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Immune alterations in lead-exposed workers
Toxicology 109 (1996) 167-172
Immune alterations in lead-exposed workers olkii hdegera,
Nuqen Bagaran
*a ,
Hande Canpmarb, Emin Kansub
“Depurrment of Phortnaceutical Toxicology, Faculty of Pharmacy, University of Hacettepe. Ankara, 06100. Turkey ‘Institute of Oncology, Faculty of Medicine, University of Haceltepe, Ankara, 06100, Turke)
Received 28 October 1995; accepted 9 February 1996
Abstract Peripheral blood lymphocytes, serum immunoglobulins (IgG, IgA and IgM), C3 and C4 complement protein concentrations of 25 male lead-exposed workers from storage-battery plants were examined and compared to 25 healthy male controls. Lead exposure was assessed using blood lead levels measured by atomic absorption spectrophotometry and zinc protoporphyrin (ZPP) levels assayed by hematofluorometry. The absolute number and the percentage of functionally different subsets of peripheral blood mononuclear lymphocytes, i.e. T, T-suppressor, B and natural killer cells, were unchanged. However, T-helper lymphocytes were significantly lower in lead-exposed workers compared to healthy controls (P < 0.05). In addition, lead-exposed workers had a significant reduction in the IgG, IgM and C3, C4 complement levels (P < 0.05). These results suggest that human chronic exposure to lead may be detrimental to the immune system. Keywords:
Lead; Cellular immunity; Humoral immunity; Natural killer cells
1. Introduction In addition to the well-documented and numerous toxic effects of lead on various target organs, a number of studies have shown that acute and chronic exposure to inorganic lead may result in impairment of immune functions in experimental systems. Depression of cellular and humoral immune functions has been recorded, even at relatively low levels of lead which were not associated with overt toxicity (Lawrence, 1981; Descotes, 1988; Koller, 1990). In animals, lead has been suggested to suppress
*Corresponding 0300-483X/96/$1
author. 5.00
delayed-type hypersensitivity and mixed lymphocyte culture reactions (Miiller et al., 1977; Faith et al., 1979) and to inhibit antibody production and response (Koller et al., 1976; Blakley et al., 1980; Trust et al., 1990). Decreased host resistance to bacterial and viral infections, have been reported in rodents (Cook et al., 1975; Exon and Keller, 1979). However, the accumulated data are not consistent and differences can be accounted for by variations in species, strains, dosages, assays, routes and the duration of exposure to different lead salts. In humans, very few and limited data about the influence of lead on the immune system are available, but it was also suggested that immunotoxic abnormalities induced by lead may
I:(> 1996 Elsevier Ireland Ltd. All rights reserved
PII: SO300-483X(96)03333-1
168
ii ihderger
et ul. I Toxicology 109 (I 996) I67- I72
exist as well. Impaired responses to mitogens (Cohen et al., 1989; Borella and Giardino, 1991; Fischbein et al., 1993) alterations in the number of lymphocytes (Fischbein et al., 1993; Coscia et al., 1987) diminished levels of immunoglobulins (Coscia et al., 1987; Evers et al., 1982; Wagnerova et al., 1986), depression of neutrophil functions (Quillard and Lauwerys, 1989; Bergeret et al., 1990; Queiroz et al., 1993) and increased incidence of colds and influenza (Evers et al., 1982; Sachs, 1978) have all been reported in workers with elevated blood levels. However, the effects of lead exposure on the human immune system have not been well-documented and no conclusive epidemiological studies, particularly in workers occupationally exposed to lead, have been published. Therefore, there is still confusion and controversy over the immunotoxic effects of lead. In the present study, in order to assess the immune competence of workers occupationally exposed to lead, several subsets of peripheral blood mononuclear lymphocytes, i.e. T, TCD4+, TCDS+, B and NK cells have been anaIysed, immunoglobulin and complement protein concentrations assayed. Results have been compared to those obtained in healthy unexposed controls. 2. Methods 2.1. Subjects Twenty-five male workers occupationally exposed to lead in storage-battery plants that were located in the city, for at least 6 months (mean exposure period 6 f 5 years; range OS-15 years) were examined. Twenty-five healthy subjects of comparable age, socio-economic lifestyle, and smoking habits, and with no history of lead exposure, were chosen from the University staff as the control group. The age range of the leadexposed workers was 22-55 years (mean, 33 f 8.5 years) and of the non-exposed controls was 22-56 years (mean, 33 f 9 years). A questionnaire designed to yield information on previous medical and occupational history, frequency of infectious diseases, drug usage, smoking and dietary habits was used for each worker and control.
