Robert M. Gogal

Maternal immune stimulation in mice decreases fetal malformations caused by teratogens

For unknown reasons, non-specific stimulation of the maternal immune system in pregnant mice has what appears to be a broad-spectrum efficacy for reducing birth defects. Immune stimulation by diverse procedures has proven effective, including footpad injection with Freunds complete adjuvant (FCA), intraperitoneal (IP) injection with inert particles to activate resident macrophages, IP injection with attenuated Bacillus Calmette-Guerin (BCG), and intrauterine injection with allogeneic or zenogeneic lymphocytes. Morphologic lesions that were significantly reduced included cleft palate and associated craniofacial defects, digit and limb defects, tail malformations, and neural tube defect (NTD). Teratogenic stimuli to induce these lesions included chemical agents (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], cyclophosphamide [CP], and valproic acid [VA]), physical agents (X-rays, hyperthermia), and streptozocin (STZ)-induced diabetes mellitus. Limited information is available regarding mechanisms by which such immune stimulation reduced fetal dysmorphogenesis. The collective literature suggests the possibility that immunoregulatory cytokines of maternal origin may be the effector molecules in this phenomenon.

Nonspecific Stimulation of the Maternal Immune System. II. Effects on Gene Expression in the Fetus

BACKGROUND Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freunds complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.

Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds

BACKGROUND Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. METHODS The present study evaluated chemical-induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microscopy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI). RESULTS The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelovs solution was unable to detect early apoptosis. CONCLUSIONS The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge.

Nonspecific stimulation of the maternal immune system. I. Effects on teratogen-induced fetal malformations

BACKGROUND Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freunds complete adjuvant (FCA). RESULTS Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Single-dose topical exposure to the pyrethroid insecticide, permethrin in C57BL/6N mice: effects on thymus and spleen

Immunomodulatory effects of single topical exposure to permethrin were evaluated in 5-week-old female C57BL/6N mice. Mice exposed to 5-25 microl permethrin (equivalent to 220-1100 mg/kg body weight) on shaved interscapular skin were evaluated for altered body weight; splenic and thymic organ weight and cellularity; thymocyte cell surface expression, cellular apoptosis; splenic macrophage phagocytosis and hydrogen peroxide production; splenic B cell antibody production and T cell cytolytic activity; and mitogen-induced proliferation of splenocytes and thymocytes after in vivo or in vitro permethrin exposure. Topical permethrin application (25 microl) caused 32% inhibition of splenic T cell proliferation; in vitro exposure to permethrin also diminished splenocyte proliferation by 72% at 25 microM and 86% at 100 microM. permethrin did not appear to affect other leukocyte functional assays. Dose-related decreases in thymic cellularity of 52 and 80% were seen in mice exposed to 15 and 25 microl permethrin, respectively. Apoptosis was significantly increased in CD4(-)8(-) and CD4(-)8(+) thymocytes, and the CD4(+)CD8(+) thymocyte subpopulation was most severely diminished, suggesting possible chemical-induced apoptotic mechanism of thymic atrophy. Permethrin also caused splenic hypocellularity by 31% at 15 microl, and by 50% at 25 microl, an effect that may relate to inhibited proliferation or reduced seeding from the hypocellular thymus.

Interferon regulatory factor‐1 gene deletion decreases glomerulonephritis in MRL/lpr mice

To investigate the role of interferon regulatory factor‐1 (IRF‐1) in the development of lupus nephritis, IRF‐1–/– genotype mice were bred onto the MRL/lpJfaslpr (MRL/lpr) background. We examined kidney mesangial cell function and disease progression. Endpoints evaluated included inflammatory mediators, autoantibody production, immune complex deposition, renal pathology, T cell subset analysis, and duration of survival. Mesangial cells cultured from IRF‐1–/– mice produced significantly lower levels of nitric oxide and IL‐12 but not TNF‐α when stimulated with LPS + IFN‐γ. IRF‐1–/– mice showed less aggravated dermatitis compared to the wild‐type mice. Anti‐double‐stranded DNA production and proteinuria were significantly decreased in IRF‐1–/– mice compared to IRF‐1+/+ mice. IgG and C3 deposition as well as glomerulonephritis were decreased in IRF‐1–/– mice at 26 wk of age compared to the IRF‐1+/+ mice. Splenic CD4–CD8–CD44+ T cells were decreased while CD4+CD25+ T cells were increased in the IRF‐1–/– mice when compared to IRF‐1+/+ mice. Survival rates (ED50) were 22 wk for IRF‐1+/+ mice and 45 wk for IRF‐1–/– mice. These findings suggest an important role of IRF‐1 in mediating renal disease in MRL/lpr mice.

Topical permethrin exposure inhibits antibody production and macrophage function in C57Bl/6N mice.

Permethrin was applied to the shaved dorsal interscapular region of C57Bl/6N mice at doses of 0.5, 1.5 or 5.0 microl/day. These doses corresponded to approximately 22-220 mg/kg/day topical insecticide. Mice were exposed to permethrin in this manner daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. The splenic macrophage chemiluminescent response was depressed in a dose-dependent manner at 2 and 10 days post-exposure to permethrin. Phagocytic ability of macrophages was not inhibited. Antibody production as shown by plaque-forming cell (PFC) assay decreased significantly after 10 consecutive days of exposure to permethrin. These data indicate that topical permethrin exposure may produce systemic immune effects.

