EHP 104(4) April 1996 (fwd)

[slester@essential.org]

> Persistence of Decreased T-Helper Cell Function in Industrial Workers
> 20 Years after Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin
> 
> Torsten Tonn(1), Charlotte Esser(1), E. Marion Schneider(2), Wolfgang
> Steinmann-Steiner-Haldenstätt(3), and Ernst Gleichmann(1)
> 
> (1) Medical Institute of Environmental Hygiene and (2) Institute for
> Hemostasis and Transfusion Medicine, University of Düsseldorf, 40225
> Düsseldorf, Germany; (3) Medical Department, Bayer AG Uerdingen,
> Krefeld, Germany
> 
> In experimentally exposed animals,
> 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) causes severe
> immunosuppression. However, the overall susceptibility of humans for
> the different pathological effects of TCDD has remained unclear. We
> examined the long-term effects of TCDD in 11 industrial workers who
> were exposed to high doses of TCDD for several years 20 years ago.
> Current TCDD body burdens were still at least 10 times higher (between
> 43 and 874 pg/g blood fat) in these exposed persons than in the
> average German population. To evaluate possible TCDD-induced changes
> in the percentage of different lymphocyte subsets, we determined a
> large panel of lymphocyte subsets in the blood by flow cytometric
> analysis. Immunocompetence of T- and B-lymphocytes was tested by
> mitogen (phytohemagglutinin, pokeweed mitogen)-induced
> lymphoproliferation assays and by assays using sensitive
> mixed-lymphocyte cultures. No significant differences could be
> detected between the individuals tested and controls for surface
> marker distribution or mitogen-induced lymphoproliferation.
> TCDD-exposed subjects showed a reduced response to human lymphocyte
> antigen-allogeneic lymphocytes and interleukin-2-boosted
> proliferation. Responder cells of the dioxin-exposed persons
> proliferated less in response to irradiated stimulator cells (p > to
> 0.05), and the "third-party" mixed lymphocyte reaction against
> unirradiated stimulator cells revealed suppressive activity in the
> responder cell fraction compared to the controls (p =/< 0.01).
> Furthermore, the capacity of a pool of T-cells isolated from
> TCDD-exposed subjects to proliferate upon interleukin-2 stimulation
> was significantly diminished (p =/< 0.05). TCDD has a long-term
> immunosuppressive effect on T-helper cell function, which is mediated
> more likely by a reduced functionality of individual cells rather than
> by a reduction in absolute cell numbers in the peripheral blood. Key
> words: allogeneic response, immunosuppression, lymphocyte subsets,
> mitogen stimulation, 2,3,7,8,-tetrachlorodibenzo-p-dioxin. --Environ
> Health Perspect 104:422-426 (1996)
> ----------------------------------------------------------------------
> 
> Address correspondence to C. Esser, Medical Institute of Environmental
> Hygiene, Auf´m Hennekamp 50, D-40225 Düsseldorf, Germany.
> 
> We appreciate the cooperation of the participants in this study. We
> thank Zhi-Wei Lai for critically reading the manuscript, Swantje
> Steinwachs and Dörte Post for technical help, and Britt Harms for help
> with the statistics. This study was supported in part by grant 01 KD
> 89030 from the Bundesministerium für Forschung und Technologie,
> Germany. The work of C.E. and E.G. is supported through SFB 503,
> "Molecular and cellular mediators of exogenous noxes" at the
> Heinrich-Heine-University of Düsseldorf.
> 
> Received 2 October 1995; accepted 2 January 1996.
> ----------------------------------------------------------------------
> 
> Halogenated aromatic hydrocarbons (HAHs) are ubiquituous in the
> environment. The most toxic and best-studied of these compounds is
> 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Numerous studies on the
> toxic effects of TCDD have been done, most of them on rodents. TCDD is
> tumor promoting, teratogenic, and embryotoxic (1). One of the earliest
> and most sensitive markers of TCDD toxicity in experimental animals is
> impairment of the immune system, which is evident at doses that do not
> lead to overt signs of general toxicity. TCDD leads to atrophy of
> lymphoid organs, such as the thymus, spleen, and lymph nodes (2).
