There is evidence from animal studies that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs immune responses, with the thymus being a principal target organ. The purpose of this study was to evaluate thymic function, through measurement of thymic hormone levels, in persons exposed to TCDD. We examined thymosinalpha-1 (Thya-1) levels in sera from a group of 94 persons who were presumed to be exposed to TCDD from living, working, or recreating in a contaminated residential area. We compared these results, along with results from in vitro and in vivo tests of immune function, with those from a group of 105 unexposed persons who were similar with regard to age, sex, and race. The exposed group had a significantly lower mean Thya-1 serum level (977.3 ± 304.1 pg/ml vs. 1148.7 ± 482.1 pg/ml, p < .01 by t-test). We also found a statistically significant trend of decreasing Thya-1 levels with increasing number of years of residence in the TCDD-contaminated area. However, Thya-1 levels were not associated with other measures of immune function in the TCDD-exposed group. Thus, while the principal findings suggest that long-term TCDD exposure may be associated with diminished secretion of Thya-1, the lack of an association with an increased prevalence of clinically diagnosed immune suppression in these TCDD-exposed persons makes the biologic significance of the findings unclear. Further studies are needed to more fully evaluate possible long-term TCDD-induced effects on the thymus and human immune function.
The authors acknowledge the assistance of Dr. John Bagby, Dr. Jack Dean, Dr. Greg Evans, Mr. Woodrow Garrett, Dr. Allan Goldstein, Dr. Michael Luster, Mr. Daryl Roberts, Ms. Cartha Naylor, and Dr. Jeanette Stehr-Green in conducting this study and preparing this manuscript.
Parts of this study were conducted under a cooperative agreement between the Missouri Department of Health and the Centers for Disease Control. It was supported, in part, by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund by interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service.
Use of trade names is for identification only and does not constitute endorsement by the U.S. Public Health Service, the U.S. Department of Health and Human Services, the George Washington University Medical Center, or the Missouri Department of Health.
Requests for reprints should be sent to Dr. Stehr-Green, Centers for Disease Control, 1600 Clifton Rd., Atlanta, GA 30333.
Journal of Toxicology and Environmental Health, 28:285–295, 1989
Copyright © 1989 by Hemisphere Publishing Corporation
INTRODUCTION
In 1983 a small pilot epidemiologic study of persons living in Missouri who were thought to be exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (hereinafter referred to simply as TCDD) was conducted (Stehr et al., 1986). Although the analyses did not produce any firm indications of increased disease prevalence directly related to the putative exposures, statistically nonsignificant indications of an increased prevalence of helper:suppressor T-cell ratios less than 1.0 and an apparent diminishing of delayed-type hypersensitivity (DTH) responses were noted in the group at highest risk of exposure. Results from a larger study of TCDD-exposed persons conducted in 1985 suggested that long-term exposure to TCDD was associated with anergy in skin testing and with several in vitro immune abnormalities, including increased lymphoproliferative responses to pokeweed mitogen stimulation and decreased T-cell subset percentages, although absolute cell counts were within normal limits (Hoffman et al., 1986). No increased reports of clinically diagnosed immune suppression or of prolonged or repeated infections in these groups of TCDD-exposed persons were noted in either study. Subsequent follow-up examinations conducted 15–17 mo later of 43 persons who were anergic or hypoergic in the 1985 study group did not show any individuals with anergy, although results of in vitro immunologic tests were consistent with those in the original study (Evans et al., 1988). Whether these observed reversions were due to a greater potency of test antigens used in the follow-up examinations, a “booster” effect of the reapplication following sensitization from the initial application of antigens, recovery of the affected persons’ immune systems in the intervening period, or technical problems in the original study remains unresolved. Immunologic testing of 24 persons with adipose TCDD levels that were known to be elevated (i.e., > 20 parts per trillion) again showed an association of TCDD body burden with some in vitro immunologic test results, but there was no association with the occurrence of anergy (Webb et al., 1987). The overall significance of these subclinical immunologic differences in some groups of TCDD-exposed persons, the underlying biochemical mechanisms of toxicity, and whether these differences may be predictive for future development of overt disease are not clear.