2.2. Blood sampling and analysis A 20-ml peripheral blood sample was taken from each individual. Of this sample, 10 ml were collected in preservative-free heparin and were used for the analysis of peripheral blood mononuclear cells (PBMC); 7 ml were collected in EDTA, for total and differential blood cell counts, blood lead and zinc protoporphyrin (ZPP) analysis; and 3 ml allowed to clot, for the measurement of serum immunoglobulin and complement concentrations. The serum samples for immunoglobulin and complement concentrations were frozen the same day and were kept at -70°C until they were analyzed. 2.3. Peripheral blood mononuclear cells (PBMC) After the separation of mononuclear cells from heparinized peripheral blood on Ficoll-Hypaque density gradient (Boyum, 1976), PBMC were washed in phosphate buffered saline (pH 7.2) (PBS) and cell concentrations were adjusted to 1 x 106/ml. Mononuclear cells were analyzed for cell surface phenotypes by the direct immunofluorescence technique. Fluorescein-isothiocyanate (FITC)-conjugated monoclonal antibodies directed against human CD3+, CD4+, CD8+ and CD20+ cells were obtained from Coulter Immunology. NK cells were determined by indirect immunofluorescence using CD56+ and FITC-conjugated antihuman polyclonal immunoglobulin antisera (Coulter Clone), respectively. The numbers of PBMC were analyzed by flow cytometry (EPICS 541-Coulter Electronics). The same batch of antibodies was used during the study and the gating in flow cytometry was done by the same person. 2.4. Immunoglobulin and complement protein determination
Serum concentrations of IgG, IgA, IgM, and the C3 and C4 components of complement proteins were determined by turbidimetry (Behringwerke). 2.5. Assessment of lead-exposure Blood lead levels were measured by electrothermal atomic absorption spectrophotometry (Hitachi Z-8100) (Tsalev and Zaprianov, 1983)
i.? tindeger et al. I Toxicology
and blood ZPP levels were assayed by hematofluorometry (AVIV-206). 2.6. Statistical analysis Results are expressed as mean f S.D., and the statistical comparison of the results from exposed and non-exposed individuals, was performed using Mann-Whitney U-test. An analysis of linear regression was used to estimate the effects of blood lead levels and the duration of expose on the immune response (Snedecor and Cochran, 1988).
3. Results Lead exposure characterictics in both populations are given in Table 1. In lead-exposed workers blood lead levels ranged from 38-100 pg/dl and the mean blood lead levels were found to be nearly four times higher than in the control group. Blood ZPP levels were also significantly higher, whereas blood haemoglobin levels significantly lower than workers. No other haematological abnormalities and also no evidence of infectious disease were observed in the lead-exposed group. Table 2 summarizes the absolute numbers of peripheral blood lymphocytes and lymphocyte subpopulations in lead-exposed workers and the controls. No significant differences were found either in the total number of circulating lymphocytes or in the number leukocytes. Although Table I The blood lead (PbB), zinc protoporphyrin (ZPP) and haemoglobin (Hb) levels in exposed and control subjects
PbB (pg/dt) ZPP (g/dl) Hb (g/dl)
*Significantly
Pb-Exposed Workers (n = 25)
Controls (n = 25)
74.8 + 17.8* (38-100) 232.3 k 95.6* (69.33443.5) 13.8 * 1.9* (12-16.9)
16.7 f 5.0 (11-30) 27.7 f 10.1 (14.4-62.7) 15.5 f 1.1
different
from controls
(13.9-19.4) (P < 0.005).