> Moreover, TCDD was shown to suppress cellular and humoral immune
> functions in experimental animals (2). Using a variety of in vivo
> exposure schemes or in vitro assays, TCDD was found to impair
> cytotoxic T-lymphocytes and natural killer cell functions, or inhibit
> antibody production by B-cells, for example (2). The capacity to
> respond to mitogenic stimuli, such as phytohemagglutinin (PHA) and
> lipopolysaccharide (LPS), is also affected by TCDD (3,4).
> 
> Susceptibility to the toxicity of TCDD is genetically determined by
> the aryl hydrocarbon receptor (AhR) locus. This gene codes for a
> cytosolic, TCDD-binding protein that is activated to a DNA-binding
> state upon ligand engagement (5) and induces the expression of a gamut
> of genes, including CYP1A1, genes of fatty acid metabolism, cytokines,
> and/or growth and differentiation factors (6). The binding affinity of
> AhR to TCDD and thus susceptibility varies between different animal
> species and also interindividually in outbred populations. Neither the
> overall toxicity of TCDD to humans nor TCDD-induced effects on the
> human immune system is known.
> 
> We examined 11 workers with defined TCDD body burdens who had been
> inadvertently exposed to TCDD for several years. We evaluated possible
> deviations in peripheral blood lymphocyte subsets using
> multi-parameter immunofluorescence of relevant markers (7) and
> examined in vitro effects of TCDD on immunocompetence of T- and
> B-cells.
> 
> Materials and Methods
> 
> Eleven workers, 45-63 years of age, participated in this study. They
> had been exposed to high doses of TCDD and other polychlorinated
> dibenzo-p-dioxins between 1966 and 1976 during production and
> maintenance operations at a chemical factory producing
> 2,4,5-trichlorophenol. Six other exposed workers declined to
> participate. The level of TCDD in blood lipids was determined in 1989
> and 1992 by the ERGO-Forschungsgesellschaft, Hamburg, Germany, or
> Bioscientia, Moers, Germany, respectively, according to standard mass
> spectrometry (8). Ten age-matched, healthy, males with no known TCDD
> exposure history, working in the same company in office jobs,
> volunteered as controls. Informed consent was obtained from all
> subjects.
> 
> Peripheral blood (50 ml) was drawn by venipuncture into a sterile
> tube, containing 100 U of heparin/ml blood. Peripheral blood
> mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Pharmacia
> Fine Chemicals, Uppsala, Sweden) density centrifugation.
> 
> For single or dual fluorescence analysis, aliquots of 1 x 10(6)
> peripheral blood lymphocytes were spun down and incubated for 10 min
> at 6-8°C with fluoresceinated antibodies as recommended by the
> manufacturer. Cells were washed two times after staining, and data
> from 10,000 cells were collected immediately in list-mode on a FACScan
> flow cytometer, using appropriate compensation settings
> (Becton-Dickinson, Mountain View, California). Forward and rectangular
> light scatter gates were used.
> 
> The following fluorochromed antibodies were used: mouse anti-CD3FITC
> (clone SK7), mouse anti-CD4FITC (clone SK3), mouse anti-CD8PE/CD8FITC
> (clone SK1), mouse anti-CD19FITC (clone 4G7), all from
> Becton-Dickinson; mouse anti-CD45ROPE (clone UCH-L1) and anti-CD45RAPE
> (clone F8-11-3), both from Serotec (Oxford, UK); mouse anti-CD56PE
> (clone B 159.5; Pharmingen, San Diego, California); and anti-CD57
> (Leu7; Becton-Dickinson).
> 
> We cultured 5x10(4) cells/well in 96-well round-bottom plates
> containing 150 microliters RPMI 1640 culture medium, supplemented with
> 5% heat-inactivated pooled human AB0 serum, L-glutamine (2 mM), sodium
> pyruvate (1 mM), nonessential amino acids, 50 U/ml penicillin, and 50
> U/ml streptomycin.
