John B. Barnett

Interleukin 12 a Key Immunoregulatory Cytokine in Infection Applications

Interleukin 12 (termed IL-12p70 and commonly designated IL-12) is an important immunoregulatory cytokine that is produced mainly by antigen-presenting cells. The expression of IL-12 during infection regulates innate responses and determines the type of adaptive immune responses. IL-12 induces interferon-γ (IFN-γ) production and triggers CD4+ T cells to differentiate into type 1 T helper (Th1) cells. Studies have suggested that IL-12 could play a vital role in treating many diseases, such as viral and bacterial infections and cancers. The unique heterodimeric structure, which IL-12 shares with its family members including IL-23, IL-27, and IL-35, has recently brought more attention to understanding the mechanisms that regulate the functions of IL-12. This article describes the structure and biological activities of IL-12 in both the innate and adaptive arms of the immune system, and discusses the applications of IL-12 in treating and preventing infections.

Cadmium Increases HIF-1 and VEGF Expression through ROS, ERK, and AKT Signaling Pathways and Induces Malignant Transformation of Human Bronchial Epithelial Cells

Cadmium is categorized as a human carcinogen especially involved in lung cancers. Angiogenesis is considered a fundamental requirement for tumorigenesis, but the mechanisms underlying the tumor angiogenesis induced by cadmium are poorly understood. Using in vitro and in vivo models, we investigated the angiogenic mechanisms of cadmium in human bronchial epithelial cells and tumor formation. Our results demonstrated that cadmium (CdCl(2)) activated extracellular signal-regulated kinases (ERK) and AKT signaling and elevated the expression of a key downstream proangiogenic molecule hypoxia-inducible factor-1 (HIF-1) in immortalized human lung epithelial BEAS-2B cells. Cadmium also induced reactive oxygen species (ROS) production, which could be inhibited by ROS scavengers, catalase and diphenyleneiodonium chloride. Inhibition of ROS generation also attenuated ERK, AKT, p70S6K1 activation, and HIF-1α expression. Similar results were obtained in normal human bronchial epithelial (NHBE) cells, showing that cadmium induced HIF-1 expression via ROS/ERK/AKT signaling pathway. Furthermore, cadmium induced vascular endothelial growth factor expression and transcriptional activation through ROS, ERK, and AKT pathways. Finally, cadmium transformed human bronchial epithelial cells in culture; the transformed cells induced tube formation in vitro, angiogenesis on chicken chorioallantoic membrane, and formed tumors in nude mice. Taken together, the results of this study provide explanation for the role and molecular mechanisms of cadmium in promoting angiogenesis in lung epithelial cells and malignant transformation and will be helpful for improved occupational protection, prevention, as well as chemotherapy of human lung cancers caused by heavy metal cadmium.

Immunoteratology of chlordane: cell-mediated and humoral immune responses in adult mice exposed in utero.

The functional status of the immune system of adult mice prenatally exposed to the chlorinated hydrocarbon pesticide chlordane was evaluated by assessing cell-mediated and humoral immune responses. Gravid BALBc mice fed 0, 0.16, or 8.0 mg/kg chlordane daily throughout gestation produced viable, overtly normal offspring. At 101 days of age cell-mediated immune (CMI) responses were measured by the contact hypersensitivity response to oxazolone. Offspring were sensitized by application of oxazolone to the denuded flank and 5 days later challenged by percutaneous application of oxazolone to the ear. Degree of induration was determined by daily micrometer measurement of ear thickness for 3 days after challenge. Offspring in the higher dose group had a significantly (p < 0.01) depressed CMI response when compared to offspring of the control or lower dose groups. The primary humoral response to sheep red blood cell (sRBC) innoculation was determined at 101 days of age using the hemolytic plaque assay. Subjects were immunized with washed sRBC. IgM antibody released from the spleen cells was detected by addition of complement which caused lysis of the sRBCs. No significant differences existed between the number of plaque-forming cells (PFC) produced by spleen cells from either chlordane-treated group or the vehicle-control group. Results of this study revealed two significant findings: (1) severe depression of the functional CMI response in apparently normal treated offspring, but (2) no effect on the T-cell-dependent humoral immune response. The effect of chlordane on CMI and PFC responses might be explained by a reduction in the activity or number of T effector cells.

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells.

