Circulating Soluble CD27 and CD30 in Workers Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

Recent prospective studies have shown a possible association between blood levels of selected cytokines and small cell-signaling protein molecules and future risk of non-Hodgkin lymphoma (NHL; refs. 1–6). Most notably soluble(s) CD30 has been found to be associated to lymphoma in three prospective studies (1, 3, 4). In addition sCD27 has been associated with lymphoma in the largest prospective studies to date (1, 3). Interleukin 1 receptor antagonist (IL1RA) has been linked to lymphoma risk in genetic studies (7) and in experimental and case–control studies (8, 9). As the immune system plays a pivotal role in lymphomagenesis, it is hypothesized that risk factors of lymphoma such as environmental and occupational exposures operate through deregulation of the immune system, which may be reflected by perturbations in these prediagnostic markers.

Exposure to phenoxy herbicides, which are often contaminated with dioxins such as 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD), has been linked to several cancers among which NHL (10–13). However, few epidemiologic studies have evaluated the possible effects of TCDD on markers of the immune system among which cytokines and chemokines. These studies have provided some evidence for a downregulation of the immune system but evidence is still limited (14–17). To date, no study has examined effects of TCDD exposure on blood soluble immune markers.

In our previous study (17), we showed that plasma levels of a large panel of cytokines, chemokines, and growth factors among workers occupationally exposed to TCDD tended to show a decrease with increasing TCDD levels. Here, we extend these analyses to the evaluation of TCDD exposure on blood levels of IL1RA and soluble markers of sCD27 and sCD30, two members of the tumor necrosis factor (TNF) receptor superfamily that play an important role in regulating cellular activity in subsets of T, B, and natural killer cells. Study subjects comprised a subset of a retrospective cohort of Dutch workers from two chlorophenoxy herbicide producing factories, part of the IARC multinational study of workers exposed to chlorophenoxy herbicides, chlorophenols, and dioxins during their working life (10, 18, 19).

A detailed description of the cohort study design and exposure assessment can be found elsewhere (18, 19). Current analyses utilized a subset of workers from one factory (labeled “A” in previous publications) who were exposed to TCDD as a byproduct of production of 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenol during 1953 to 1969, and/or during an occupational accident in 1963 (17). Subjects were selected for blood collection based on stratified sampling of (assumed) high-exposed and low-exposed workers. A-priori exposure status was based on a detailed occupational history including periods of employment in different departments and positions held and a detailed industrial hygiene evaluation of jobs and departments (17). Fifty high-exposed workers who had been exposed due to the industrial accident (both factory workers and contract workers involved in the clean-up after the accident) or working in departments of main production and 43 low-exposed workers employed in non-production departments who were matched by factory, sex, age, and current residence were selected for blood collection. All study subjects were male. Written informed consent was obtained from each study subject after the study was explained. Participants were asked to complete a self-administered questionnaire, which included questions on occupational and medical history, lifestyle factors and anthropometric characteristics (17, 19).

Plasma level of sCD27, sCD30, and IL1RA was measured by ELISA (Bender Medsystems: BMS286INST, BMS240, and BMS2080 kits respectively). A subset of samples (n = 68; 34 high-exposed and 34 low-exposed) was analyzed in duplicate. The median coefficient of variation was 7.01%, 4.05%, and 7.14% for sCD30, sCD27 and IL1RA respectively. Intraclass of correlation coefficients (ICC) was 0.95, 0.99, and 0.99 for sCD30, sCD27, and IL1RA, respectively. IL1RA measurement was below the limit of detection for one subject and this value was imputed based on min/√2.

Heparin plasma samples were analyzed for TCDD, at the Centers for Diseases Control and Prevention (CDC) using high-resolution gas chromatography/isotope-dilution high-resolution mass spectrometry. As we measured current levels of TCDD (TCDD_current) approximately 35 years since last exposure (lag) and TCDD is highly persistent with a long half-life in blood and human tissues, a one-compartment first-order kinetic model with a TCDD half-life (t_1/2) of 7.1 years was used to estimate TCDD blood levels at the time of last exposure (TCDD_max; refs. 17, 19). Current TCDD levels and estimated maximum TCDD levels were subsequently used to investigate exposure-response relations between TCDD levels and sCD27, sCD30, and IL1RA. Individual TCDD levels (high-exposed, n = 15; low-exposed, n = 20), which were below the limit of detection, were imputed using a maximum likelihood estimation method as previously described (19).

The two-sample t test was used to test for statistical significance of differences in the blood marker concentrations between high- and low-exposed subjects. Linear regression models adjusted for potential confounders, i.e., body mass index, alcohol intake, smoking status, medication, and chronic and acute medical conditions were used to explore exposure–response relations between sCD27, sCD30 and ILRA and TCDD_current or TCDD_max. Moreover, exposure–response relations were also investigated using subjects free of chronic disease at the time of blood draw as a sensitivity analysis as previous analyses showed that subjects with chronic disease unduly influenced the analyses. All P values were two-sided, with P < 0.05 considered statistically significant.