Immunomodulation by diethylstilbestrol is dose and gender related: effects on thymocyte apoptosis and mitogen-induced proliferation.

It is believed, but not proven, that the immunomodulatory effects of DES may vary with the dose and/or gender. To address these critical gaps in the literature, diethylstilbestrol (DES) was administered to female and male CD-1 mice as four subcutaneous injections for 1 week at 0, 5, 15, and 30 microg/kg bw doses, and immunological and reproductive effects examined a day after the last injection. Female thymuses were significantly larger than their male counterparts. Short-term administration of DES to female or male mice neither induced thymic atrophy nor altered the relative percentages of thymic subsets. Nevertheless, DES treatment of female or male mice induced a dose-related apoptosis of CD4(+)8(+), CD4(+)8(-) and CD4(-)8(+) subsets as analyzed by 7-amino-actinomycin D (7-AAD). Immature CD4(-)8(-) subset of thymocytes from females was also affected by high dose DES. The pattern of mitogen-induced proliferation of splenic lymphocytes varied with the dose of hormone and the gender. In females, splenic lymphocytes from low dose DES (5 microg/kg bw)-treated mice exhibited an increased proliferative response to Con-A, LPS or PMA/ionomycin compared with controls. Similar cultures from mice treated with higher doses of DES (15 or 30 microg/kg bw) did not manifest an increased proliferative response, but rather showed a trend for suppressed proliferation, especially in response to Con-A. In males, DES had minimal effects with the exception of increased proliferative response to Con-A in splenocytes from medium-dose-DES-treated mice. The changes in mitogen-induced proliferation in DES-treated female mice were not mirrored by similar changes in the relative numbers of CD90(+) or CD45R(+) cells, or in ratios of anti-apoptotic Bcl-2 to apoptotic Bax proteins. Con-A-activated splenocytes from DES-treated mice, particularly from females, had a decreased ability to secrete interferon-gamma compared with controls. Taken together, these findings suggest that short-term exposure to DES has differential immunological effects depending upon the dose of hormone and sex.

Tilapia (Oreochromis niloticus) dosed with azathioprine display immune effects similar to those seen in mammals, including apoptosis

Azathioprine, an anti-neoplastic drug and therapeutic immunosuppressant, was administered intraperitoneally at 10.0 and 50.0 mg/kg to 3-6-month-old tilapia (Oreochromis niloticus). Consistent alterations in immune cellular parameters of the blood, pronephros (hematopoietic kidney) and spleen were observed. Peripheral blood total cellularity decreased as the azathioprine dose increased, to approximately half that of the control. Differential analysis of white blood cells indicated a decline in lymphocyte number, in particular, with increased dosage of azathioprine. Pronephric total cellularity was depressed in fish receiving the 10.0 or 50.0 mg/kg dose. In contrast, both splenic weight and splenic total cellularity increased proportionately with the increase in the drug dosage. Histopathologic examination of the spleens showed normal patterns for both control and 10.0 mg/kg dose groups. At 50.0 mg/kg, spleens were characterized by marked expansion of the white pulp, although lymphocytes were rare. Melanomacrophage centers at the higher dose were also larger and more numerous than in the control group. Evaluation of splenic and pronephric leukocytes with apoptotic markers showed an increase in apoptotic cells in the pronephros with increasing drug dose. These changes in fish are consistent with those seen in humans and laboratory rodents dosed with azathioprine, suggesting that fish may be potentially useful as preliminary models for detecting immunosuppressive compounds.

Maternal immune stimulation reduces both placental morphologic damage and down-regulated placental growth-factor and cell cycle gene expression caused by urethane: are these events related to reduced teratogenesis?

Activation of the maternal immune system in mice decreased cleft palate caused by the chemical teratogen, urethane. Direct and indirect mechanisms for this phenomenon have been suggested, including maternal macrophages that cross the placenta to find and eliminate pre-teratogenic cells, or maternal immune proteins (cytokines) that cross placenta to alleviate or partially alleviate toxicant-mediated effects in the developing fetus. A third mechanism to explain improved fetal developmental outcome in teratogen-challenged pregnant mice might involve beneficial effects of immune stimulation on the placenta. In the present experiments, urethane treatment altered placental morphology and impaired placental function, the latter indicated by down-regulated activity of cell cycle genes and of genes encoding cytokines and growth factors. Maternal immune stimulation with either Freunds complete adjuvant (FCA) or interferon-gamma (IFNgamma) reduced morphologic damage to the placenta caused by urethane and normalized expression of several genes that were down-regulated by urethane. Urethane treatment also shifted placental cytokine gene expression toward a T cell helper 1 (Th1) profile, while immunostimulation tended to restore a Th2 profile that may be more beneficial to pregnancy and fetal development. These data suggest that the beneficial effects of maternal immune stimulation on fetal development in teratogen-exposed mice may, in part, result from improved placental structure and function.