T Lymphocytes in Mercury-Exposed Workers

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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 19(4), 499-5 10 (1997). T LYMPHOCYTES IN MERCURY- EXPOSED WORKERS. Mary L. S. ...
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 19(4), 499-5 10 (1997)

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T LYMPHOCYTES IN MERCURY- EXPOSED WORKERS

Mary L. S. Queiroz* and Denise C. M . Dantas Department of Pharmacology and Hemocentre, Faculty of Medical Sciences, State University of Campinas, UNICAMP, P.O. Box 61 11- CEP13083- 970, Campinas- SP, Brazil.

ABSTRACT In this work we have investigated the changes in T- helper and T- suppressor cells and T- cell proliferative response to phytohemagglutinin ( PHA ) in mercuryexposed workers. The study group consisted of 33 workers from a mercury- producing plant with a mean age of 29 years and a mean exposure period of 19 months. At the time of testing, and for the three previous months, the exposed population had urinary mercury levels below the currently accepted limit of 50 ug / g creatinine. A reverse CD4' / CD8' ratio was observed in the mercury- exposed individuals which was characterized by a reduction in the number of CD4+ lymphocytes. No changes were observed in the proliferative response of lymphocytes from exposed individuals to PHA. Similarly, no proliferative response was observed when lymphocytes from normal individuals were cultivated in the presence of serum from the exposed workers. We found no correlations between lymphocytes changes and urinary mercury concentrations, time of exposure or the age of the workers.

INTRODUCTION Exposure to heavy metals has the potential of disrupting the body's normal

.

immune homeostasis either by acting directly on the cells of the immune system or by

Dantas, D. C. M. is the recipient a M. Sc. grant from FundaGBo de Amparo h Pesquisa do Estado de SBo Paul0 ( FAPESP ) * Corresponding author 499 Copyright 0 1997 by Marcel Dekker, Inc.

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interacting with other cells or organ systems so as to render them immunologically reactive. A direct effect of toxicant exposure on the immune system is quite obvious in that there would likely be changes in lymphocyte subset ratios, altered morphology of

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immune tissue, decrease in total immune cell numbers, or altered immune functions with or without the above changes (1 7).

In man, a well- known immunological alteration induced by exposure to mercury compounds is contact dermatites, but in addition there are reports of Hg- induced immune glomerulonephritis and oral lichen planus (10). In genetically susceptible rats and mice, a systemic autoimmune disease can be experimentaly induced by repeated injections of low doses of HgC12 (12). Mercury- induced autoimmunity appears to involve an imbalance between T- helper and T- suppressor cells (24). T cytotoxid suppressor cells have more cell surface thiols, are more sensitive to permeant thiol blockers than helper T- cells (17). A decline in suppressor cell activity and/or an increase in B cell activity has been suggested to account for the higher incidence of autoimmunity with age (2). Mercury, with its high affinity for sulfhydryl groups, m a y alter cytotoxic / suppressor cells, resulting in immunopathophysiological changes ( 17). Therefore, considering that T cells play a central role in the development of mercury- induced disease, we have designed the present study to find out whether occupational exposure to mercury reflects any changes in T- helper and T- suppressor cells.

SUBJECTS AND METHODS Population The study group consisted of 33 workers from a mercury producing plant. The mean age of the workers was 29 years (range 19-46 years) and the mean exposure period

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to mercury was 19 months (range 3-72 months). In this plant, mercury is separated from diatomaceous earth contamined with mercury in the sulphate form, using a wastecleaning process of chloro- alkali plants that use mercury electrodes to produce chlorine

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and soda. This diatomaceous earth is roasted in shaft furnaces and metallic mercury is obtained by subsequente condensation. Control group of comparable age with no history of mercury exposure were chosen from donors arriving at the University Hospital blood bank. Each worker was examined in a standard fashion by a physician including a complete neurological examination. A complete occupational history was noted and included the occurence in the preceding six months of symptoms possible related to mercury exposure as well as observations on past episodes of micromercurialism, of the incidence of infections and alcohol and drug ingestion. A similar protocol was applied to the control group. At the time of this study, there was no clinical evidence of infection in any of the workers. Urine samples from each worker were collected for determination of mercury. To avoid errors arising from inaccurate collection of 24h urine samples, use was made of spot samples voided during the period of blood sampling. Urinary mercury levels were determined by atomic absorption spectrophotometry ( Varian model AA 175 equipped with a Hg hollow- cathode lamp ) according to the hybrid generator method (1) and the results expressed in terms of the urinary creatinine content of the same sample. Urinary creatinine was determined by Jaff6 method using spectrophotometry (14). Venous blood samples were collected for the evaluation of T cell populations. Sampling was always performed between 8:OO- 9:OO A M , when the subjects had been fasting for at least 12h.

