Letter to the Editor

Janine Ezendam1,2,3, Joseph G. Vos2,3 and Raymond Pieters1

1 Institute for Risk Assessment Sciences, Utrecht University,P.O. Box 80.176, 3508 TD, Utrecht, The Netherlands 2 National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands 3 Faculty of Veterinary Medicine, Department of Pathobiology, Utrecht University, P.O. Box 80.150, 3508 TD, Utrecht, The Netherlands

To the Editor:

We have read the comments of Dr. Nanne Bloksma on our paper, "Hexachlorobenzene-induced immunopathology in Brown Norway rats is partly mediated by T cells" (Ezendam et al., 2004aGo).

We appreciate her comments and would like to respond to them. The study mentioned by Bloksma indeed showed that in genetically athymic WAG/Rij rats and in thymectomized Brown Norway (BN) rats HCB-induced immune effects still occurred. Skin lesions in the athymic rats could not be assessed properly because of preexisting inflammatory skin lesions in these rats, but in the thymectomized BN rats skin lesions appeared later and in one of the ten rats no lesions developed at all. In addition, by using adult thymectomy, lethal irradiation, and bone marrow reconstitution, not all peripheral T cells were depleted. Michielsen and colleagues have shown that in the spleen the number of T cells falls from 14.5% (±1.2%) to 4.2% (±1.2%) (Michielsen et al., 1999Go), meaning that T cell numbers were only decreased to around 29%. Such an amount of T cells is very likely to induce at least some T cell-dependent immune effects. Even lower amounts of T cells have been shown to be able to do so in thymectomized mice (Sado et al., 1980Go). Together with the above-mentioned delay or even complete prevention of HCB-induced skin pathology, we felt that this previous study was not conclusive enough. Therefore, we decided to investigate the role of T cells in HCB-induced immunopathology further by this additional study with cyclosporin A (CsA). The advantage of this approach is that CsA not only reduces peripheral T cell numbers, but also inhibits antigen-induced T cell activation. CsA treatment has successfully prevented mercuric chloride-induced autoimmune disease in BN rats, which is a T cell-dependent disease (Aten et al., 1988Go; Baran et al., 1986Go).

In our study we have shown that CsA treatment delayed the onset of HCB-induced skin lesions. Furthermore lung eosinophilia, the increase in auricular lymph node weight, and humoral responses were prevented completely, but HCB-induced splenomegaly and infiltrations of macrophages in spleen and lung were not affected by CsA. Moreover, in our study macrophage infiltrations in lung and spleen were not affected by CsA treatment (Ezendam et al., 2004aGo), which is in contrast with the observations of Satonaka et al., who have shown that CsA inhibits macrophage infiltrations in rat femoral arteries (Satonaka et al., 2004Go). More importantly, we also show that the HCB-induced increase of TNF-{alpha} production after lipopolysaccharide (LPS) stimulation was not affected by CsA treatment. Together with the presence of macrophage infiltrations in the lungs, this demonstrates that in our model CsA does not influence the function of macrophages. Furthermore, we have shown in this paper that the function of T cells is impaired, because ConA-induced IL-2 production is decreased in CsA-treated rats (Ezendam et al., 2004aGo). Direct effects of CsA on other cells are not excluded, but many of the studies mentioned by Bloksma on the effects of CsA on other cell types have been performed in vitro (Hamalainen et al., 2002Go; Kaye et al., 1992Go), and it is not known if the in vitro concentrations used will be reached in vivo. Notably, other in vitro studies have shown that CsA does not influence other cell types. Hidalgo et al. have shown that CsA does not affect the function of macrophages (Hidalgo et al., 1992Go), and another paper shows that CsA primarily inhibits T cells and not accessory cells, such as dendritic cells and monocytes (Granelli-Piperno et al., 1988Go).

The effects of CsA on calcineurin in relation to vascular inflammation and monocyte chemoattractant protein-1 expression (Satonaka et al., 2004Go) could indeed also explain the prevention of lung eosinophilia and the skin lesions that are characterized by perivascular inflammation, as mentioned by Bloksma. However, cell migration is probably not affected (as macrophage influx was still seen in lung and spleen) and eosinophilia also depends on T cell-derived cytokines, such as IL-5. In other words we favor impairment of T cells by CsA as the cause of decreases in eosinophilia.

