Gary R. Burleson

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on Influenza Virus Host Resistance in Mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes numerous immunotoxic effects including thymic involution and an immunosuppression of nonspecific as well as specific cell- and humoral-mediated immunity. TCDD administration to laboratory animals also results in a decreased resistance to numerous bacteria, viruses, and parasites. Effects on virus host resistance appear to be among the most sensitive effects of TCDD immunotoxicity. However, previous studies have not achieved a no effect level. The present studies utilized an influenza virus host resistance model in mice to quantify the sensitivity of this model to TCDD and to determine the NOAEL (no observed adverse effect level) of TCDD for influenza virus. Results indicated that a single dose of TCDD at 0.10, 0.05, or 0.01 microgram/kg resulted in an increased mortality to Hong Kong influenza virus when mice were challenged 7 days after TCDD administration. Increased mortality was not correlated with increased virus titers in the lungs. TCDD at 0.005 or 0.001 micrograms/kg had no effect on influenza-induced mortality. TCDD alone did not affect thymus weight at any dose administered in this study. TCDD also did not alter the virus-enhanced increase in lung weight:body weight ratio nor the virus-induced decrease in thymus weight. Thus, low levels of TCDD exposure lead to enhanced mortality to influenza virus; however, the mechanism of this effect remains to be elucidated. Nonetheless, enhanced mortality to influenza virus in mice following a single dose of 10 ng TCDD/kg represents the most sensitive adverse effect yet reported for TCDD.

Examination of immune parameters and host resistance mechanisms in B6C3F1 mice following adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Adult female B6C3F1 mice were given a single ip dose of 0, 01, 1.0, or 10.0 micrograms/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and examined for immune function and host resistance 7-10 d later. Exposure to TCDD resulted in a significant dose-related decrease in induction of both IgM and IgG antibody-forming cells. This suppression was noted for both T-dependent and T-independent antigens. TCDD at a dosage of 10 micrograms/kg was shown to suppress production of antibody to viral hemagglutinin. In contrast, TCDD exposure had no significant effect on natural killer cell function, production of interferon, or various parameters of macrophage function. Assessment of host resistance revealed a significant increase in susceptibility to fatal infection with influenza virus, but no significant alteration in susceptibility to infection with the bacterium Listeria monocytogenes.

Enhanced and prolonged pulmonary influenza virus infection following phosgene inhalation

Animal infectivity models have been important in the demonstration of enhanced susceptibility to viral and bacterial infection as a result of low-level toxicant exposure. This study demonstrated an enhanced and prolonged viral infection using an influenza virus infectivity model in the rat following exposure to the toxicant gas phosgene. Fischer-344 rats exposed to either air or a sublethal concentration of phosgene demonstrated peak pulmonary influenza virus titers 1 d after infection. Virus titers in rats exposed to air declined rapidly falling below detectable levels by 4 d after infection. However, a significantly enhanced and prolonged pulmonary influenza virus infection was observed on d 3 and 4 after infection in rats exposed to phosgene. Virus was cleared below detectable limits on d 5 after infection in animals exposed to phosgene. Thus, inhalation of sublethal concentrations of phosgene resulted in an increased severity of pulmonary influenza virus infection. This study provides a demonstration of the effective use of a rat viral infectivity model to detect the immunotoxicity of inhaled pollutants. This model will allow future studies to focus on the immunological mechanism(s) responsible for the enhanced and prolonged pulmonary influenza virus infection.

Natural Killer Activity in Fischer-344 Rat Lungs as a Method to Assess Pulmonary Immunocompetence: Immunosuppression by Phosgene Inhalation