109 (I 9%)
167-l
72
Table 2 The measured values of peripheral blood mononuclear cells of lead-exposed workers and controls
Leukocytes Lymphocytes B-Lymphocytes T-Lymphocytes
Pb-Exposed Workers* (n = 25)
Controls* (n = 25)
8320 + 2286.7 (43OG I5 200) 2762.2 + 840.6 (1247-4559) 545.5 k 296.1 (115.2- 1427.6) 1644 f. 615.3
8564 + 1972.9 (580&13000) 3173 + 1521 (1491-7490) 635.9 f 289.7 (253.5- ll273.3) 2044.7 + 1147 (760.4-6066.9) 1140.3 f 68 I .2 (462.2-3 520.3) 977.6 f !il3.5 (348-2381) 1.3 + 0.4 (0.7-2.4) 692.7 + 420.4 (138.2-2010.2)
(160.6-2826.6) T-Helper (Th)
858.8 & 341.2** (386.6-1955.4) T-Suppressor (Ts) 829.1 f 315.9 (274.3- 1733.2) Th/Ts Ratio 1.1 * 0.3 (0.7-2) NK 793.1 f 511.8 (108.6-2116.7)
*The values were given in absolute means of cell counts/ mm3 & standard error. **Significantly different from controls (P < 0.05).
the total number and the percentage of B, T, and TCD8+ lymphocytes were slightly depressed in workers compared to controls, the differences are not significantly important but the absolute number and the percentage of TCD4+ cells was significantly lower (P < 0.05) in lead-exposed workers. Serum immunoglobulin and complement levels in workers were found to be more markedly depressed as presented in Table 3. Significantly lower IgG and IgM levels were observed in workers than in controls (P < 0.05) while a statistically non-significant decrease in serum IgA levels were also seen in the lead-exposed group. The mean levels of C3 and C4 complement proteins were also significantly lower in workers compared to controls (P < 0.005 and P < 0.05, respectively). The correlations between blood lead levels and the altered immunological parameters, i.e. TCD4+, IgM, C4, C3 and IgG were; -0.20 P > 0.05, -0.33 P > 0.05, --0.17 p > 0.05, -0.52 P < 0.05 and -0.65 P < 0.05, respectively. Only a significant negative correla-
ti. iiiiderger
170
et al. IToxicology
Table 3 The serum immunoglobulin and complement concentrations of lead-exposed workers and controls
IgG IgA IgM c3 c4
Pb-Exposed Workers* (n = 25)
Controls* (n = 25)
854.6 f 415.6** (230- I 170) 168.1 f 99.6 (46.2-498) 93.3 k 39.6** (29.4- 162) 45.1 + 18.5*** (20-80.4) 17.8-8.5** (5-43.6)
1202.1 f 393.6 (371-2310) 210.3 f 118.3 (76.7-684) 140.4 * 66.1 (48.7-278) 61 + 17.4 (33.3- 106) 22.1 + 7.8 (11.8-54.2)
*The levels are expressed as mg/dl. **Significantly different from controls (P < 0.05). ***Significantly different from controls (P < 0.005).
between blood lead levels and C3 and IgG concentrations were observed whereas the correlations between blood lead levels and the other altered immunological parameters were low. No correlation between duration of exposure and the immune parameters has been observed. tion
4. Discussion It has been well documented in experimental systems that exposure to lead is associated with suppression of the immune response. Circulating antibody titers to infectious agents from animals exposed to lead were significantly lower than those from control animals (Koller et al., 1976; Blakley et al., 1980; Trust et al., 1990). Chronic exposure to lead produced a significant decrease in antibody synthesis, particularly IgG indicating that memory cells are involved. The reduced antibody synthesis has been suggested to be responsible for the increased mortality from bacterial and viral diseases in animals chronically exposed to lead (Koller, 1973; Koller and Kovacic, 1974; Luster et al., 1978). But the information about the effects of lead in human immunoglobulin levels is still limited and inconsistent. Reigart and Graber (1976) compared the
109 (1996)
167-172
