Constitutive Expression of FasL in Thyrocytes

C. Giordano et al. (1) reported evidence that the ligand for Fas antigen (FasL) is constitutively expressed on thyroid follicular cells from both normal and Hashimoto’s thyroiditis (HT) tissue, and that normal thyrocytes express Fas antigen only after induction with interleukin-1b (IL-1b). Giordano et al. conclude that their results suggested a possible mechanism for thyrocyte cytotoxicity in autoimmune thyroiditis. The absence of Fas antigen expression on the surface of normal thyrocytes has been supported by one study (2); however, other studies have found Fas expression by normal thyrocytes both in situ (3) and in primary cultured cells (4, 5). The findings by Giordano et al. raised the possibility of the expression of both Fas antigen and FasL on normal thyrocytes. To clarify this issue, we examined the expression of both FasL and Fas antigen mRNA in primary cultured thyrocytes with the use of reversetranscriptase polymerase chain reaction (RTPCR) (6) and ribonuclease protection (7) techniques. Neither assay, performed on RNA isolated from normal human thyrocytes, demonstrated mRNA for FasL (Fig. 1, A and B). To assure that this result was not unique to this sample, RNA samples from five different normal and Graves’s diseased thyrocytes were screened by ribonuclease protection assay; these also did not show mRNA for FasL (Fig. 1C). In contrast, Fas antigen mRNA was detected in all five specimens (Fig. 1C) and has also been detected by RT-PCR (5). Treating the thyroid cells with TSH, IL-1b, or gINF for up to 48 hours (before harvest and RNA isolation) also did not induce the expression of FasL mRNA or alter the expression of Fas antigen mRNA. We also examined the expression of FasL protein in thyroid follicular cells with the use of immunohistochemical staining (8) and Western blotting (9) techniques similar to those employed by Giordano et al. (1). Immunostaining of normal thyrocytes with a polyclonal, rabbit antibody to FasL did not detect FasL protein (10), but, consistent with the other studies, these cells demonstrated significant amounts of Fas antigen staining (2–4). Unexpectedly, a protein immunoblot of thyroid cell lysates performed with the mouse monoclonal antibody used by Giordano et al. (clone 33, Transduction Laboratories, Lexington, Kentucky) yielded an intense band at the appropriate size for FasL (Fig. 2). However, there was no difference in intensity of this band in lysates of PMA stimulated as opposed to untreated Jurkat cells, and this finding did not correlate with the changes in mRNA concentration observed in these cells (Fig. 1, A and B). This result brings into question the specificity of the clone 33 antibody for FasL. Although there may be differences in the tissues examined by ourselves and Giordano et al., we have not observed the expression of mRNA for FasL in primary cultured thyrocytes from over 20 normal and thyroiditis tissue samples, unless there was also evidence of mRNA for rearranged immunoglobulin genes (10). The latter result suggests that, in those situations, the message for FasL came from lymphocyte contamination of the thyroid cells. More importantly, FasL-induced programmed cell death in thyroiditis is questioned by our recent finding that the Fas pathway in thyroid follicular cells is blocked by a labile protein inhibitor (5). It has also been found that the in vitro induction of the Fas pathway with soluble ligand or antibody may be less efficient than that achieved by cytotoxic T cells (11). Together, these considerations make it difficult to predict the relative importance of Fas-mediated apoptosis in thyroiditis. We hope that the findings presented in this comment will promote the research and discussion necessary to clarify the potential role of the Fas pathway in the pathogenesis of thyroiditis. Theophil A. Stokes Michal Rymaszewski Patricia L. Arscott Su He Wang James D. Bretz Jeffery Bartron James R. Baker, Jr. Division of Allergy, Department of Medicine, University of Michigan Medical School, Ann Arbor, MI 48109–0666, USA