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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 8, Num. 1, 2004, pp. 63-65

Journal of Applied Sciences & Environmental Management, Vol. 8, No. 1, June, 2004, pp. 63-65

Evaluation of Hybridoma B9 cell line handling of Cadmium in the presence of Interleukin-6

ORUPABO, I

Dept. of Chemical Pathology, University of Port Harcourt, Uniport P. O. Box 449, Choba, Port Harcourt, Nigeria
*Corresponding author E-mail:

Code Number: ja04013

ABSTRACT:

Induction of metallothionein synthesis in the presence or absence of Interleukin –6 (IL-6) was one of the responses examined to identify the the mechanism by which IL-6 (at high concentration) was able to reverse cadmium inhibition of B9 cell line proliferation. Cellular metallothionein was assayed by Cd-haem method.  The results revealed a Cd-induced increase in cellular metallothionein synthesis.  IL-6 alone was not sufficient to induce expression of metallothionein in this cell line. The Kinetics of B9 cells handling of cadmium (using radio active  109Cd  as tracer) was another response examined. Cells were pre-loaded with 109Cd then transferred to 109Cd-free medium containing IL-6.   The results shows the efflux of Cd from IL-6 stimulated B9 cells which suggest that there is no functional linkage  between IL-6 stimulation and cellular handling of Cd.  The  efflux of cadmium out of the cells is however an  important  observation in that many function assays used in immunotoxicology  are carried out in-vitro after cellular exposure to the chemical in-vivo.  In the interpretation of such data, the ability of the cells to recover as a result of cellular efflux of the chemical should always be considered. @JASEM

Interleukin-6 (IL-6) is a multifunctional cytokine involved in the regulation of immune and inflammatory responses.   The functions of IL-6 include induction of haematopoietic cell proliferation this function can be mimicked in-vitro using hybridoma B9cell line (Lansdorp et al., 1986).  A number of studies have reported the involvement of cytokines in metal-mediated immunomodulation.   In one of such studies, it was demonstrated that mercury-induced increase in IgE and IgG2 in mouse was abrogated with specific antibody to interleukin – 4 (IL-4) (Ochel et al., 1991). The observation suggests that the mercury-induced increase in IgE and Ig G1, in vivo, was mediated by an increase in IL-4 production.  In vitro, Cadmium, lead, copper and zinc (each at 10 or 100 µM) have been shown to inhibit antigen-induced interleukin-2 production  by D11.10 cell line (Smith and Lawrence, 1998).   The report by Scuderi, (1990) showed inhibitory effect of copper on interleukin-6 secretion by human monocytes.  Kowolenko and his colleagues (1988) have demonstrated that lead acetate (50 or 100 µM) inhibited, the response of macrophage to colony stimulating factor-1 (CSF-1). Orupabo and associates (1992) have also reported the inhibitory effect of cadmium on B9 cell proliferation in response to interleukin-6. In the same study, high concentration of IL-6 reversed the inhibitory effect of cadmium. This study examines hybridoma (B9) cell line handling of Cadmium in the presence of Intreleukin-6 to identify how IL-6  (at high concentration) was able to reverse cadmium inhibition of B9 cell proliferation.

MATERIAL AND METHODS

The IL-6 dependent B9 hybridoma cell line was propagated as established by Dr. L. A. Aarden  (Lansdorp et al., 1986),   Cell cultures were incubated in a humidified incubator at 5%  CO2 and  37oC.  B9 cells (5 x 105/well) were treated in 24 well culture plates with varying concentrations of IL-6 (0 – 1ng/ml) with or without CdC12 (`10µM) in RPMI basal medium.   Cell growth was allowed to proceed for 72 hours.  At the end of the incubation period, aliquot (100m1) of the cell suspension were transferred to a 96 well plate and cell proliferation measured by a bio-assay procedure using tetrazolium bromide (Mosmann, 1983). Metallothionein assay is based  on the procedure  as described by Easton and Toal (1982). 

Measurement of 109Cd Efflux: B9 cells were suspended at 5 x 105/ml in basal medium containing CdC12 (10 µM) and 1 µCi/ml 109Cd in 250 ml culture flasks.   After two-hour incubation period, the cells were washed three times in 10ml aliquot of normal saline before re-suspension in Cd-free medium containing IL-6 (100 pg/ml).  At specified time interval, cells were harvested and efflux was determined. Cells were wash with two 3ml aliquots of normal saline then lysed with one millilitre of 1% sodium dodecyl sulphate (SDS) and then transferred to a scintillation vial for gamma counting. 109Cd content at the indicated time was expressed relative to isotope present in cultures, which received the wash protocol without any intervening efflux interval. In this way, a time course of 109Cd efflux was constructed by preloading sets of cultures with radioisotopes, subjecting individual cultures to defined efflux intervals, and measuring the corresponding fractional retention of tracer ion.