> 
> Cells were cultured in triplicate either with medium alone or in the
> presence of 9 micrograms/ml PHA (Sigma, St. Louis, Missouri) or 6
> micrograms/ml pokeweed mitogen (PWM; Sigma) for 3 days at 37°C in a
> water-saturated 5% CO2 atmosphere. On day 4 of culture, cells were
> pulsed with 1 microCi of 3H-thymidine and then harvested 20 hr later
> to determine incorporated radioactivity (beta counter LS 6000 IC,
> Beckman Instruments, München, Germany).
> 
> The human lymphocyte antigen (HLA) phenotype of peripheral blood
> mononuclear cells was determined by standard microlymphotoxicity
> assays using A,B,C plates (One-lambda, Canoga Park, California) and
> HLA-class II plates (Biotest, Dreieich, Germany). For mixed lymphocyte
> cultures, washed cells were resuspended in serum-free CG-medium
> (Vitronex, Vilshofen, Switzerland), supplemented with 60 micrograms/ml
> gentamcycin. We seeded 5 x10(4) responder cells in triplicate into
> 96-well round-bottom microtiter plates. For "one-way" mixed lymphocyte
> cultures, the same number of irradiated (30 Gy) stimulator cells were
> added. Stimulator cells were from a pool of cells from 20 allogeneic,
> HLA-unrelated individuals. For "third-party" suppressor assays, the
> lymphocytes from dioxin-exposed or control persons were irradiated (30
> Gy) and added to the pooled allogeneic cells (in this case
> unirradiated).
> 
> Interleukin-2 (IL-2)-inducible proliferation of T-cells was measured
> after adding 30 U/ml recombinant IL-2 per 5 x104 cells. Recombinant
> IL-2 was a gift of P. Loeliger (Sandoz, Switzerland).
> 
> Results
> 
> The concentration of TCDD in the blood of 11 workers exposed between 2
> and 11 years before 1976 (with one exception, see Table 1) was
> determined in 1989 or 1992, i.e., 13-15 years after the last exposure.
> Table 1 summarizes the exposure parameters and the health status of
> the TCDD-exposed workers determined at a thorough general medical
> examination in 1992. The TCDD values in blood fat differed up to
> 20-fold between individuals, yet even the lowest burdens were well
> above the average level of the German population, which is about 4
> pg/g blood fat (9). At the time of the study, five persons still
> suffered from chloracne, of which one had chronic gastritis and one
> hyperthyreosis. Two subjects displayed a disturbance of fatty acid
> metabolism. The others appeared healthy.
> 
> [Image]
> 
> The frequencies of various lymphocyte subsets were determined by flow
> cytometry. The percentages of B-cells (CD19), T-cells (CD3), and
> subsets thereof were determined, e.g., T-helper cells (CD4), cytotoxic
> T-cells (CD8), virgin helper/inducer T-cells (CD4CD45RA) as well as
> primed helper/inducer (CD4CD45RO). Moreover, natural killer cells
> (CD56 and CD57) and HLA-DR expression were measured. Some of the
> subset markers were previously shown to be sensitive parameters of
> TCDD-exposure in marmosets, a new world primate species (7). Table 2
> shows the result of the analysis for each of the 11 TCDD-exposed
> individuals and for the controls. No difference between controls and
> the mean values of the TCDD-exposed group was evident for any of the
> lymphocytes subsets analyzed. Note that the percentage of
> CD56-expressing cells (i.e., natural killer cells) is comparatively
> lower than reported in the literature for younger individuals,
> possibly a phenomenon of age (27).