immunological status of 12 low-level lead-exposed children to seven controls, and found no differences with respect immunoglobulin and C3 complement levels. Also no differences in the serum concentrations of IgG, IgA and IgM in individuals occupationally exposed to low levels of lead were observed by Kimber et al. (1986). On the other hand, Evers et al. (1982) reported low serum IgM, IgG, IgA and C3 complement levels in 72 lead-exposed workers. Several other recent studies on humoral immunity showed an IgM reduction and IgA, IgG variations according to lead blood concentrations (Coscia et al., 1987; Wagnerova et al., 1986). In the present study, we have shown that serum IgG and IgM levels were significantly depressed in lead-exposed workers. We have also observed low levels of C3 and C4 complement proteins in these workers. The effects of lead-exposure on the complement protein levels have been examined less often. In two studies (Evers et al., 1982; Reigart and Graber, 1976) where only C3 complement protein concentrations were measured, a significant reduction was found in one study (Evers et al., 1982) whereas no difference was noted in the other study (Reigart and Graber, 1976). In another report where only C4 complement levels were determined, a significant increase was found (Coscia et al., 1987). Our results suggest that lead exposure have immunosuppresive effects both on C3 and C4 complement proteins. In lead-exposed workers, only a significant reduction in the absolute number and the percentage of TCD4+ cells was found. However, no other alterations in leukocytes and peripheral blood lymphocyte subsets were observed in our study group. Available data about the effects of lead exposure on peripheral lymphocytes appeared to be limited. In workers following toxic high-lead level exposures, a marked reduction in the percentage and the absolute numbers of circulating T and TCD4+ lymphocytes were noted by Fischbein et al. (1993), whereas Coscia et al. (1987) have observed significant increases in the numbers of B and TCD8+ lymphocytes in 38 lead-exposed workers. There is also little information available regarding the influence of lead exposure on the
ii. findejer
et al. IToxicology
function of NK cells which have been implicated in host resistance to malignant and infectious diseases. In our lead-exposed workers, no difference in the absolute number of NK cells was found and these findings are consistent with the available limited human data that lead-exposed population exhibited levels of peripheral blood NK cells comparable with control subjects (Fischbein et al., 1993). It is undoubtedly difficult to assess the toxicological relevance of the results of the present study which indicated that industrial exposure to lead resulting in a group mean blood lead concentrations of 74.8 _t 17.8 pg/dl, is associated with a depression of TCD4+ lymphocytes, IgG, IgM and C3, C4 complement levels, but they support the hypothesis that the immune system could be a target for lead toxicity. However, studies on larger lead-exposed groups are needed. Acknowledgements
This study was supported by a grant from Research Foundation of Hacettepe University 94 01 013 004. References Bergeret, A., Pouget, E., Tedone, R., Meygret, T., Cadot, R. and Descotes, J. (1990) Neutrophil functions in leadexposed workers. Hum. Exp. Toxicol. 9, 231. Blakley, B.R., Sisodia, C.S. and Mukkur, T.K. (1980) The effect of methylmercury, tetraethyl lead and sodium arsenite on the humoral immune response in mice. Toxicol. Appl. Pharmacol. 52, 245. Borella. P. and Giardino, A. (1991) Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes. Environ. Res. 55, 165. Boyum, A. (1976) Isolation of lymphocytes, granulocytes and macrophages. &and. J. Immunol. 5, 9. Cohen. N., Modai, D., Golik, A., Weissgarten, J., Peller, S., Katz, A., Averbukh, Z. and Shaked, V. (1989) Increased concanavalin-A induced suppressor cell activity in humans with occupational lead exposure. Environ, Res. 48. I. Cook, J.A., Hoffmann, E.O. and Di Luzio, N.R. (1975) Influence of lead and cadmium on the susceptibility of rats. Proc. Sot. Exp. Biol. Med. 150,741. Coscia. G.C., Discalzi, G. and Ponzetti, C. (1987) Immu-
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nological
aspects
I71
of occupational
lead exposure.
Med.