By comparison, experimental animals exposed to TCDD have shown depressed DTH responses, reduced allograft rejection, and increased susceptibility to infection (Faith and Luster, 1979). Among the most consistent findings of these laboratory studies were thymic atrophy with cortical thymic depletion and depletion of T-dependent areas in spleen and lymph nodes (Clark et al., 1981; Vos et al., 1974, 1980). In light of the effect of TCDD on the thymus in experimental animals and the consistent in vitro immunologic test results in humans exposed to TCDD, we undertook this study to evaluate the association of TCDD exposure with thymic function, as measured by the levels of the thymic peptide, thymosinalpha-1 (Thya-1), in human sera, and to examine the possible role of differences in Thya-1 levels in explaining observed TCDD-associated subclinical effects on human immune responses.
MATERIALS AND METHODS
For the present study, we reevaluated some of the data collected as part of the 1985 study of persons who were presumed to be exposed to TCDD from living, working, or recreating in a contaminated residential area; participant selection criteria and the methods of specimen collection and laboratory analysis for the various in vivo and in vitro immunologic tests have been previously described (Hoffman et al., 1986). Sera for this study had been stored at -40ºC during the intervening years. Thya-1 in serum has been shown to be stable for at least 6 yr when stored under these conditions (P. H. Naylor, personal communication, 1988). We tested all specimens containing a sufficient quantity of serum for measurement of Thya-1 levels [i.e., 94 (61.0%) of the original 154 TCDD-exposed participants and 105 (67.7%) of the 155 unexposed persons] using a radioimmunoassay that has been previously described (Goldstein et al., 1977). For this study, the assay was modified by absorbing heterologous rabbit antiserum against a synthetic peptide containing the 14C-terminal amino acids of Thya-1, which was coupled to sepharose 4B (Wada et al., 1988); the resulting antiserum was specific for the N-terminal region of Thya-1, yielding a more sensitive assay. We randomly assigned serum specimens from TCDD-exposed and unexposed participants into batches and analyzed them in 15 separate runs conducted over 2 consecutive days. Results were evaluated relative to standard plasma used as an internal reference, which was inserted at the beginning, middle, and end of each analytical run. Overall, the intra- and interassay coefficients of variation were 10% and 25%, respectively. As with all other laboratory tests and clinical examinations, technicians performing the Thya-1 assays were not aware of the exposure status of any participant.
Qualitative data (e.g., percentage of males and females) were examined by using a contingency table approach (chi-squared or two-tailed Fisher’s exact analyses). We initially compared means (e.g., serum Thya-1 levels by exposure group), using t-tests and analysis of variance. We also evaluated associations between variables with continuous distributions (e.g., Thya-1 levels and total white blood cell counts), using correlation coefficients. Analysis of covariance was used to compare differences between exposure groups, controlled for possible confounding factors. Covariates used in regression models included age, sex, and socioeconomic level (two-factor Hollingshead index; Hollingshead, 1957). In addition, to assess the possible influences of stress and other neurobehavioral factors on Thya-1 and on immune function, we examined results of the Profile of Mood States psychological battery (Derogatis, 1982), which had been administered to each participant older than 7 yr of age. For all tests of hypotheses, differences were considered statistically significant if the p value was less than .05.
RESULTS
To evaluate whether the present study population (i.e., the subset of participants for whom Thya-1 serum levels were measured) was representative of the original study population, we compared their demographic characteristics and immune status. There were no differences within exposure groups in distributions by sex, race, T-cell subsets, lymphoproliferative responses, serum IgG, or DTH responses between the present study group and those excluded from the original study population because of insufficient amounts of reserve serum. Among the TCDD-exposed persons, the current study group was slightly older than those excluded (reflecting the fact that reserve serum was not collected from children), had a higher average socioeconomic level, and had lower leukocyte and lymphocyte counts; the only differences noted in the unexposed group between the current study participants and those excluded were somewhat lower lymphocyte counts.
For the 199 participants who comprise the present study population, there were no differences between the TCDD-exposed and unexposed groups with regard to age, sex, race, or socioeconomic level. In addition, differences between TCDD-exposed and unexposed groups for in vitro and in vivo immune function measurements in this subset were similar to those reported in the original 1985 study (Hoffman et al., 1986).