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Quantitation of T- cell populations For the determination of T- cell populations, DAKO (USA) monoclonal antibodies were used in indirect immunofluorescence. Heparinized human blood was

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diluted 1 :1 with PBS and mononuclear cells purified by centrifugation over Ficollhypaque (Pharmacia Chemical). Samples containing 5x lo6 viable cells were incubated 4"C, 60 min. - using lO0ul monoclonal mouse anti-human CD4+, helper (FTTC

conjugated) and monoclonal mouse anti- human CD8+, suppressor The preparation after this incubation, was washed with RPMI medium and prepared to verify the reactivity with rnAb by indirect immunofluorescence. Cell suspension was observed on ZEISSFluorescence JENAMED microscopy, and a minimum of 200 lymphocytes were examined and the percentage of positive fluorescent cells were compared with a preparation of T lymphocytes in individuals non- exposed to mercury.

Measurement of Phytohaemagglutinin-Induced Lymphocytes Blastogenesis Peripheral blood mononuclear cells were adjusted to 1 x lo6 / ml in RPMI 1640 supplemented with 10 % human AB serum and 200 ul aliquots were seeded onto microtest plates (well diameter 6.4 mm, corning). Purified phytohaemagglutiniri (PHA) (Wellcome Diagnostics Ltd) was added to a final concentation of 2.5 ug / ml. Control cultures received 200 ul of RPMI supplemented with 10 % human AB serum. All tests were established in triplicate and cultured in a humidified atmosphere of 5 % CO2 in air for 48 h at 37" C . All cultures were then pulsed with lu Ci of 3H- methylthymidine (specific activity 5.0

Ci / mmol)

(Amersham International) and incubation

continued for a further 24 h. Cultures were then terminated by atomic cell harvesting and 'HTdR incorporation determined by beta- scintillation counting.

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Statistical analysis The results from exposed and non- exposed individuals were compared using the nonparametric Mann-Whitney U-test. Association between variables were evaluated

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based on the Pearson correlation coeficient (3).

RESULTS Urinary mercury concentrations which were used as an indicator of metal exposure, were below the accepted safety threshold level of 50 ug/ g creatinine (32) in all the workers studied. A restropective study demonstrated that these workers presented safe urinary mercury concentrations for at least three months prior to the initiation of this study. The average urinary mercury levels for the workers was 19.8 k 10.3 ug/ g creatinine. These concentrations ranged from 1.4- 46.0 ug Hg I g creatinine and the distribution among the workers was as follow: < 10 ug I g, 4 workers; 10- 20 ug /g, 15 workers; 20- 30 ug I g, 9 workers; 30- 40 ug I g, 3 workers; 40- 50 ug I g, 2 workers. None of the controls had urinary mercury concentrations above 5 ug/ g creatinine, which is considered the safe limit of mercury exposure for non- exposed populations.

No changes in phytohemagglutinin- induced T-cell proliferation were observed,

as compared to controls. In addition, the incubation of normal human lymphocytes with serum from mercury- exposed workers produced no differences in the proliferative response of these cells. On the ther hand, a low CD4+ I CD8+ ratio was found in 15 workers (45.4 % ) Fig. 2. This reversed ratio was produced by a decrease in T- helper lymphocytes compared with controls Fig. 1. T suppressor cell concentrations were not altered in comparison to the control group. We found no correlations between lymphocyte changes and urinary mercury concentrations, length of exposure or the age of the workers.

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TABLE 1

Length of exposure, urinary mercury concentration and age of workers and controls.

EXPOSED

18.5

14.3

19.8

10.3

mercury- exposed

29

8 .o

28

7.6

in=33)

CONTROL

< 5 ug Hg / g ___--____________-------Creatinine

~(n=13)

I

CD4+

CD8+

Figure 1: The p e r c s n b g e of lymphocylss in b e peripheral blood ( C o d + ,CO8*) In workers exposed lo mercury (0) (ni33) and controls (a ) (0-13). p < 0.05 (Mann-Whilney U-\es\). Rmsu\Ls arc prraenlsd a s medbans

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‘1

Figure 2: CD4+ I COB+ (T-helper I T.supprsssor) cell ralio In mercury-exposed workers (0) (n.33) and conbola ( = ) (n-13). p < 0.05 (Mann-Vlhllncy U.lest). Resultr are presenled a s medians.