Lab. 78, 360. Descotes, J. (1988) lmmunotoxicology of Drugs and Chemicals, Elsevier, Amsterdam, p. 297. Evers, U., Stiller-Winkler. R. and Ided, H. (1982) Serum immunoglobulin, complement C3, and salivary IgA levels in lead workers. Environ. Res. 29, 351. Exon, J.H. and Koller, L.D. (1979) Lead-cadmium interaction: effects on viral-induced mortality and tissue residues in mice. Arch. Environ. Health 34, 469. Faith, R.E., Luster, M.I. and Kimmel, C.A. (1979) Effect of chronic developmental lead exposure on cell-mediated immune functions. Clin. Exp. Immunol. 35, 415. Fischbein, A., Tsong, P., Lou, J.C.J., Roboz, J.P., Jiang, J.D. and Bekesi, J.G. (1993) Phenotypic aberrations of CD3’ and CD41 cells and functional impairments of lymphocytes at low-level occupational exposure to lead. Clin. Immunol. Immunopathol. 66, 163. Guillard, Q. and Lauwerys, R. (1989) In vitro and in viva effect of mercury, lead and cadmium on the generation of chemiluminescence by human whole blood. Biochem. Pharmacol. 38, 2819. Kimber, I., Stonard, M.D., Gidlow, D.A. and Niewola, Z. (1986) Influence of chronic low-level exposure to lead on plasma immunoglobulin concentration and cellular immune function in man. Int. Arch. Occup. Environ. Health 57, 117. Keller, L.D. (1973) lmmunosupression produced by lead. cadmium and mercury. Am. J. Vet. Res. 34. 1457. Koller, L.D. (1990) The immunotoxic elfects of iedd in leadexposed laboratory animals. Ann. N.Y. Acad. Sci. 587. 160. Koller, L.D. and Kovacic, S. (1974) Decreased antibody formation in mice exposed to lead. Nature 250, 148. Koller, L.D., Exon, J.H. and Roan, J.G. (1976) Humoral antibody response in mice after single dose exposure to lead or cadmium. Proc. Sot. Exp. Biol. Med. 151, 339 Lawrence, D.A. (1981) In vivo and in vitro effects of lead on humoral and cell-mediated immunity. Infect. Immunol 31. 136. Luster, M.I., Faith. R.E. and Kimmel, C.A. (1978) Depression of humoral immunity in rats following chronic developmental lead exposure. J. Environ. Pathol. Toxicol. I. 397. Miiller, S.. Gillert. K.E., Gross, U.. L’Age-Stehr. J. and Diamantstein, T. (1977) Supression of delayed type hypcrsensitivity of mice by lead. Experientia 33. 667. Queiroz, M.L.S., Almeida, M., Gallho, M.I. and Hoehr. N.F (1993) Defective neutrophil function in workers, occupationally exposed to lead, Pharmacol. Toxicol. 72. 73. Reigart. J.R. and Graber, C.D. (1976) Evaluation of the humoral immune response of children with low level lead exposure. Bull. Environ. Contam. Toxicol. 16. I I!. Sachs. H.K. (1978) Intercurrent infection in lead poisoning. Am. J. Dis. Child. 132, 315. Snedecor. G.W. and Cochran, W.G. (1988) Statistical Methods, Iowa State University Press. Iowa. Trust. K.A., Miller. M.W.. Ringelman. J.K. and Orme. I.M
*
e
0 rl 0
0
% 6
2 i E
17*
u. ihderjer
et al. I Toxicology 109 (I 9961 167- 172
(1990) Effects of ingested lead on antibody protuction in mallards. J. Wild]. Dis. 26, 316. Tsalev, D.L. and Zaprianov, Z.K. (Ed.) (1983) Atomic Absorption Spectrometry in Occupational and Environmental Health Practice, CRC Press, Florida.
Wagnerova, M., Wagner, V., Madlo, Z., Zavazal, V., Wokunova, D.. Kric J. and Mohyla, 0. (1986) Seasonal variations in the level of immunoglobulins and serum proteins of children differing by exposure to air-borne lead. J. Hyg. Epidemiol. Microbial. Immunol. 30, 127.
Immune alterations in lead-exposed workers olkii hdegera,
Nuqen Bagaran
*a ,
Hande Canpmarb, Emin Kansub
“Depurrment of Phortnaceutical Toxicology, Faculty of Pharmacy, University of Hacettepe. Ankara, 06100. Turkey ‘Institute of Oncology, Faculty of Medicine, University of Haceltepe, Ankara, 06100, Turke)
Received 28 October 1995; accepted 9 February 1996
Abstract Peripheral blood lymphocytes, serum immunoglobulins (IgG, IgA and IgM), C3 and C4 complement protein concentrations of 25 male lead-exposed workers from storage-battery plants were examined and compared to 25 healthy male controls. Lead exposure was assessed using blood lead levels measured by atomic absorption spectrophotometry and zinc protoporphyrin (ZPP) levels assayed by hematofluorometry. The absolute number and the percentage of functionally different subsets of peripheral blood mononuclear lymphocytes, i.e. T, T-suppressor, B and natural killer cells, were unchanged. However, T-helper lymphocytes were significantly lower in lead-exposed workers compared to healthy controls (P < 0.05). In addition, lead-exposed workers had a significant reduction in the IgG, IgM and C3, C4 complement levels (P < 0.05). These results suggest that human chronic exposure to lead may be detrimental to the immune system. Keywords:
Lead; Cellular immunity; Humoral immunity; Natural killer cells
1. Introduction In addition to the well-documented and numerous toxic effects of lead on various target organs, a number of studies have shown that acute and chronic exposure to inorganic lead may result in impairment of immune functions in experimental systems. Depression of cellular and humoral immune functions has been recorded, even at relatively low levels of lead which were not associated with overt toxicity (Lawrence, 1981; Descotes, 1988; Koller, 1990). In animals, lead has been suggested to suppress
*Corresponding 0300-483X/96/$1