RESULTS

Metallothionein level was measured in B9 cells after 72 hours in culture stimulated with varying concentrations of IL-6 in the presence or absence of CdC12 (10 µM). IL-6 alone did not enhance  metallothionein synthesis in this cell line. Cadmium induced   metallothionein synthesis in the cell line at the same time inhibit cell proliferation. Cell proliferation was measured to correct for cell number (Fig. 1). The relationship between IL-6 stimulation and the kinetics of Cd efflux from B9 cells was investigated. Cells were pre-loaded with 109Cd under standard growth conditions followed by transfer to Cd-free medium containing IL-6.   The results are presented as changes relative to isotopes present in the cell culture, which received the wash protocol without any intervening efflux interval.   The result shows that by 24 hours, about 80% of the cellular Cd had diffused out of the cells (Fig. 2).  This suggests that IL-6 stimulation does not control cellular handling of cadmium in this cell line

DISCUSSION

IL-6 alone did not induce metallothionein synthesis in this study. The observation in this study, is supported by the fact that the metallothionein mRNA analysis conducted in the report by Schroeder and Cousins, (1990) also identified zinc and glucocorticoid dependency for IL-6 stimulation of metallothionein expression. This indicate that IL-6 signal alone is not sufficient for induce expression of metallothionein. As expected, Cd induced metallothionein synthesis was demonstrated in this cell line independent of  IL-6 function. 

Efflux of cellular Cd from B9 cells was observed when the cells were transferred from Cd-containing medium with IL-6 to Cd-free medium. This observation is important in that many function assays used in immunotoxicology (Luster et al., 1988) are carried out in-vitro after cellular exposure to the chemical in-vivo.  In the interpretation of such data, the ability of the cells to recover as a result of cellular efflux of the chemical should always be considered. . This study has demonstrated the lack of interaction between IL-6 function and cellular handling of Cadmium.

ACKNOWLEDGEMENT:

The association of commonwealth universities sponsored this work.

REFERENCES

  • Eaton, D L; Toal, B F (1982). Evaluation of Cd-haemoglobin affinity assay for the rapid determination of metallothionein in biological tissues.   Toxicol.  Appl. Parmacol. 66: 134 – 142.
  • Kowolenko, M; Tracey, L; Lawrence, DA (1989).  Lead induced alteration of in-vitro bone marrow response to colony stimulating factor – 1 (CSF – 1).  J. Leucocyte Biol. 45.198.
  • Lansdorp, PM; Aarden, LA;  Calafa,  J;  Zeejimaker,  WP (1986). Growth factor dependent B-cell hybridoma.  Curr. Top. Microbiol. Immunol. 132: 105-113.
  • Luster, ML;   Munson, HE;  Thomas, P;  Holsapple, MP;   Fenters, J;  White, KL;  Lauer, LD;  Dean, JH (1988).   Development of a testing battery to assess chemical induced immunotoxicity.  Fund.  Appl. Toxicol. 10: 2-19.
  • Mosmann, T (1983). Rapid colorimetric assays for cellular growth and survival:  Application to proliferation and cytotoxicity assays.  J. Immunol Methods 65: 55 – 63.
  • Ochel, M; Vohr, H;  Pfeiffer, C; Gleichmann, E  (1991).  IL-4 is required for the 1gG1  autoantibody formation in mice treated with mercuric chloride. J. Immunol 146: 3006 – 3011.
  • Orupabo, I; Hay, AWM; Evans, SW (1992). Effect of metal cations on interleukin-6 mitogenic stimulation of murine hybridoma cells. Immunopharmacol.  Immunotoxicol.  4: 23 – 28.
  • Schroeder, JJ; Cousins, RJ (1990).  Interleukin-6 regulates metallothionein genes expression and zinc metabolism in hepatocyte monolayer cultures.  Proc. Natl. Acad. Sci. 87: 3137 – 3141.
  • Smith, KL; Lawrence, DA;  (1988).  Immunomodulation of in-vitro antigen presentation by cations. Toxicol. Appl. Pharmacol. 96: 476-484.

Copyright 2004 - Journal of Applied Sciences & Environmental Management


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