DISCUSSION T cells play a pivotal role in the development of mercury- induced autoimmunity, as shown by the absence of pathological changes in T- cell deprived animals (35,23,33). In the susceptible BN rat, the hyper IgE and enhanced serum IgG1 concentration imply a T cell dependent B cell policlonal activation (12,15,29,20,19). Data on human immune reactions to inorganic mercury exposure are scarce and sometimes contradictory. We have previously demonstrated that neutrophil functions such as chemotaxis, lytic activity and respiratory burst are significantly reduced in mercury- exposed workers presenting mercury urinary concentrations below the biological limit values of 50 ug Hg / g creatinine (27,28). Renal immunopathology has been observed in many individuals using lightening creams containing mercury (16). In mercury- exposed workers excreting more than 50 ug Hg / g creatinine, increased concentrations of total immunoglobulins (4,6) and anti- DNA antibodies have been reported (6). Some investigations on the levels of IgG, IgA and IgM have also been performed in workers chronically exposed to mercury. Most of these studies reported an

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increase in the levels of these immunoglobulins (4,6,31) even in workers with urinary mercury concentrations below the accepted biological threshold limit value of 50 ug / g creatinine (31). In one of these studies, however, the levels of these immunoglobulins

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were normal (18). The IgE concentration and the titers of autoantibody were also investigated in the latter work and the authors have found no changes in the exposed population. We have recently observed increased IgE serum levels in mercury exposed workers presenting mercury urinary concentrations below the biological limit values. Morever, we have also found a negative correlation between the levels of IgE and the time of exposure to mercury ( Dantas & Queiroz, 1997-submitted to publication j, thus suggesting that the mechanism of autoregulation described in animals exposed to mercury (7,8,9,19,5) may also occur in man. Despite enhanced humoral immunity, mercury- exposed workers presented reduced numbers of B cells (22; Dantas and Queiroz, 1997- submitted to publication ).

In this study, a reversed CD4' / CD8' ratio was observed in mercury- exposed workers presenting mercury absorption parameters within '' safe" levels. The reduced ratio was characterized by a reduction in the number of CD4+ lymphocytes. Of the 33 workers studied, none presented urinary mercury concentrations above the biological limit values of 50 ug Hg / g creatinine, and this limit had not been exceeded in at least the three months prior to the initiation of this study. No changes were observed in the prolifera.tiveresponse of lymphocytes from exposed workers in the presence of PHA. Similarly. no proliferative activity was observed when normal lymphocytes were cu1tivate.d in the presence of serum from these workers. We found no correlations between lymphocyte changes and urinary mercury concentrations, the time of exposure to mercury or the age of the workers. There was another study in the literature that

investigated the changes in T lymphocytes in mercury- exposed workers (21). 'The

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authors have also observed a decrease of the helper / suppressor ratio which was determined by a lower increase of the helper T- cell compartment. In contradiction to our findings, however, the authors observed a positive correlation between the length of

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exposure and the changes in T cells. It seems therefore that the polyclonal activation of

B lymphocytes observed in mercury- exposed workers (6,4,31)might not be due to an increase in the number of T helper lymphocytes. The characteristics of HgC12- induced autoimmunity, in experimental models, indicate preferential activation of the T helper 2 subset of T helper cells; and upregulation of interleukin- 4 has been shown in response to HgC12 in vivo (1 1) and in vitro (30). Moreover, it was suggested that CD8+ T cells might be one of the resistance factors in the resistance of LEW rats to mercury- induced disease (25,26). A defect at the T helper level has been suggested in human diseases such as lepromatous leprosy (1 3). Functional subsets of human T cells were delineated by analysing patterns of lymphokines produced by clones from individuals with leprosy. From these studies, it was found that interleukin- 4 was also produced by CD8+ suppressor clones from immunologically unresponsive individuals with leprosy and that it was necessary for suppression in vitro (34). The authors suggested that both the classic reciprocal relation between antibody formation and cell- mediated immunity and resistance or susceptibility to certain diseases may be explained by T cells subsets differing in patterns of lymphokine production. Experiments are in progress in our laboratory to elucidate the lymphokine profile in mercury- exposed workers.

Acknowledgements- This work was supported by grants from the Fundasgo de Amparo 5 Pesquisa do Estado de Siio Paulo ( FAPESP ) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico ( CNPq ).

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