cigarette smoke condensate is able to transform AhR to an active form which is capable of initiating gene transcription. Micronucleus formation in two hepatoma cell lines was used as an index of genotoxicity. Cigarette smoke condensate was observed to induce a higher frequency of micronuclei in Hepa1c1c7 cells relative to TAOc1BP(r)c1 cells, which express approximately 10-fold less AhR. Furthermore, the frequency of micronuclei was potentiated when Hepa1c1c7 cells were pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a high affinity ligand of AhR. These in vitro studies were followed by an in vivo experiment with Ahr+/+ and Ahr-/- mice. Animals were dosed for three consecutive days with cigarette smoke condensate (0.5-10 microg/kg/day, i.p. injection). The frequency of micronuclei in reticulocytes and total erythrocytes was determined in peripheral blood samples collected 24 h after the last administration. While condensate was found to increase the incidence of micronucleated reticulocytes in Ahr+/+ mice, no increase was observed in the null allele animals. Furthermore, the frequency of micronucleated erythrocytes, a measure of basal chromosome-damaging activity, was slightly but significantly higher in Ahr+/+ relative to Ahr-/- mice. Together, these data suggest that cigarette smoke contains chemicals which transform the AhR to an active transcription factor and AhR-regulated enzyme induction plays an important role in mediating the genotoxicity of this complex environmental pollutant.

Alternate immune system targets for TCDD: lymphocyte stem cells and extrathymic T-cell development.

We here summarize evidence that thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated, at least in part, by damage to extrathymic T-cell precursors in bone marrow and fetal liver. This atrophy induction does not involve apoptotic mechanisms in thymocytes affected by the bcl-2 proto-oncogene. TCDD mediates atrophy induction through its specific receptor (the AhR) and not through effects on the estrogen receptor. Both TCDD and estradiol induce extrathymic T-cell differentiation in the liver. These extrathymic T-cell populations include cells expressing elevated levels of V beta T-cell receptors that are normally deleted in thymic development.

Lymphocyte stem cell alterations following perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Perinatal exposure of experimental animals to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to thymic atrophy and a suppression of cell-mediated immunity that is more severe and persistent than that caused by adult exposure, suggesting that events involved in the maturation of the immune system are particularly sensitive to TCDD. We report here that perinatal TCDD exposure produces an alteration in the lymphocyte stem cell population in the fetus and neonate, as evidenced by a significant reduction in the lymphocyte stem cell-specific enzyme terminal deoxynucleotidyl transferase (TdT). After maternal treatment with a single dose of TCDD (10 micrograms/kg of body weight) on gestational day (gd) 14, TdT biosynthesis and TdT-specific mRNA were reduced more than 50% in fetal liver lymphoid cells on gd 18. An even more extensive reduction was seen in neonatal bone marrow through postnatal day 18. In contrast, thymic TdT synthesis appeared to be relatively unaffected on a per cell basis by perinatal TCDD exposure, although the actual number of TdT-synthesizing thymocytes was diminished due to extensive thymic atrophy. These effects occurred at concentrations of 1-31 fg of TCDD/mg of thymus. Flow cytometric analysis of thymocyte surface marker expression revealed a slight decrease in the percentage of Lyt-2+L3T4+ thymocytes on gd 18 and postnatal day 4. This alteration was no longer apparent by postnatal day 11, when marrow TdT biosynthesis was most suppressed. These results suggest that TCDD-induced thymic atrophy during the perinatal period may be due, in part, to an effect on the prothymocyte.