In all, we are in the opinion that our data support the conclusion that T cells are responsible for some of the HCB-induced immune effects, but we also stated that T cells do not account for all HCB-induced effects (Ezendam et al., 2004aGo). The importance of other inflammatory cells and mediators is strengthened by a recent paper by us (Ezendam et al., 2004bGo) in which we assessed gene expression profiles of several organs after subchronic HCB exposure. Transcriptome profiles show that after HCB exposure many genes in macrophages, mast cells, and granulocytes were upregulated, just as pro-inflammatory cytokines, chemokines, antioxidants, and acute phase proteins were. Furthermore, recently experiments were performed in which macrophages were depleted with clodronate-liposomes, and these experiments showed that macrophages are indeed important in HCB-induced skin lesions, lung eosinophilia, and humoral responses (unpublished data). This supports our view that HCB-induced immune effects are initiated by pro-inflammatory cells and mediators, including macrophages, and that T cells are important in the acceleration of the immunopathology.

REFERENCES

Aten, J., Bosman, C. B., De Heer, E., Hoedemaeker, P. J., and Weening, J. J. (1988). Cyclosporin A induces long-term unresponsiveness in mercuric chloride-induced autoimmune glomerulonephritis. Clin. Exp. Immunol. 73, 307–311.[ISI][Medline]

Baran, D., Vendeville, B., Vial, M. C., Cosson, C., Bascou, C., Teychenne, P., and Druet, P. (1986). Effect of cyclosporine A on mercury-induced autoimmune glomerulonephritis in the Brown Norway rat. Clin. Nephrol. 25, S175–180.[ISI][Medline]

Ezendam, J., Hassing, I., Bleumink, R., Vos, J. G., and Pieters, R. (2004a). Hexachlorobenzene-induced immunopathology in Brown Norway Rats is partly mediated by T cells. Toxicol. Sci. 78, 88–95.[Abstract/Free Full Text]

Ezendam, J., Staedtler, F., Pennings, J., Vandebriel, R. J., Pieters, R., Harleman, J. H., and Vos, J. G. (2004b). Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats. Environ. Health Perspect. 112, 782–791.[ISI][Medline]

Granelli-Piperno, A., Keane, M., and Steinman, R. M. (1988). Evidence that cyclosporine inhibits cell-mediated immunity primarily at the level of the T lymphocyte rather than the accessory cell. Transplantation 46, 53S–60S.[Medline]

Hamalainen, M., Lahti, A., and Moilanen, E. (2002). Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines. Eur. J. Pharmacol. 448, 239–244.[CrossRef][ISI][Medline]

Hidalgo, H. A., Helmke, R. J., German, V. F., and Mangos, J. A. (1992). The effects of cyclosporine and dexamethasone on an alveolar macrophage cell line (NR8383). Transplantation 53, 620–623.[ISI][Medline]

Kaye, R. E., Fruman, D. A., Bierer, B. E., Albers, M. W., Zydowsky, L. D., Ho, S. I., Jin, Y. J., Castells, M. C., Schreiber, S. L., Walsh, C. T., et al. (1992). Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: Resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12. Proc Natl Acad Sci U.S.A. 89, 8542–8546.[Abstract]

Michielsen, C. C., Bloksma, N., Klatter, F. A., Rozing, J., Vos, J. G., and van Dijk, J. E. (1999). The role of thymus-dependent T cells in hexachlorobenzene-induced inflammatory skin and lung lesions. Toxicol. Appl. Pharmacol. 161, 180–191.[CrossRef][ISI][Medline]

Sado, T., Kamisaku, H., and Aizawa, S. (1980). Nature of T-cells resident in spleens of thymectomized, lethally irradiated, bone marrow-reconstituted mice. Cell. Immunol. 49, 51–63.[ISI][Medline]

Satonaka, H., Suzuki, E., Nishimatsu, H., Oba, S., Takeda, R., Goto, A., Omata, M., Fujita, T., Nagai, R., and Hirata, Y. (2004). Calcineurin promotes the expression of monocyte chemoattractant protein-1 in vascular myocytes and mediates vascular inflammation. Circ. Res. 94, 693–700.[Abstract/Free Full Text]





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