Helga H. Timmerman

Suppression of natural killer cell activity in harbour seals (Phoca vitulina) fed Baltic Sea herring

Mass mortalities among marine mammal populations in recent years have raised questions about a possible contributory role of contaminants accumulated through the marine food chain. While viruses were shown to be the primary cause of the outbreaks, an immunotoxic action by organochlorine chemicals in affected animals could not be ruled out. We carried out a 212-year immunotoxicological experiment in which two groups of 11 harbour seals each were fed herring from either the relatively contaminated Baltic Sea or the relatively uncontaminated Atlantic Ocean. Seals in the Baltic Sea group accumulated 3–4 times higher levels of Ah-receptor-mediated 2,3,7,8-TCDD Toxic Equivalents in blubber than did their Atlantic counterparts following 2 years on the respective diets. Blood was sampled a total of 17 times during the course of the experiment for immunological evaluation, during which time the natural cytotoxic activity of peripheral blood mononuclear cells isolated from seals fed Baltic Sea herring declined to a level approximately 25% lower than that observed in seals fed Atlantic herring (P < 0.01). Natural killer (NK) cell activity has not been previously described for a marine mammal species. We characterized the natural cytotoxic activity of harbour seal peripheral blood mononuclear cells (PBMC), and found this to be interleukin-2 (IL-2) responsive, sensitive to antibody anti-asialo GM1, and it was higher against a virus-infected target cell, like NK cells described for other mammals. As NK cells are leukocytes which play an important role in the first line of defence against viruses, the observed impairment of NK cell activity in the seals feeding on the Baltic Sea herring suggests that exposure to contaminants may have an adverse effect on the defence against virus infections in seals inhabiting polluted waters in Europe. This may therefore have affected the severity of the infections, the survival rates and the spread of infections during recent epizootics.

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.

Impaired cellular immune response in harbour seals (Phoca vitulina) feeding on environmentally contaminated herring.

In a 2.5‐year immunotoxicological study, two groups of captive harbour seals (Phoca vitulina) were fed herring from the heavily polluted Baltic Sea or from the relatively uncontaminated Atlantic Ocean. Blood samples were collected at regular intervals, and functional immunological parameters were monitored. T cell mitogen and mixed lymphocyte‐induced proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from seals fed Baltic herring were significantly reduced over the course of the experiment. Upon immunization with rabies virus antigen (RV) and tetanus toxoid (TT), specific proliferative responses of PBMC from the seals fed Baltic herring were also significantly reduced. Impairment of T cell‐mediated immune responses became especially apparent during the second year on the respective diets, and correlated significantly to 2,3,7,8‐tetrachloro‐dibenzo‐p‐dioxin toxic equivalent levels in blubber biopsies taken from the seals after 2 years on the respective diets. Humoral immune responses, including lipopolysaccharide (LPS)‐induced lymphoproliferative responses, in vitro immunoglobulin production by PBMC, as well as RV‐, TT‐ and poliovirus‐specific serum antibody responses following immunization, remained largely unaffected. We conclude that suppression of the cellular immune response in the seals fed Baltic herring was induced by the chronic exposure to immunotoxic environmental contaminants accumulated through the food chain. Since cellular immune responses are known to be of crucial importance in the clearance of morbillivirus infections, these results suggest that environmental pollution‐related immunosuppression may have contributed to the severity and extent of recent morbillivirus‐related mass mortalities among marine mammals.

Short term fasting does not aggravate immunosuppression in harbour seals (Phoca vitulina) with high body burdens of organochlorines

Two groups of 11 harbour seals (Phoca vitulina) with different body burdens of organochlorines were subjected to an experimental 15-day fasting period, during which they lost an average 16.5% of their body weights. Blood levels of the most persistent organochlorines showed an approximate twofold increase, while levels of aryl hydrocarbon receptor-binding organochlorines remained largely unaffected. Few differences in immunological parameters were observed between the two dietary groups. Numbers of circulating lymphocytes dropped to about 65% of the initial values and NK cell activity showed a slight increase in both groups. Mitogen- and antigen-induced lymphoproliferative responses of the Baltic group of seals remained within normal ranges. These results suggest that relatively short-term fasting periods do not present an additional immunotoxicological risk to seals with high body burdens of organochlorines.

HLA Class I-Restricted Cytotoxic T-Cell Epitopes of the Respiratory Syncytial Virus Fusion Protein

ABSTRACT Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. We have generated cytotoxic T-cell clones (TCC) from two infants who had just recovered from severe RSV infection. These TCC were functionally characterized and used to identify HLA class I (B57 and C12)-restricted CTL epitopes of RSV.

Two distinct pathways for histamine H2 receptor down-regulation.

Pretreatment of Chinese hamster ovary cells expressing the histamine H receptor (CHOrH cells) with histamine resulted in a time-dependent (t ≈ 7 h) and dose-dependent (EC = 18 nM) H receptor down-regulation measured as [I]iodoaminopotentidine binding (44 ± 10% down-regulation). Pretreatment of CHOrH cells with cholera toxin or forskolin also led to H receptor down-regulation. Forskolin time-dependently (t ≈ 7 h) and dose-dependently (EC = 0.3 μM) induced H receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O., Lameh, J., Hogger, P., and Sadée, W.(1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H receptor from the G-protein, we introduced the Leu Ala mutation in the second intracellular loop of the H receptor. The H Leu Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH cells, H receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.

