laboratory-estimation-net-trophic-transfer-efficiencies-pcb-congeners

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (<em>Salvelinus namaycush</em>) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

Grand Valley State University

Peripheral CD8(+) T cells circulate in a quiescent naive state until they are primed by specific antigen and differentiate into effector cells. In the effector state, CD8(+) T cells acquire cytolytic activity and produce increased levels of cytokines such as interferon-gamma. They also exhibit increased T cell receptor sensitivity, decreased CD28 dependence, and become inhibitable by CTLA-4 and other negative regulatory pathways. We hypothesized that one mechanism by which these two states are regulated is via differential expression of specific genes. To this end, basal gene expression profiles of naive and effector 2C TCR transgenic x RAG2(-/-) CD8(+) T cells were analyzed using Affymetrix arrays representing 11,000 genes. Of the 177 differentially expressed known genes, 68 were expressed at higher levels in effector cells, but 109 were more abundant in naive cells, supporting the notion that the naive state is not passive. Expression of genes related to metabolism, actin cytoskeletal dynamics, and effector function increased with priming, whereas expression of putative anti-proliferative genes decreased. Semiquantitative reverse transcription-PCR was utilized as a secondary validation for selected transcripts, and Western blot analysis was used to examine protein expression for molecules of interest. Surprisingly, for 24 genes examined, 12 showed discordant protein versus mRNA expression. In summary, our study indicates that: 1) not only does the expression of some genes in naive CD8(+) T cells become up-regulated upon priming, but the expression of other genes is down-regulated as well and 2) the complexities of T cell differentiation include regulation at the post-transcriptional level.

Gene array and protein expression profiles suggest post-transcriptional regulation during CD8+ T cell differentiation.

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration.

Although both naive and effector T lymphocytes interact with antigen-expressing cells, the functional outcome of these interactions is distinct. Naive CD8(+) T cells are activated to proliferate and differentiate into effector cytolytic T lymphocytes (CTL), whereas CTL interact with specific targets, such as tumor cells, to induce apoptotic death. We recently observed that several molecules linked to actin cytoskeleton dynamics were up-regulated in effector vs. naive CD8(+) T cells, leading us to investigate whether T cell differentiation is accompanied by changes in actin-dependent processes. We observed that both naive and effector CD8(+) T cells underwent T cell receptor capping and formed stable conjugates with antigen-specific antigen-presenting cells. However, the characteristics of the immunological synapse were distinct. Whereas accumulation of signaling molecules at the T cell/antigen-presenting cell contact site was detectable in both naive and effector CD8(+) T cells, only effector cells developed a central supramolecular activation cluster as defined by punctate focusing of PKC theta, phospho-PKC theta, and phospho-ZAP70. Extended kinetics, CD28 costimulation, and high-affinity antigenic peptide did not promote PKC theta focusing in naive cells. Nonetheless, naive CD8(+) T cells polarized the microtubule organizing center, produced IL-2, proliferated, and differentiated into effector cells. Our results suggest that the formation of a central supramolecular activation cluster is not required for activation of naive CD8(+) T cells and support the notion that one role of an organized immune synapse is directed delivery of effector function.

Formation of a central supramolecular activation cluster is not required for activation of naive CD8+ T cells.

The functional implication of molecular segregation within the immunological synapse remains uncertain. We recently reported that effector but not naive TCR transgenic murine CD8+ T cells formed immunological synapses containing a central supramolecular activation cluster (cSMAC), suggesting that execution of effector functions such as cytolytic activity might be facilitated by the cSMAC structure. We have now explored this hypothesis using two approaches. First, by simultaneously imaging cSMAC formation and mobilization of cytotoxic granules to the synapse, we observed no correlation between the presence of a cSMAC and granule reorientation. Second, we took advantage of the observation that CD28 costimulation markedly enhances cSMAC formation. Granule polarization to the contact site was indistinguishable with B7-1+ and B7-1- target cells, and cytolytic activity against B7-1+ or B7-1- targets was similar and granule-dependent. Together, our results indicate that the formation of a cSMAC is not required for cytolytic activity in CD8+ effector T cells.

Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells.

Metastasis suppressor proteins: discovery, molecular mechanisms, and clinical application.

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.

Induction of cytotoxic granules in human memory CD8+ T cell subsets requires cell cycle progression.

Lake whitefish (Coregonus clupeaformis) were fed rainbow smelt (Osmerus mordax) in four laboratory tanks over a 133-d experiment. At the start of the experiment, 10 to 14 of the fish in each tank were sacrificed, and the concentrations of 40 polychlorinated biphenyl (PCB) congeners within these fish were determined. Polychlorinated biphenyl congener concentrations were also determined in the 15 lake whitefish remaining in each of the four tanks at the end of the experiment as well as in the rainbow smelt fed to the lake whitefish. Each lake whitefish was weighed at the start and the end of the experiment, and the amount of food eaten by the lake whitefish during the experiment was tracked. Using these measurements, net trophic transfer efficiency (gamma) from the rainbow smelt to the lake whitefish in each of the four tanks was calculated for each of the 40 PCB congeners. Results showed that gamma decreased exponentially as log K(OW) for the congeners increased from 6 to 8. Further, gamma averaged 0.70 for the tetrachloro congeners but averaged only 0.45 for the higher chlorinated congeners.

Net trophic transfer efficiencies of polychlorinated biphenyl congeners to lake whitefish (Coregonus clupeaformis) from their food.

We recently reported that differentiation of CD8(+) T cells from the naïve to the effector state involves the upregulation of glucose-dependent metabolism. Glucose deprivation or inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) selectively inhibited production of IFN-gamma but not of IL-2. To determine a more global role of glucose metabolism on effector T-cell function, we performed gene array analysis on CD8(+) effector T cells stimulated in the presence or absence of 2-DG. We observed that expression of only 10% of genes induced by TCR/CD28 signaling was inhibited by 2-DG. Among these were genes for key cytokines, cell cycle molecules, and cytotoxic granule proteins. Consistent with these results, production of IFN-gamma and GM-CSF, cell cycle progression, upregulation of cyclin D2 protein, cytolytic activity, and upregulation of granzyme B protein and also conjugate formation were exquisitely glucose-dependent. In contrast to glucose, oxygen was little utilized by CD8(+) effector T cells, and relative oxygen deprivation did not inhibit these CTL functional properties. Our results indicate a particularly critical role for glucose in regulating specific effector functions of CD8(+) T cells and have implications for the maintenance of the effector phase of cellular immune responses in target tissue microenvironments such as a solid tumor.

Glucose deprivation inhibits multiple key gene expression events and effector functions in CD8+ T cells.

Pacific salmon (Oncorhynchus spp.) can transport bioaccumulated organic pollutants to stream ecosystems where they spawn and die. We quantified PCBs, DDE, and PBDEs in resident fishes from 13 Great Lakes tributaries to assess biotransport of pollutants associated with introduced Pacific salmon. Resident fishes sampled from salmon spawning reaches had higher mean pollutant concentrations than those from upstream reaches lacking salmon (93.5 and 4.1 μg x kg(-1) [PCB], 24.0 and 3.1 μg x kg(-1) [DDE], 8.5 and 1.0 μg x kg(-1) [PBDE], respectively), but differences varied substantially among lake basins. In Lake Michigan tributaries, PCB concentrations in resident fishes from salmon reaches were over four times higher than those from salmon reaches in Lake Huron and over 30 times higher than those from Lake Superior. Moreover, resident fish pollutant concentrations were better explained by pollutant inputs from salmon (μg x m(-2); R(2) = 0.76 [PCB], 0.64 [DDE], 0.64 [PBDE]) than by land development/agriculture, watershed area, resident fish species, body length, or lipid content. These results suggest that pollutant dispersal to stream ecosystems via biotransport is an often overlooked consequence of salmon stocking and historical food web contamination in the Great Lakes. Our findings have implications for Great Lakes management, including dam removal and wildlife conservation.