author. 5.00
delayed-type hypersensitivity and mixed lymphocyte culture reactions (Miiller et al., 1977; Faith et al., 1979) and to inhibit antibody production and response (Koller et al., 1976; Blakley et al., 1980; Trust et al., 1990). Decreased host resistance to bacterial and viral infections, have been reported in rodents (Cook et al., 1975; Exon and Keller, 1979). However, the accumulated data are not consistent and differences can be accounted for by variations in species, strains, dosages, assays, routes and the duration of exposure to different lead salts. In humans, very few and limited data about the influence of lead on the immune system are available, but it was also suggested that immunotoxic abnormalities induced by lead may
I:(> 1996 Elsevier Ireland Ltd. All rights reserved
PII: SO300-483X(96)03333-1
168
ii ihderger
et ul. I Toxicology 109 (I 996) I67- I72
exist as well. Impaired responses to mitogens (Cohen et al., 1989; Borella and Giardino, 1991; Fischbein et al., 1993) alterations in the number of lymphocytes (Fischbein et al., 1993; Coscia et al., 1987) diminished levels of immunoglobulins (Coscia et al., 1987; Evers et al., 1982; Wagnerova et al., 1986), depression of neutrophil functions (Quillard and Lauwerys, 1989; Bergeret et al., 1990; Queiroz et al., 1993) and increased incidence of colds and influenza (Evers et al., 1982; Sachs, 1978) have all been reported in workers with elevated blood levels. However, the effects of lead exposure on the human immune system have not been well-documented and no conclusive epidemiological studies, particularly in workers occupationally exposed to lead, have been published. Therefore, there is still confusion and controversy over the immunotoxic effects of lead. In the present study, in order to assess the immune competence of workers occupationally exposed to lead, several subsets of peripheral blood mononuclear lymphocytes, i.e. T, TCD4+, TCDS+, B and NK cells have been anaIysed, immunoglobulin and complement protein concentrations assayed. Results have been compared to those obtained in healthy unexposed controls. 2. Methods 2.1. Subjects Twenty-five male workers occupationally exposed to lead in storage-battery plants that were located in the city, for at least 6 months (mean exposure period 6 f 5 years; range OS-15 years) were examined. Twenty-five healthy subjects of comparable age, socio-economic lifestyle, and smoking habits, and with no history of lead exposure, were chosen from the University staff as the control group. The age range of the leadexposed workers was 22-55 years (mean, 33 f 8.5 years) and of the non-exposed controls was 22-56 years (mean, 33 f 9 years). A questionnaire designed to yield information on previous medical and occupational history, frequency of infectious diseases, drug usage, smoking and dietary habits was used for each worker and control.
2.2. Blood sampling and analysis A 20-ml peripheral blood sample was taken from each individual. Of this sample, 10 ml were collected in preservative-free heparin and were used for the analysis of peripheral blood mononuclear cells (PBMC); 7 ml were collected in EDTA, for total and differential blood cell counts, blood lead and zinc protoporphyrin (ZPP) analysis; and 3 ml allowed to clot, for the measurement of serum immunoglobulin and complement concentrations. The serum samples for immunoglobulin and complement concentrations were frozen the same day and were kept at -70°C until they were analyzed. 2.3. Peripheral blood mononuclear cells (PBMC) After the separation of mononuclear cells from heparinized peripheral blood on Ficoll-Hypaque density gradient (Boyum, 1976), PBMC were washed in phosphate buffered saline (pH 7.2) (PBS) and cell concentrations were adjusted to 1 x 106/ml. Mononuclear cells were analyzed for cell surface phenotypes by the direct immunofluorescence technique. Fluorescein-isothiocyanate (FITC)-conjugated monoclonal antibodies directed against human CD3+, CD4+, CD8+ and CD20+ cells were obtained from Coulter Immunology. NK cells were determined by indirect immunofluorescence using CD56+ and FITC-conjugated antihuman polyclonal immunoglobulin antisera (Coulter Clone), respectively. The numbers of PBMC were analyzed by flow cytometry (EPICS 541-Coulter Electronics). The same batch of antibodies was used during the study and the gating in flow cytometry was done by the same person. 2.4. Immunoglobulin and complement protein determination
Serum concentrations of IgG, IgA, IgM, and the C3 and C4 components of complement proteins were determined by turbidimetry (Behringwerke). 2.5. Assessment of lead-exposure Blood lead levels were measured by electrothermal atomic absorption spectrophotometry (Hitachi Z-8100) (Tsalev and Zaprianov, 1983)
i.? tindeger et al. I Toxicology
and blood ZPP levels were assayed by hematofluorometry (AVIV-206). 2.6. Statistical analysis Results are expressed as mean f S.D., and the statistical comparison of the results from exposed and non-exposed individuals, was performed using Mann-Whitney U-test. An analysis of linear regression was used to estimate the effects of blood lead levels and the duration of expose on the immune response (Snedecor and Cochran, 1988).