Host resistance to rat cytomegalovirus (RCMV) and immune function in adult PVG rats fed herring from the contaminated Baltic Sea

Abstract The immunotoxic potential of many classes of environmental contaminants has been well established in laboratory studies, with much attention being focussed on aryl hydrocarbon (Ah)-receptor binding polychlorinated biphenyl (PCB), polychlorinated dibenzo-p-dioxin (PCDD), and polychlorinated dibenzofuran (PCDF) congeners. In a semi-field study, we previously showed that harbour seals (Phoca vitulina) fed herring from the contaminated Baltic Sea had lower natural killer cell activity, T-lymphocyte functionality and delayed-type hypersensitivity responses than seals fed herring from the relatively uncontaminated Atlantic Ocean. While ethical and practical constraints preclude in-depth studies in seals, specific reagents and a wider array of immune function tests allow such studies in laboratory rats. We therefore carried out a feeding study in rats aimed at extending our observations of contaminant-induced immunosuppression in harbour seals. The same two herring batches used in the seal study were freeze-dried, supplemented and fed to female adult PVG rats for a period of 4 12 months. Daily contaminant intakes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) were estimated to be 0.3 ng/kg body weight and 1.6 ng/kg in the Atlantic and Baltic groups, respectively. At the end of the feeding experiment, no contaminant-related changes in spleen CD+4/CD+8 cellularity, natural killer cell activity, or mitogen-induced proliferative responses of thymus or spleen cells could be detected. However, total thymocyte numbers and thymus CD+4/CD+8 ratios were reduced in the Baltic group. A novel model was established to assess the specific T-cell response to rat cytomegalovirus (RCMV). When applied to the feeding study, no differences between the Atlantic and Baltic groups in the RCMV-induced proliferative T-lymphocyte responses could be detected, but virus titres in salivary glands of infected rats of the Baltic Sea group were higher. These elevated RCMV titres and changes in thymus cellularity suggest that the dietary exposure to low levels of contaminants may have been immunotoxic at a level which our immune function test could not otherwise detect. While the herring diet per se appeared to have an effect on several immune function parameters, lower plasma thyroid hormone levels in the Baltic Sea group of rats confirmed that exposure to the environmental mixture of contaminants led to adverse PHAH-related health effects.

Time-Dependent Differential Changes of Immune Function in Rats Exposed to Chronic Intermittent Noise

Noise is a highly relevant environmental and clinical stressor. Compared to most other experimental stressors, noise is a modest activator of neuroendocrine pathways that mimic the situation in human health where neuroendocrine activation by environmental stressors is often absent or difficult to establish. Little is known about the effects of noise exposure on the immune system. In the present work, the effects of a low-intensity chronic intermittent unpredictable noise regimen on various parameters of immune function was studied. Male wistar rats were exposed to a randomized noise protocol (white noise, 85 dB, 2-20 kHz) for 10 h per day, 15 min per h over a total period of 3 weeks. Control animals were exposed to ambient sound only. Immune function was monitored after 24 h, 7 days, and 21 days of noise exposure. Noise induced several significant changes in immune function in a time-dependent differential pattern involving both immunosuppression and immunoenhancement. After 24 h, serum IgM levels were increased and peripheral phagocytic activity was decreased. Splenic lymphocytic proliferation to mitogens was significantly decreased after 7 days, but slightly elevated after 3 weeks. The activity of splenic NK cells was increased significantly after 24 h and 7 days, but suppressed after 3 weeks. These results show that various parameters of immune function are affected differentially over time in a period of chronic mild noise stress, possibly due to sequential activation of different physiological mechanisms.

Comparison of the in vivo effects of morphine and methadone on natural killer cell activity in spleen, peritoneal cavity, and lungs in rats

Opiates and opioid agents are known to affect the immune system. In humans this includes alterations in natural killer (NK) cell activity. Morphine is reported to reduce in vivo spleen NK activity in rats, whereas for methadone only in vitro data have been described. In the present paper we describe a systematic study on the chronic effects of well-known opiates, comparing for the first time the effects of morphine and methadone on NK cell activity in various organs: in addition to spleen, also in the peritoneal cavity, and lungs. In all organs the NK activity was determined using three effector: target cell ratios. Morphine and methadone given by food during 6 weeks decreased the NK cell activity in rat spleen, supporting published data on morphine. The role of the opiate receptor is discussed. However, the overall action of morphine could not be described as suppressive because stimulation of NK cell activity in the peritoneal cavity and lungs by morphine was found. In contrast, methadone induced a decrease in the NK cell activity in these organs. Apart from these differential expressions of morphine- and methadone associated effects on NK cell activity, the findings demonstrate the potential adverse effects of these opiates on an important antiviral defence mechanism.