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A pro-cancer gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

The effect of prenatal chlordane exposure on the delayed hypersensitivity response of BALB/c mice

Previous studies in our laboratory have indicated that in utero chlordane exposure caused a significant enhancement in the survival of the offspring to influenza virus infection. Further studies, reported here, show that the non-specific delayed type hypersensitivity (DTH) response to oxazolone at 100 days of age, but not at 30 days of age, was significantly depressed. In contrast, the Con A-induced blastogenic response of spleen cells from chlordane-treated offspring was not depressed and was, in fact, significantly enhanced. However, neither the response to PHA nor to LPS mitogens was significantly altered. In utero exposure to chlordane significantly depressed the mixed lymphocyte reactivity (MLR) of spleen cells from male offspring, whereas females showed no significant alteration of MLR. The significant depression of the DTH and MLR responses supports our previous reports of enhanced survival of influenza virus infection following in utero exposure to chlordane, since active DTH contributes to the pathology of influenza virus infection in mice. The normal or enhanced T-cell mitogen response suggested that the chlordane-induced depression of DTH and MLR was not due to overt toxicity to T-cells.

Developmental Immunotoxicity of Atrazine in Rodents

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.

The role of calcium release activated calcium channels in osteoclast differentiation.

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m‐CSF and the TNF‐family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca2+. Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m‐CSF and RANKL, revealing significant loss in both the expression and function of the required components of store‐operated Ca2+ entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4‐dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down‐regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states. J. Cell. Physiol. 226: 1082–1089, 2011.

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1−/− mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1−/− mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1−/− mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1−/− animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1−/− and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.

Effect of nutritional copper deficiency on adjuvant arthritis and immunocompetence in the rat

Both severe and marginal copper deficiency were produced in male Sprague Dawley rats prior to induction of adjuvant arthritis. Degree of copper deficiency was confirmed by analysis of plasma, liver, and brain samples prior to adjuvant injection. Incidence of adjuvant arthritis was the same in both copper deficient and control animals although the severity was slightly but not statistically less in the former. However, recovery from foot edema was impaired in copper-deficient animals, while marginally copper-deficient animals recovered at the same rate as did controls. Plasma copper concentration increase in response to the injection of adjuvant and the increase was directly related to dietary copper content. Plasma zinc concentration was decreased in arthritic animals and the decrease was inversely correlated to paw edema. Liver copper, zinc, and iron concentrations in arthritic animals remained unchanged or increased slightly in comparison to the corresponding non-injected controls. Copper-deficient rats were immunosuppressed as demonstrated by impaired responsiveness to the T-cell dependent contact sesitizing agent oxazolone and diminshed capacity to respond to the T-cell independent antigen Type III pneumococcal polysaccharide. Although a statistical difference in paw volumes was not found for group of animals fed diets differing in copper content, it is postulated that copper deficiency may alter the severity and kinetics of adjuvant arthritis by impairing aspects of the immune response and the tissue repair processes subsequent to injury.

Comparison of the immunotoxicity of propanil and its metabolite, 3,4-dichloroaniline, in C57Bl/6 mice

Propanil (3,4-dichloropropionaniline), used extensively as a postemergence herbicide in rice and wheat, has as its major metabolite, 3,4-dichloroaniline (DCA). Propanil has previously been shown to affect the T cell-dependent antibody response. To determine the immunotoxicity of DCA, as well as extend the previous immunotoxicity studies, several T cell-dependent and -independent immune responses were determined after DCA or propanil exposure. Unlike propanil, DCA caused a significant reduction in T-dependent antibody production (anti-SRBC response) only at a high dose (150 mg/kg). DCA or propanil at 150 or 200 mg/kg, respectively, caused a significant reduction in the number of anti-DNP antibody producing cells. However, doses of 37 or 50 mg/kg of DCA or propanil, respectively, caused an increase in the number of anti-DNP antibody producing cells. These data indicate that both propanil and DCA have a differential effect on the T-independent antibody response depending on the dose. Similar to propanil, DCA (at 150 mg/kg) caused a significant increase in spleen weight and cellularity. The effect of DCA or propanil on selected cellular immune functions was also determined. DCA caused a significant decrease in the natural killer (NK) cell activity at doses of 75 or 150 mg/kg, and propanil caused a significant decrease at 100 or 200 mg/kg. Cytotoxic T lymphocyte activity, however, was unaffected even at 150 or 200 mg/kg DCA or propanil, respectively. Thus, it appears that T cells are relatively resistant to the effects of propanil and DCA, whereas, other immune cell types, e.g., NK cells are sensitive to its effects.