Resident fishes display elevated organic pollutants in salmon spawning streams of the great lakes.

When vigorous exercise becomes too much of a good thing.

Lake trout (Salvelinus namaycush) were fed bloater (Coregonus hoyi) in eight laboratory tanks over a 135-d experiment. At the start of the experiment, four to nine fish in each tank were sacrificed, and the concentrations of 75 polychlorinated biphenyl (PCB) congeners within these fish were determined. Polychlorinated biphenyl congener concentrations were also determined in the 10 lake trout remaining in each of the eight tanks at the end of the experiment as well as in the bloater fed to the lake trout. Each lake trout was weighed at the start and the end of the experiment, and the amount of food eaten by the lake trout was recorded. Using these measurements, net trophic transfer efficiency (γ) from the bloater to the lake trout in each of the eight tanks was calculated for each of the 75 congeners. Results showed that γ did not vary significantly with the degree of chlorination of the PCB congeners, and γ averaged 0.66 across all congeners. However, γ did show a slight, but significant, decrease as log K(OW) increased from 6.0 to 8.2. Activity level of the lake trout did not have a significant effect on γ.

Net trophic transfer efficiencies of polychlorinated biphenyl congeners to lake trout (Salvelinus namaycush) from its prey.

Concentrations of 86 congeners of polychlorinated biphenyls (PCBs) were determined for 25 adult female and 25 adult male burbot (Lota lota) from Lake Erie. Significant differences in mean proportions of total PCB concentration between males and females ages 14-17 were found for 26 congeners. Males generally had higher mean proportions of hexachloro congeners, whereas females had higher mean proportions of more highly chlorinated congeners. In contrast, only four congeners exhibited differences between males and females ages 6-13 in mean proportions of total PCB. Therefore, the sexual difference in PCB congener distribution widened for older burbot. Males ages 14-17 also had higher proportions of certain hexachloro congeners than the other three demographic groups (males ages 6-13, females ages 6-13, and females ages 14-17) we examined. The reverse was true for more highly chlorinated congeners. The results supported a previous hypothesis that older male burbot spent a substantial amount of time feeding in the vicinity of mouths of rivers with sediment contaminated with PCBs. However, additional studies are needed to clarify and corroborate this apparent "hot spot" effect, such as seasonal movements, spatial distributions, and diet of burbot; and PCB congener distributions of various species of prey fishes captured at various locations throughout the eastern basin of Lake Erie and at different locations in the contaminated rivers.

Sexual difference in PCB congener distributions of burbot (Lota lota) from Lake Erie.

We compared the distributions of the congeners of polychlorinated biphenyls (PCBs) detected in whole-body samples of burbot (Lota lota) from Great Slave Lake and Lake Erie. Total PCB concentrations in Great Slave Lake burbot were about 1/60 of the concentrations in Lake Erie burbot. Burbot from Great Slave Lake contained a higher proportion of lower-chlorinated (2-6 chlorines) congeners than did burbot from Lake Erie; the reverse occurred for more highly chlorinated (7-9 chlorines) congeners. Hexachloro congeners, followed by pentachloro congeners, dominated the proportions of total PCB in burbot from both lakes. There were no differences between sexes in whole-body samples or between gonad and somatic tissues in the proportions of the 39 congeners and three sets of co-eluters detected in burbot from Great Slave Lake. In contrast, there were distinct sex differences in congener distributions for older burbot from Lake Erie. Our results generally supported a prediction of higher proportions of lower-chlorinated PCB homologs in organisms in remote polar areas. However, the latitudinal effect on PCB congener distribution may be more complex than that portrayed in previous studies. Environ Toxicol Chem © 2014 SETAC.

James P. O'Keefe

Annis Water Resources Institute

Grand Valley State University

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

James P. O'Keefe

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Grand Valley State University

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

Annis Water Resources Institute