The shapes of the frequency distributions of Thya-1, levels in serum were similar for both groups (Fig. 1), but the entire distribution for the TCDD-exposed group was shifted to the left (i.e., toward lower Thya-1 levels). The TCDD-exposed persons had a significantly lower mean Thya-1 serum level than the unexposed persons (Table 1). This difference persisted for both males and females, across all age groups and most socioeconomic class strata, and remained statistically significant after simultaneously controlling for age, sex, and socioeconomic status by using analysis of covariance. None of the study subjects had any clinically recognized immunologic disorders that could explain this observed difference in Thya-1 levels. In a multivariate linear regression model controlled for age, sex, and socioeconomic status, Thya-1 levels were inversely associated with the number of years spent living in the contaminated areas (i.e., a surrogate for TCDD dose that ranged up to
FIGURE 1. Distribution of thymic hormone levels, by TCDD-exposure group.
a maximum of 7 yr); the final form of this model was Thya-1 = 1295.9 + 1.7(age) - 146.8(sex) - 0.1(Hollingshead index) - 37.7(years lived in TCDD area), with estimated beta-coefficients for (sex) and (years lived in TCDD area) being significantly different from zero (p < .05).
Serum levels of Thya-1 within the TCDD-exposed group were not statistically significantly associated with age, sex, socioeconomic level, smoking or other current use of tobacco products, current consumption of alcohol, frequency of outdoor recreation, body weight, or any of the psychological measures of confusion/bewilderment, depression/dejection, tension/anxiety, vigor/activity, or fatigue/inertia. There were also no significant correlations between serum Thya-1 levels in the TCDD-exposed group and any of the measures of T-cell subset populations, lymphocyte proliferative responses, serum immunoglobulin, or delayed-type hypersensitivity (Table 2). Nor was there a clustering of abnormal immune test results among those persons in the lowest decile of Thya-1 serum levels. In contrast, statistically significant higher Thya-1 levels in males and positive correlations of serum Thya-1 levels were found with total leukocyte count, number of granulocytes, number of positive DTH responses to the seven recall antigens, and total average induration response to the recall antigens among the 105 persons in the unexposed comparison group (Table 2).
DISCUSSION AND CONCLUSIONS
The thymus gland plays a key role in the development of T-cell immune function and accomplishes many of its effects through the elaboration of thymic peptides (Goldstein et al., 1966, 1981). Thya-1 is a 28-amino-acid
TABLE 1.Comparison of Thyal-1 Levels in Serum, by Exposure Group, Age Group, Sex, and Socioeconomic Class
Level of thymosinalpha-1 (pg/ml) |
|||||||||||
TCDD-exposed group |
TCDD-unexposed group |
||||||||||
| Demographic strata |
N |
Median |
Mean |
N |
Median |
Mean |
Difference |
||||
|
|||||||||||
| Total group | 94 |
961.0 |
977.3 |
(304.1) |
105 |
1115.0 |
1148.7 |
(482.1) |
p < .01 |
||
| Age | |||||||||||
| 0–18 yr | 23 |
970.0 |
996.0 |
(292.5) |
17 |
1168.0 |
1170.9 |
(432.4) |
p = .13 |
||
| 19–39 yr | 54 |
950.0 |
971.9 |
(311.6) |
68 |
1089.0 |
1115.9 |
(492.5) |
p = .05 |
||
| 40–64 yr | 13 |
846.0 |
941.4 |
(330.5) |
19 |
1102.0 |
1228.1 |
(512.6) |
p = .09 |
||
| ≥65 yr | 4 |
1010.5 |
1060.3 |
(263.7) |
1 |
1492.0 |
1492.0 |
(—) |
— |
||
| Difference with exposure groupb |
p = .90 | p = .71 | |||||||||
| Sex | |||||||||||
| Male | 43 |
973.0 |
1021.9 |
(318.2) |
44 |
1197.5 |
1273.4 |
(612.5) |
p = .02 |
||
| Female | 51 |
904.0 |
939.7 |
(289.6) |
61 |
1062.0 |
1058.7 |
(338.1) |
p = .05 |
||
| Difference within exposure groupb |
p = .19 | p = .02 | |||||||||
| Hollingshead socioeconomic classc |
|||||||||||
| Class I | 0 |
— |
— |
(—) |
2 |
1293.5 |
1293.5 |
(61.5) |
— |
||
| Class II | 3 |
1255.0 |
1368.7 |
(302.0) |
1 |
807.0 |
807.0 |
(—) |
— |
||
| Class III | 18 |
817.0 |
909.3 |
(239.6) |
24 |
1033.0 |
1112.5 |
(563.4) |
p = .12 |
||
| Class IV | 55 |
996.0 |
1001.7 |
(301.6) |
63 |
1143.0 |
1163.6 |
(472.5) |
p = .03 |
||
| Class V | 15 |
805.0 |
920.5 |
(355.2) |
15 |
1104.0 |
1147.5 |
(448.9) |
p = .14 |
||
| Difference within exposure groupb |
p = .08 | p = .93 | |||||||||
|
|||||||||||
ap Values calculated for comparisons of means by TCDD-exposure groups within demographic strata by using t-tests.