After excluding two subjects with missing TCDD results and 6 subjects with a previous (non-skin) cancer diagnosis, a total of 85 subjects were available for analysis. High- (n = 47) and low-exposed workers (n = 38) had identical distributions of covariate variables (Table 1) except a significant higher alcohol consumption in low-exposed workers compared with high-exposed workers (P = 0.05). Geometric mean of TCDD_current and TCDD_max were significantly higher in high-exposed workers (TCDD_current: 3.25 ± 7.43 ppt; TCDD_max: 79.82 ± 33.28 ppt) compared with low-exposed workers (TCDD_current: 1.07 ± 6.42 ppt; TCDD_max: 7.53 ± 32.14 ppt). Blood concentration of all markers did not significantly differ between the two exposure groups although marker levels tended to be lower in high-exposed workers as compared to low-exposed workers (Table 2). We found no significant association between blood levels of sCD27, sCD30, and IL1RA and TCDD_current (Table 2) or TCDD_max (data not shown) in the confounder adjusted linear regression analyses; although as previous indicated a tendency of lower marker levels were found. Sensitivity analyses based on subjects without chronic disease (n = 41) showed significant association for IL1RA [Estimate = −0.11, 95% confidence interval (CI) = −0.183–0.038; P = 0.004, adjusted for age, BMI, and alcohol). However, due to small sample size, this finding should be interpreted with caution.

Both sCD27 and sCD30, members of the TNF receptor superfamily, are considered markers of lymphocyte activation (20, 21). Studies have shown that levels of sCD27 and sCD30 decreased following chemotherapy, which induces an immunosuppressive status (22–24). The observed general reduction in blood levels of measured markers with increasing TCDD levels is consistent with the earlier observation among the same study subjects that indicated a general decrease in most cytokine-, chemokine-, and growth factors levels with increasing TCDD exposure levels (17) and provides more evidence that TCDD exposure could lead to a general suppression of the immune system. Therefore, given that immunosuppression is an established risk factor for NHL (25) and given the suggested (animal, experimental, and human) evidence for an association between TCDD exposure and immune suppression (26, 27), these results support the biologic plausibility that TCDD could be involved in the development of lymphomas and might explain the observed increased NHL risk in this cohort of workers exposed to chlorophenoxy herbicides, chlorophenols, and contaminants (19).

We found a significant negative association between IL1RA and TCDD level among workers without chronic disease. There were no significant differences between workers with and without chronic diseases in TCDD exposure levels and baseline characteristics except that workers with chronic disease used more anti-inflammatory drugs (22.4% compared with 1.2%). The higher use of inflammatory drugs among the subjects with chronic diseases may have masked the observed IL1RA-TCDD association that was observed among the subjects without a chronic disease. IL1RA has been shown to play a crucial role in the prevention of various inflammatory diseases and counteract inflammatory effects of IL1 family members (28). Moreover, chronic inflammation has been linked to several cancers including NHL, gastric cancer, hepatocellular carcinoma, and adenocarcinoma of the large intestine (29). A recent genetic study found IL1RN to be associated with NHL risk (7). IL1RN is known to alter IL1β expression levels (30), leading to a modulation of a variety of IL1–related immune and inflammatory responses. In addition, a case–control study showed that pretreatment serum level of IL1RA was associated to increased risk of diffuse large B-cell lymphoma and follicular lymphoma (9). However, in a recent nested case-control study examining IL1RA in prediagnosed blood samples, no association was found between IL1RA and NHL (6). However, taken together, it would suggest that inflammatory pathways may be another relevant mechanism for the association between TCDD exposure and NHL development.

Our study is the first investigation that examined the effect of TCDD exposure on blood markers of sCD27, sCD30, and IL1RA in human, markers that have been linked to lymphoma risk (1–4, 7–9). Together with our previous study on immune markers the results suggest a decrease in several components of the immune system in relation to TCDD exposure.

No potential conflicts of interest were disclosed.

Conception and design: H.B. Bueno-de-Mesquita, R. Vermeulen

Development of methodology: L. Portengen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.B. Bueno-de-Mesquita, D. Heederik, R. Vermeulen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Saberi Hosnijeh, L. Portengen, D. Heederik, R. Vermeulen

Writing, review, and/or revision of the manuscript: F. Saberi Hosnijeh, L. Portengen, H.B. Bueno-de-Mesquita, D. Heederik, R. Vermeulen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.B. Bueno-de-Mesquita, R. Vermeulen

Study supervision: F. Saberi Hosnijeh, R. Vermeulen

The authors thank Wayman E. Turner for blood plasma analyses of TCDD (Division of Environmental Health Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA), and Nena Burger for immune markers analyses (Institute for Risk Assessment Sciences, Utrecht University, the Netherlands). The authors also thank all workers for participation in the study.