3. Results Lead exposure characterictics in both populations are given in Table 1. In lead-exposed workers blood lead levels ranged from 38-100 pg/dl and the mean blood lead levels were found to be nearly four times higher than in the control group. Blood ZPP levels were also significantly higher, whereas blood haemoglobin levels significantly lower than workers. No other haematological abnormalities and also no evidence of infectious disease were observed in the lead-exposed group. Table 2 summarizes the absolute numbers of peripheral blood lymphocytes and lymphocyte subpopulations in lead-exposed workers and the controls. No significant differences were found either in the total number of circulating lymphocytes or in the number leukocytes. Although Table I The blood lead (PbB), zinc protoporphyrin (ZPP) and haemoglobin (Hb) levels in exposed and control subjects
PbB (pg/dt) ZPP (g/dl) Hb (g/dl)
*Significantly
Pb-Exposed Workers (n = 25)
Controls (n = 25)
74.8 + 17.8* (38-100) 232.3 k 95.6* (69.33443.5) 13.8 * 1.9* (12-16.9)
16.7 f 5.0 (11-30) 27.7 f 10.1 (14.4-62.7) 15.5 f 1.1
different
from controls
(13.9-19.4) (P < 0.005).
109 (I 9%)
167-l
72
Table 2 The measured values of peripheral blood mononuclear cells of lead-exposed workers and controls
Leukocytes Lymphocytes B-Lymphocytes T-Lymphocytes
Pb-Exposed Workers* (n = 25)
Controls* (n = 25)
8320 + 2286.7 (43OG I5 200) 2762.2 + 840.6 (1247-4559) 545.5 k 296.1 (115.2- 1427.6) 1644 f. 615.3
8564 + 1972.9 (580&13000) 3173 + 1521 (1491-7490) 635.9 f 289.7 (253.5- ll273.3) 2044.7 + 1147 (760.4-6066.9) 1140.3 f 68 I .2 (462.2-3 520.3) 977.6 f !il3.5 (348-2381) 1.3 + 0.4 (0.7-2.4) 692.7 + 420.4 (138.2-2010.2)
(160.6-2826.6) T-Helper (Th)
858.8 & 341.2** (386.6-1955.4) T-Suppressor (Ts) 829.1 f 315.9 (274.3- 1733.2) Th/Ts Ratio 1.1 * 0.3 (0.7-2) NK 793.1 f 511.8 (108.6-2116.7)
*The values were given in absolute means of cell counts/ mm3 & standard error. **Significantly different from controls (P < 0.05).
the total number and the percentage of B, T, and TCD8+ lymphocytes were slightly depressed in workers compared to controls, the differences are not significantly important but the absolute number and the percentage of TCD4+ cells was significantly lower (P < 0.05) in lead-exposed workers. Serum immunoglobulin and complement levels in workers were found to be more markedly depressed as presented in Table 3. Significantly lower IgG and IgM levels were observed in workers than in controls (P < 0.05) while a statistically non-significant decrease in serum IgA levels were also seen in the lead-exposed group. The mean levels of C3 and C4 complement proteins were also significantly lower in workers compared to controls (P < 0.005 and P < 0.05, respectively). The correlations between blood lead levels and the altered immunological parameters, i.e. TCD4+, IgM, C4, C3 and IgG were; -0.20 P > 0.05, -0.33 P > 0.05, --0.17 p > 0.05, -0.52 P < 0.05 and -0.65 P < 0.05, respectively. Only a significant negative correla-
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170
et al. IToxicology
Table 3 The serum immunoglobulin and complement concentrations of lead-exposed workers and controls
IgG IgA IgM c3 c4
Pb-Exposed Workers* (n = 25)
Controls* (n = 25)
854.6 f 415.6** (230- I 170) 168.1 f 99.6 (46.2-498) 93.3 k 39.6** (29.4- 162) 45.1 + 18.5*** (20-80.4) 17.8-8.5** (5-43.6)
1202.1 f 393.6 (371-2310) 210.3 f 118.3 (76.7-684) 140.4 * 66.1 (48.7-278) 61 + 17.4 (33.3- 106) 22.1 + 7.8 (11.8-54.2)
*The levels are expressed as mg/dl. **Significantly different from controls (P < 0.05). ***Significantly different from controls (P < 0.005).