bp Values calculated for comparisons of means across age groups and socioeconomic classes within TCDD-exposure groups by using analysis of variance, and for comparisons of means by sex within TCDD-exposure classes, by using t-tests.
cHollingshead two-factor index is calculated baed on education level and occupation, with Class I representing the highest socioeconomic class and Class V representing the lowest socioeconomic class.
biologically active peptide that was originally isolated from thymus tissue extracts termed "thymosin fraction five" (Goldstein et al., 1977). The major source of production of Thya-1 is in the mediatory epithelioid cells of the thymus (McClure et al., 1982; Hirakawa et al., 1982; Oates et al., 1987), although additional studies have suggested that secondary lymphoid tissues also produce Thya-1 (Panneerselvam et al., 1987). Thya-1 influences cell-mediated immune function at several stages, by modulating the differentiation of prothymocytes to mature thymocytes (Schulof and Goldstein, 1983; Goldstein et al., 1983), as well as the function of peripheral blood lymphocytes through effects on lymphokine expression, especially gamma-interferon, and lymphokine
TABLE 2. Correlation of Serum Thya-1 Levels and Selected Immune System Parameters, by TCDD-Exposure
Group
Simple correlation coefficients |
||
| TCDD-exposed (n = 94) |
TCDD-unexposed |
|
| Complete blood counts (cells/mm3) | ||
| White Blood Cell Count | -0.01 |
0.25a |
| Lymphocytes | -0.10 |
0.02 |
| Monocytes | -0.10 |
0.16 |
| Granulocytes | 0.02 |
0.26a |
| Non-T-lymphocytes | -0.02 |
0.09 |
| T-Cell subset populations (cells/mm3) | ||
| OKT3 | -0.12 |
-0.06 |
| OKT4 | -0.13 |
-0.04 |
| OKT8 | -0.10 |
-0.07 |
| OKT11 | -0.13 |
-0.02 |
| OKT4/OKT8 ratio | 0.08 |
0.04 |
| Lymphocyte proliferative responses (cpm) | ||
| Phytohemagglutinin | -0.03 |
0.08 |
| Conconavalin A | -0.06 |
-0.13 |
| Pokeweed mitogen | 0.08 |
-0.10 |
| Tetanus toxoid | -0.05 |
-0.08 |
| Allogeneic T-cell cytotoxicity (% activity) |
-0.03 |
0.13 |
| Serum IgG (mg/dl) | 0.21b |
-0.05 |
| Measures of delayed-type hypersensitivityc |
||
| Average induration (mm) | -0.28 |
0.26a |
| Number of "positive" antigens |
-0.28 |
0.27a |
a Coefficient significantly different from zeo, p < .05.
bCoefficient different from zero, p = .05.
cBecause of concerns about unreliable DTH skin-test measurements for some participants, results for them
were considered to be invalid; thus, these analyses represent only a subset of the study group (n = 31 for
TCDD-exposed participants and n = 71 for TCDD-unexposed participants).
receptors, especially for interleukin-2 (Svedersky et al., 1982; Sztein et al., 1986; Zatz et al., 1984).