Phosgene, also known as carbonyl chloride, carbon oxychloride, and chloroformyl chloride, is a toxic air pollutant and a potential occupational health hazard. Studies were initiated (a) to evaluate the measurement of pulmonary natural killer (NK) activity as a method to assess pulmonary immunocompetence, and (b) to determine whether exposure to phosgene resulted in local pulmonary or systemic immune dysfunction. Fischer-344 male rats were exposed either to filtered air or to 1.0 ppm phosgene gas for four hours. The effect of phosgene on lung NK activity was quantified at different times after acute phosgene exposure. Pulmonary NK activity was measured by mincing lung tissue into small pieces prior to incubation with collagenase. Whole-lung homogenate was assayed for NK activity utilizing a 4 hour 51-Cr-release assay with YAC-1 cells as target cells. Acute phosgene exposure resulted in a suppressed pulmonary NK activity on days 1, 2, and 4 after exposure; however, normal levels of biological activity were observed 7 days after exposure. The suppressed NK activity was not restored after removal of adherent cells from the lung homogenate, thus indicating that the effect of phosgene on NK activity was not due to immunosuppression via mobilization of suppressor alveolar macrophages. Pulmonary immunotoxicity was also observed after exposure at 0.5 ppm, while no adverse effects were observed at 0.1 ppm phosgene. Systemic immunotoxic effects were observed for NK activity in the spleen, but not in the peripheral blood. It is thus important in pulmonary immunotoxicology to evaluate systemic immune functions, since secondary effects--distant to the original interaction--may occur with potentially serious consequences. Cells exhibiting natural killer activity comprise a part of the nonspecific innate immunity that is important in defense against both neoplastic and viral diseases. Any perturbation of this important nonspecific immunological mechanism may result in a compromised host more susceptible to infectious and neoplastic disease.

Effects of 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and cyclosporin A on murine cytomegalovirus infection: Studies of resistance mechanisms☆

Susceptibility to murine cytomegalovirus (MCMV) was enhanced by treating B6C3F1 and CD-1 mice subcutaneously with 100 mg 7,12-dimethyl-benz[a]anthracene (DMBA)/kg fractionated over a 2 week period prior to sub-lethal infection. Virus-augmented natural killer cell (NKC) activity was depressed in B6C3F1 mice treated with 100 mg DMBA/kg, while serum interferon (IFN) levels were unaffected. Treatment with 50 mg DMBA/kg had no effect on susceptibility to virus or virus-augmented NKC activity. Susceptibility to MCMV was not affected by treating mice with 400 mg benzo[a]pyrene (B[a]P)/kg using the same exposure regimen. Virus-augmented NKC activity was suppressed in B[a]P-treated mice, but the magnitude of the suppression (18%) was much less than that for DMBA-treated mice (39%). Susceptibility to MCMV, virus-augmented NKC and IFN induction were not affected in mice treated intraperitoneally with 50 mg cyclosporin A (CSA)/kg/day for 5 days and infected on the 5th day of treatment. In contrast, enhanced susceptibility to MCMV and depressed NKC activity were observed in mice treated by the same exposure regimen on days 1-5 post infection. Susceptibility was not affected by CSA given on days 5-9 post infection. The data are useful not only because they show that DMBA and appropriately-timed CSA treatments suppress virus augmented NKC and enhance susceptibility to MCMV, but also because they help to define the relative importance of certain immune responses in defending against the infection, thus improving the usefulness of MCMV as a host resistance model for immunotoxicity testing. The data suggest that chemicals which depress NKC are likely to enhance susceptibility to MCMV, and conversely that effects on NKC should be suspected when chemical exposure enhances susceptibility to MCMV.

Effects of NiCl2 and CdCl2 on susceptibility to murine cytomegalovirus and virus-augmented natural killer cell and interferon responses

Female C3H/HeJ or CD-1 mice were infected with a sublethal dose of murine cytomegalovirus (MCMV) and then exposed to nickel chloride (NiCl2) or cadmium chloride (CdCl2) intramuscularly (im) or by inhalation. Effects of these treatments on disease susceptibility, virus-augmented and spontaneous natural killer (NK) cell activity, and virus induction of interferon (IFN) were determined. NiCl2 (20 mg/kg, im) enhanced mortality due to MCMV in both mouse strains, and a reduction in virus-augmented NK cell activity was seen at doses as low as 10 mg NiCl2/kg im. At 6.25 mg CdCl2/kg im there was a significant depression of NK cell activity, but there was no effect on mortality due to infection. Effects on NK activity did not appear to be due to effects on IFN production since neither of the metal treatments caused depression of this response. Neither metal when given by inhalation had any effect on these parameters.