Impairment of prothymocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure of experimental animals to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in severe thymic atrophy and suppression of cell-mediated and humoral immune functions. However, despite much effort the mechanism by which TCDD produces these responses, particularly thymic atrophy, remains unclear. In this report, we have examined the effect of acute TCDD exposure on lymphocyte stem cells in young adult BALB/c mice to determine whether alterations to events early in T lymphopoiesis contribute to TCDD-induced thymic atrophy. TCDD produced a dose-dependent reduction in thymic weight and cellularity following a single dose of 5 to 120 micrograms TCDD/kg. This thymic atrophy correlated with a dose-dependent suppression of the biosynthesis and mRNA levels of the lymphocyte stem cell-specific DNA polymerase terminal deoxynucleotidyl transferase in bone marrow and thymus. However, the reduction in thymic terminal deoxynucleotidyl nucleotidyl transferase synthesis, on a per cell basis, was less than that observed in bone marrow. Intrathymic CD4/CD8 and IL-2R expression demonstrated only mild alterations after exposure to 30 micrograms TCDD/kg. These data suggest that thymocytes are more refractory to TCDD than are pre-T cells. To assess this possibility directly, bone marrow prothymocytes from TCDD-treated donor mice were examined for their capacity to reconstitute the thymuses of adoptive, irradiated recipients. Our results indicate that prothymocyte activity was severely impaired by TCDD exposure and that this effect occurred at low tissue levels of TCDD. In contrast, we observed no reduction in the number of colony-forming unit-granulocyte macrophage and a moderate decrease in colony-forming unit-spleen. These data suggest that TCDD-induced thymic atrophy is the result, at least in part, of impaired thymic seeding by prothymocytes.

Catabolite-insensitive revertants of lac promoter mutants.

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.

Isolation of a subset of thymocytes inducible for terminal transferase biosynthesis.

A new technique using direct binding of nucleated hematopoietic cells to PNA-coated rabbit red blood cell monolayers was used to separate PNA + and PNA - cells from murine thymus. The rigorously purified PNA - thymocyte population was found to lack TdT and to be low in TL. Incubation of the negative fraction with TP 5 resulted in the synthesis of TdT in a large number of cells in the fraction, and the appearance of TL on the surface of about 20% of the cells. Isolation of this inducible population has led us to propose a new class of cells in intrathymic T cell development.

Mouse fzd4 maps within a region of chromosome 7 important for thymus and cardiac development.

The cardiac neural crest (CNC) plays a central role in development of the thymus gland and cardiovascular system. Through morphological and histological characterization of embryos homozygous for the Del(7)Tyr(c-112K) and Del(7)Tyr(c-3H) albino deletions, we identified abnormalities that are consistent with aberrant development of tissues requiring CNC contributions. The defects include incompletely penetrant heart and great vessel patterning defects and hypoplastic thymus glands. The CNC phenotype is complemented by the partially overlapping deletion Del(7)Tyr(c-23DVT). Combined, these results suggest that a functional region necessary for development of CNC derived tissues is located between the Del(7)Tyr(c-23DVT) and Del(7)Tyr(c-112K) distal deletion breakpoints. This interval encompasses a functional region previously identified as important for juvenile survival (juvenile development and fertility, jdf). Using deletion mapping, we localized the Frizzled4 (Fzd4) gene to the jdf/thymus and cardiac development intervals.

The thymus does not mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited alterations in bone marrow lymphocyte stem cells.

Our previous studies have shown that bone marrow lymphocyte stem cells are affected following perinatal or adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These alterations may, in part, be responsible for thymic atrophy that is also observed following TCDD exposure. However, other investigators have suggested that the thymus or thymic-derived lymphocytes can affect bone marrow stem cell development. The purpose of these studies was to determine whether the TCDD-elicited effects that we have observed on lymphocyte stem cells in bone marrow were secondary to the actions of this chemical on the thymus. A single intraperitoneal dose of TCDD (30 micrograms/kg) to sham-operated or neonatally thymectomized female BALB/c mice reduced the levels of mRNA in the bone marrow for the lymphocyte stem cell-specific enzymes terminal deoxynucleotidyl transferase (TdT) and recombinase activating gene (RAG-1). TdT biosynthesis was also reduced by TCDD treatment. Thus, neonatal thymectomy had no effect on the TCDD-elicited reduction of TdT or RAG-1 mRNAs or TdT biosynthesis. Genetically athymic (nu/nu) mice were used to further determine if the actions of TCDD on the thymus or long-lived T-cells altered lymphocyte stem cell development. As observed in BALB/c mice, TCDD treatment decreased the expression of TdT and RAG-1 mRNAs in bone marrow from athymic nu/nu and intact nu/+ littermates. We conclude that TCDD-elicited alterations in bone marrow lymphocyte stem cells are not secondary to any actions, direct or indirect, that TCDD has on the thymus or thymic-derived T-cells.