> 
> [Image]
> 
> Peripheral blood lymphocytes were tested for their capacity to respond
> to mitogens PHA and PWM. As shown in Figure 1 [not shown], lymphocytes
> from TCDD-exposed and control persons responded equally well to
> mitogen stimulation, with some individual variation. There was no
> statistically significant difference between the response of the two
> groups (p = 0.5641). Moreover, when we corrected the data for the
> interindividual differences in T-cell frequencies in the blood (see
> Table 2), it becomes even more clear that TCDD exposure does not
> affect the mitogen-induced proliferative capacity (p = 0.7823; data
> for PHA stimulation not shown). No correlation existed between
> individual TCDD levels in the blood nor the age of the persons and the
> respective proliferative capacity of their lymphocytes (not shown).
> 
> We tested the capacity of T-cells ("responder cells") from
> dioxin-exposed persons to specifically react against irradiated,
> HLA-different, allogeneic lymphocytes ("stimulator cells") in a
> mixed-lymphocyte culture. In another experiment, we added irradiated
> responder cells as a third party to a pool of unirradiated peripheral
> blood mononuclear cells from 20 different donors. Whereas the former
> assay measures the response to allo-major histocompatibility complex,
> the latter is used to detect suppressive factors/cells in the
> responder cell population, which would inhibit the respective response
> of the unirradiated cells (10). As shown in Figure 2 [not shown], the
> responder cells of the TCDD-exposed persons proliferated less in
> response to irradiated stimulator cells (p < 0.05 by Student's
> t-test). Moreover, the third-party mixed lymphocyte reaction against
> unirradiated stimulator cells revealed a small amount of suppressive
> activity in the responder cells of dioxin-exposed individuals,
> resulting in a decreased overall proliferation of T-cells (Fig. 2).
> This significant suppression (p < 0.01 by Student's t-test) is
> indicative of a reduced T-helper cell response (11). However, the
> actual number of T-helper cells was unaffected by TCDD (see Table 2).
> 
> The capacity of a pool of T-cells to proliferate upon IL-2 stimulation
> is a parameter of normal T-cell function and correlates with the
> presence of preactivated T-cells in the pool, which would result in a
> higher overall proliferative response. Peripheral blood mononuclear
> cells of TCDD-exposed individuals and control persons were co-cultured
> with a low dose of IL-2 for 4 days. The cells of TCDD-exposed persons
> revealed a reduced capacity to proliferate with IL-2 (Fig. 2; p <
> 0.05). The values for one exposed individual were not included in the
> data due to an excessively high proliferation rate. Those particular
> values reflected an extreme preactivation of the T-cells, which is
> typical for the beginning of an acute, but undetected, infection. No
> atopic status is known for that person. However, it must be noted that
> the statistically significant differences between the TCDD-exposed and
> control persons disappear if this one value is included.
> 
> Discussion
> 
> Ever since the major accident at Seveso, Italy, the danger of dioxin
> has been recognized. The acute toxicity dose of TCDD is among the
> lowest for any known chemical substance. Fortunately, no people died
> at Seveso, and most scientists believe humans to be at the lower end
> of the susceptibility scale. However, TCDD continues to be
> inadvertently released into the environment, and little is known on
> the long-term effects of low doses. In Germany, the body burden of the
> general population is about 4 ppt TCDD in blood fat, with an estimated
> daily uptake, mostly by food, of 26 pg (9).
> 
> The immune system seems to be the most sensitive target of TCDD action
> in experimental animals. Humoral as well as cellular components of
> immune responses are suppressed by TCDD (2,12,13). The interaction of
> the human immune system with TCDD has remained controversial. Studies
> with an evolutionarily closely related species, the primate Callithrix
> jacchus (marmosets), revealed sensitive parameters for TCDD-induced
> alterations in peripheral blood lymphocyte subsets. Using combinations
> of surface markers, changes in the frequency of memory (CD4+, CDw29+),
> suppressor-inducer (CD4+, CD45RA+) T-cells and B-cells (CD20+) were
> found (7). In vitro studies on human blood leukocytes after accidental
> or occupational exposure to TCDD were ambiguous with respect to the
> immunological alterations induced (14). Some authors reported
> suppressed responses (15), whereas others reported enhanced
> mitogen-induced lymphoproliferative responses (16) and increased
> percentages in suppressor/cytotoxic T-cells and the absolute number of
> natural killer cells in the peripheral blood (17,18). Others found no
> effect on mitogen stimulation (19) and no deviations in peripheral
> blood subsets (20,21). With the exception of one of these studies
> (21), the actual body burden had not been determined. It is
> interesting to note that also in experimental animals the
> immunosuppressive effects of TCDD are commonly detected by functional
> tests (i.e., following antigen-specific stimulation), rather than
> unspecifically, by mitogen stimulation, for example. Thus, it was
> important to choose an appropriately sensitive test system.
