Immune stimulation in urethane-exposed pregnant mice increases expression level of spleen leukocyte genes for TGFbeta3 GM-CSF and other cytokines that may play a role in reduced chemical-induced birth defects.

For unknown reasons, activation of the maternal immune system in mice reduces morphologic defects caused by diverse teratogenic agents. Such immune stimulation of the maternal animal has been correlated with altered cytokine mRNA transcripts in the placenta (e.g., TGFbeta2) as well as in fetal target tissues of the teratogen (e.g., TNFalpha in fetal heads of cyclophosphamide-exposed pregnant mice). The teratogen urethane was reported to down-regulate cell cycle and apoptotic regulatory genes in fetal mouse heads that displayed cleft palate, an effect that was also reversed by maternal immune stimulation. The molecular mediators of the above phenomena have not been identified, however proteins synthesized and released by activated maternal immune cells have been suggested. The present studies therefore evaluated the effects of maternal immune stimulation in urethane-exposed mice on thymus and spleen leukocyte populations, in an attempt to identify events that may correlate with protection against birth defects. Immune stimulation did not change the hypocellularity of the thymus nor the altered T cell differentiation caused by urethane. A limited and transient increase in splenic leukocyte number, including increased T and B lymphocytes and macrophages, was caused by immune stimulation and was not felt to play a significant role in reduced morphologic defects. Urethane treatment caused down-regulated expression of numerous genes involved in cell-cycle control, while maternal immune stimulation caused comparative up-regulation of many of these genes. Coordinate shifts in gene expression by treatment were evaluated using principal component analysis, which identified several growth factor genes that were differentially expressed in mice receiving urethane alone as compared to urethane plus immune stimulation. Up-regulated expression of TGFbeta3 and GM-CSF genes, in particular, was observed in leukocytes of urethane-exposed mice receiving immunostimulation. Interestingly, the cytokine products of these two genes were recently suggested as growth factors that may be related to reduction of fetal defects caused by teratogens. Genes for growth factors IGF-I, IGF-II and IL-2 were also identified as differentially expressed in urethane vs. urethane+immune stimulation mice, suggesting that these proteins should be considered for a potential contributing effect to reduced birth defects caused by immunostimulation.