As mentioned above, the most consistent findings of laboratory studies of experimental animals exposed to TCDD were thymic atrophy with cortical thymic depletion (Clark et al., 1981; Vos et al., 1974, 1980). More recent in vitro studies have shown that both mouse (Greenlee et al., 1985) and human (Cook et al., 1987) thymic epithelial cell cultures treated with TCDD showed diminished T-cell maturation activity. Our findings of a lower mean Thya-1 serum level in TCDD-exposed persons and a trend of decreasing Thya-1 with increasing number of years of residence in the TCDD-contaminated residential area, along with the previously observed differences in in vivo and in vitro immune tests in this same cohort (Hoffman et al., 1986), suggest that early mild insult to the thymus and/or lymphoid tissues may have occurred.
Analogous, but larger, decreases in serum Thya-1 levels have been observed in pediatric immunodeficiency diseases where thymic function is abnormal (Wara et al., 1982) and following irradiation immuno-suppression accompanied by lymphoid involution in mice (P. H. Naylor, personal communication, 1988). The clinical significance of the diminution in Thya-1 levels among this group of TCDD-exposed persons is somewhat difficult to interpret because normative data representative of thymic hormone levels in the general population are not available for comparison; our unexposed group (i.e., a sample of presumably normal, healthy persons) represents the first population-based group to be evaluated using this assay. The lack of association of Thya-1 levels with other immune system parameters within the TCDD-exposed group, however, provides no evidence for concomitant effects on other aspects of immune function mediated through TCDD-induced lower Thya-1 levels. Similarly, the absence of an increased prevalence of clinically diagnosed immune suppression and/or prolonged or repeated infections in these TCDD-exposed persons make the biologic significance of our findings unclear, as does the unexplained reversion to normal DTH responses in some persons from this cohort who had initially been anergic. Overall, the apparently TCDD-induced differences detected in this and previous studies may fall within the physiologic reserve capacity of the human immune system. Nonetheless, the long-term implications, if any, of diminished levels of thymic hormone and the other observed immune system differences in these TCDD-exposed persons require further elucidation. Important questions remain, such as delineating the point at which this immunologic reserve may be exhausted or whether effects on the developing immune systems of younger persons who are exposed to TCDD may be more pronounced, similar to the effects seen in TCDD-dosed experimental animals (Vos and Luster, 1989).
The possibility of diminished precision of statistical estimates or the introduction of biases due to misclassification of exposure status is also a concern. Results from a study of persons in Missouri for whom TCDD concentrations were measured in adipose biopsy specimens (Patterson et al., 1986) showed some misclassification of presumably exposed persons who were selected by using ecological models of exposure. However, the validity and accuracy of these models in properly identifying exposure groups were supported by the overall results of the adipose-TCDD study, which showed statistically significant mean differences in adipose-TCDD levels between "exposed" and "unexposed" groups selected a priori on the basis of ecological criteria that were identical to those used in the current study. In any case, misclassification errors usually tend to mitigate against detecting small differences between exposure groups by inappropriately including truly unexposed persons in the "exposed" group, and vice versa. Thus, even if misclassification errors (or other factors, such as the relatively small sample size) have lowered the statistical power of this epidemiologic study, or diminished the precision of the estimates, the true magnitude of any excess risks of effects due to TCDD exposure may actually be larger.
Additional studies might evaluate biological mechanisms by which effects of diminished Thya-1 levels may become manifest, such as through incomplete differentiation of T-lymphocytes in the peripheral circulation and/or alterations in the production and expression of related lymphokines. Ideally, simultaneous measurement of Thya-1 and TCDD levels in serum, along with the other in vitro and in vivo measurements of immune function, would provide the best estimates of the effects of exposure and permit evaluation of a dose-response relationship. It would be especially interesting to follow persons exposed to TCDD as children, since they would potentially be at higher risk of thymic effects resulting in long-term immune abnormalities. Finally, appropriate animal models should also be developed to more fully evaluate TCDD-induced effects on thymic function and cell-mediated immune responses.
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Received March 18, 1989
Accepted July 5, 1989
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