between blood lead levels and C3 and IgG concentrations were observed whereas the correlations between blood lead levels and the other altered immunological parameters were low. No correlation between duration of exposure and the immune parameters has been observed. tion
4. Discussion It has been well documented in experimental systems that exposure to lead is associated with suppression of the immune response. Circulating antibody titers to infectious agents from animals exposed to lead were significantly lower than those from control animals (Koller et al., 1976; Blakley et al., 1980; Trust et al., 1990). Chronic exposure to lead produced a significant decrease in antibody synthesis, particularly IgG indicating that memory cells are involved. The reduced antibody synthesis has been suggested to be responsible for the increased mortality from bacterial and viral diseases in animals chronically exposed to lead (Koller, 1973; Koller and Kovacic, 1974; Luster et al., 1978). But the information about the effects of lead in human immunoglobulin levels is still limited and inconsistent. Reigart and Graber (1976) compared the
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immunological status of 12 low-level lead-exposed children to seven controls, and found no differences with respect immunoglobulin and C3 complement levels. Also no differences in the serum concentrations of IgG, IgA and IgM in individuals occupationally exposed to low levels of lead were observed by Kimber et al. (1986). On the other hand, Evers et al. (1982) reported low serum IgM, IgG, IgA and C3 complement levels in 72 lead-exposed workers. Several other recent studies on humoral immunity showed an IgM reduction and IgA, IgG variations according to lead blood concentrations (Coscia et al., 1987; Wagnerova et al., 1986). In the present study, we have shown that serum IgG and IgM levels were significantly depressed in lead-exposed workers. We have also observed low levels of C3 and C4 complement proteins in these workers. The effects of lead-exposure on the complement protein levels have been examined less often. In two studies (Evers et al., 1982; Reigart and Graber, 1976) where only C3 complement protein concentrations were measured, a significant reduction was found in one study (Evers et al., 1982) whereas no difference was noted in the other study (Reigart and Graber, 1976). In another report where only C4 complement levels were determined, a significant increase was found (Coscia et al., 1987). Our results suggest that lead exposure have immunosuppresive effects both on C3 and C4 complement proteins. In lead-exposed workers, only a significant reduction in the absolute number and the percentage of TCD4+ cells was found. However, no other alterations in leukocytes and peripheral blood lymphocyte subsets were observed in our study group. Available data about the effects of lead exposure on peripheral lymphocytes appeared to be limited. In workers following toxic high-lead level exposures, a marked reduction in the percentage and the absolute numbers of circulating T and TCD4+ lymphocytes were noted by Fischbein et al. (1993), whereas Coscia et al. (1987) have observed significant increases in the numbers of B and TCD8+ lymphocytes in 38 lead-exposed workers. There is also little information available regarding the influence of lead exposure on the
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function of NK cells which have been implicated in host resistance to malignant and infectious diseases. In our lead-exposed workers, no difference in the absolute number of NK cells was found and these findings are consistent with the available limited human data that lead-exposed population exhibited levels of peripheral blood NK cells comparable with control subjects (Fischbein et al., 1993). It is undoubtedly difficult to assess the toxicological relevance of the results of the present study which indicated that industrial exposure to lead resulting in a group mean blood lead concentrations of 74.8 _t 17.8 pg/dl, is associated with a depression of TCD4+ lymphocytes, IgG, IgM and C3, C4 complement levels, but they support the hypothesis that the immune system could be a target for lead toxicity. However, studies on larger lead-exposed groups are needed. Acknowledgements
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