> 
> We tested possible immunosuppression in TCDD-exposed workers using
> three parameters of lymphocyte competence. We found no difference
> compared to the age-matched control persons for the distribution of
> lymphocyte subsets in the blood. This result is in accordance with the
> more elaborate study of Neubert and co-workers (7), in which they
> applied the experience gained in Callithrix jacchus on TCDD-sensitive
> lymphocyte subsets to humans, but failed to detect any decrease in
> leukocyte subsets associated with elevated dioxin body burdens. In the
> present study, we demonstrated a small but significant difference in
> the allo-response of T-cells and in their proliferative response to
> IL-2. Moreover, the lymphocytes of TCDD-exposed persons displayed a
> suppressive activity, which inhibited an ongoing allo-response of
> HLA-unrelated lymphocytes. This is probably due to an increased
> proliferation response of T-cells counteracting the CD4+ T-helper-1
> cells, representing the primary responder type in mixed lymphocyte
> cultures.
> 
> As reported previously (22), the changes in immunocompetence we
> observed in vitro did not correlate with obvious diseases related to
> severe immunodeficiency such as certain cancers and infections. The
> workers were generally healthy and, with one exception, had no history
> of increased susceptibility to infections. The functional reserve of
> the immune system is enormous, so that impaired immune responsiveness
> need not have pathological consequences (23). Indeed, the term
> "immunotoxic" has not been properly defined (24). Only a large,
> well-controlled epidemiological study might reveal the actual health
> effects of subtle changes in immunocompetence.
> 
> The reduced immunocompetence in vitro observed here fails to correlate
> with a reduced number of lymphocyte subsets. Thus, TCDD-induced
> immunosuppression is more likely mediated by a reduced functionality
> of individual cells rather than by a reduction in cell numbers
> circulating in the blood.
> 
> Thymus involution, a reduction of thymus weight and cellularity, is a
> hallmark of TCDD exposure. In mice, TCDD skews the distribution of
> lymphocyte subsets (25,26). However, although a link between thymus
> events and peripheral immunosuppresssion is often implicitly assumed,
> nothing is known about the balance between thymocyte/T-cell generation
> and migration to the periphery under TCDD treatment. The thymus
> normally begins to atrophy at about the time of sexual maturity in
> mice and humans (27). T-cell numbers and other leukocytes in the
> periphery remain unaffected in TCDD-exposed versus nonexposed persons
> (28), thus an additional atrophic effect on the thymus by TCDD might
> not be detectable in people exposed to TCDD as adults over about 30
> years of age.
> 
> We found no correlation between TCDD levels in blood and performance
> of peripheral blood mononuclear cells in the assays. This is not
> surprising, since the development of chloracne, the major effect of
> dioxin exposure on humans, in individuals affected by the Seveso
> accident did not correlate to the severity of TCDD exposure. It is now
> generally accepted that the Ah receptor mediates dioxin toxicity, and
> the reason for variability in individual responses is likely due to
> genetic differences in Ah receptor alleles in different individuals.
> 
> We have presented here data where the actual body burden of the
> individuals analyzed were known. We know of only two other such
> studies. In accordance with our data, no phenotypic difference between
> lymphocyte subsets in the exposed and unexposed groups was found, with
> the exception of CD8 cells, which were slightly increased (21,22).
> However, the present study is the first to demonstrate reduced
> immunocompetence of lymphocytes from TCDD-exposed humans.
> ----------------------------------------------------------------------
> 
> References
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> ----------------------------------------------------------------------
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