Effects Of Acute Exposure To Phosgene On Pulmonary Host Defenses And Resistance To Infection

AbstractPhosgene is a toxic gas widely used in industrial processes. The most sensitive endpoint for phosgene toxicity in mice is decreased resistance to challenge with bacterial infection or tumor cells. These effects were attributed to impaired alveolar macrophage (AM) and pulmonary natural killer cell (NK) function. The purpose of this study was to investigate whether similar effects occurred in Fischer 344 rats. Intrapulmonary killing of bacteria was impaired and the inflammatory response enhanced in rats infected by aerosol with Streptococcus zooepidemicus immediately after a 6-/1 exposure to 0.1 or 0.2 ppm phosgene as compared to air controls. Also, ingestion of latex beads in vitro by AM obtained from bronchoalveolar lavage (BAD fluid of rats exposed to 0.2 ppm phosgene was significantly less than controls. If infection or BAL were delayed until 18 h after exposure, there was no difference between phosgene and air exposed rats. In uninfected rats polymorphonuclear leukocytes were increased in BAL f...

Effects of Phosgene Exposure on Lung Arachidonic Acid Metabolism

AbstractPhosgene can acylate macromolecules and may react with and affect enzymes involved in arachidonic acid metabolism. We examined the effects of an acute phosgene exposure in vivo and in vitro on lung arachidonic acid metabolism. Fischer 344 rats were exposed either to air or to phosgene (0.05–7.0 ppm) for 4 h and the lungs were lavaged at 0, 4, 20, and 44 h postexposure. Leukotriene B, (LTBJ, peptide leuko-trienes C4 D4 and E4 (LTC4/D/E4, and prostaglandin E2 (PCE2) were measured in la-vage fluid by radioimmunoassay Phosgene exposure in vivo (0.7–7.0 ppm for 4 b) produced significant decreases in concentrations of PGE, (maximal decrease of 74%), LTB, (maximal decrease of 59%), and LTC4/D4/E4 (maximal decrease of 97%) measured in rat lavage fluid immediately postexposure. Associated with this decrease in eicosa-noid production was a decrease in the number of alveolar macrophages and an increased number of neutrophils recovered in the lavage fluid of phosgene-exposed rats. Lung lavage eicosanoid conce...

Enhancement of natural killer cell activity and interferon production by manganese in young mice.

The effect that MnCl2 has on murine splenic natural killer (NK) cell activity was investigated in infant (10 days old), pre-weanling (18 days old) and weanling (24 days old) C57BL/6J mice. A single intraperitoneal injection of 10, 20 or 40 micrograms MnCl2/g body weight caused a significant enhancement in NK activity, as determined by the in vitro 51Cr release assay. Comparable enhancement of NK activity was observed for age-matched mice injected intraperitoneally with polyinosinic polycytidylic acid (Poly I:C). Both MnCl2 and Poly I:C caused elevations in serum interferon levels. Time-course studies revealed that interferon levels returned to normal within 48 hours following injection with either MnCl2 or Poly I:C; however enhanced NK activity persisted for up to 48 hours in Poly I:C-injected mice and 72 hours in MnCl2-injected mice. The administration of rabbit anti-asialo GMl to MnCl2-injected mice completely abrogated the enhanced NK activity. In addition, the injection of rabbit anti-mouse interferon alpha, beta but not gamma completely abrogated the enhanced NK activity. In addition, the injection of rabbit anti-mouse interferon alpha, beta but not gamma completely abrogated the enhancement of NK activity by MnCl2 and to a lesser extent the enhancement of NK activity by Poly I:C. These results indicate that despite low levels of NK activity in pre-weanling mice, MnCl2 is capable of enhancing this activity by 8-9 fold. Furthermore, Mn-enhanced NK activity in these young mice appears to be mediated by the production of interferon alpha, beta.

Poly(I):poly(C)-Enhanced Alveolar and Peritoneal Macrophage Phagocytosis: Quantification by a New Method Utilizing Fluorescent Beads

Abstract Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess (1) the baseline levels of phagocytosis for rat alevolar and peritoneal macrophages from the same animal and (2) the kinetics and level of enhanced phagocytosis for alevolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.