Technical Comments

Constitutive Expression of FasL in Thyrocytes

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Science  27 Mar 1998:
Vol. 279, Issue 5359, pp. 2015
DOI: 10.1126/science.279.5359.2015a

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-1β (IL-1β). 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 reverse-transcriptase polymerase chain reaction (RT-PCR) (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-1β, or γINF 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.

Figure 1

(A) Ribonuclease protection assay for FasL mRNA. Protected fragment of FasL mRNA yielded a 353-bp band, while a 96-bp band from a protected fragment of GAPDH mRNA was used to standardize RNA concentrations. RNA from two different individual's primary cultured thyrocytes (lanes 1 and 2) showed no message for FasL. An equal amount of RNA isolated from Jurkat cells stimulated with PMA (lane 3) demonstrated FasL mRNA and served as a positive control. RNA isolated from untreated Jurkat cells (lane 4) also was negative for FasL mRNA. (B) RT-PCR assay for FasL mRNA. Primers were designed to yield a 663-bp product corresponding to bp 796 to 1459 of Fasl mRNA. RNA from normal thyrocytes that were either TSH-deprived (lane 1) or TSH-supplemented (100 mU/ml) (lane 2) showed no FasL mRNA. RNA from Jurkat cells stimulated with PMA served as a positive control (lane 3), while no message was observed in RNA from untreated Jurkat cells (lane 4). RT-negative amplifications showed no product in the positive control (data not shown). (C) Ribonuclease protection assay for FasL and Fas mRNA. RNA from two additional normal (lanes 1 and 2) and three Graves (lanes 3 to 5) primary cultured thyrocytes was analysed. Although message for FasL was not detected in any of the thyrocyte samples, a 316-bp protected fragment of Fas mRNA was detected in all of the thyrocyte RNA preparations. A small amount of RNA from peripheral blood lymphocytes stimulated with PMA (lane 6) served as a positive control for FasL 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.

Figure 2

Protein immunoblot analysis of cell lysates probed with a monoclonal antibody for FasL (clone 33, Transduction Technologies). Cell lysates correspond to the following lane numbers: (1) unstimulated Jurkat cells; (2) activated Jurkat cells; (3) thyroid goiter, cultured with TSH; (4) thyroid goiter, cultured without TSH; (5) normal thyroid cultured with TSH; and (6) normal thyroid cultured without TSH. A heavy band is visible in all samples at roughly 37 kD, the reported size of FasL. Presence of the bands in all samples, including resting Jurkat samples that were from cells devoid of FasL mRNA, is inconsistent with the data obtained from the immunohistochemical staining, RT-PCR, and nuclease protection assays.

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.

REFERENCES AND NOTES

Giordano et al. describe the constitutive expression of Fas ligand (CD95L) by thyrocytes that they isolated from the glands of patients with Hashimoto's thyroiditis or nontoxic goiter (1). On the basis of this unexpected result, Giordano et al. propose that the concommitant expression of Fas and its ligand induces programmed cell death of thyrocytes and that this might be a major pathological mechanism underlying many forms of hypothyroidism. To detect FasL expression, Giordano et al. used polymerase chain reaction (PCR), immunohistochemical stainings, and FACS analysis. For the latter experiments, they used two commercially available antibodies against FasL, C-20 and mAb33. C-20 (Santa Cruz Biotechnology, Santa Cruz, California) is a rabbit polyclonal IgG antibody against an extracellular FasL epitope corresponding to amino acid residues 260 to 279, and it is recommended for use in protein immunoblots and immunohistochemistry. Monoclonal antibody mAb33 (Transduction Laboratories, Lexington, Kentucky), is an IgG1 monoclonal antibody against the extracellular part (216-277) of human FasL. It is made for the study of human FasL by protein immunoblots, immunoprecipitation, and immunofluorescence.

With the use of monoclonal antibody 33 (mAb33), we analyzed FasL expression with protein immunoblots in a panel of human tumor cell lines covering different B cell, monocyte, and T cell lines. Unexpectedly, we found that all these cell lines express FasL, as shown by a single band on the blot at about 37 kD. To confirm these results, we tested these cell lysates with a FasL-specific rabbit polyclonal antiserum PE62 against an extracellular peptide of FasL (2). In contrast to the results obtained with mAb33, none of these cell lines now showed a FasL-specific signal (Fig.11A).

Figure 1

FasL expression in cell lines. Total cell lysates prepared from 16 different tumor cell lines (eight are not shown) were tested by Western blotting for FasL expression with the use of either mAb33 (A) or the rabbit polyclonal antibody PE62 (B). Arrows indicate the position of the signal obtained with mAb33. Molecular weight markers were run in parallel as indicated. Cells were lysed in PBS containing 1% NP40 and a mixture of protease inhibitors. Total cell lysates were separated on a 12% SDS-PAGE, blotted against nitrocellulose filter, and analyzed with the antibodies as indicated with the use of HRPO-coupled goat antibody to mouse, rat, or rabbit IgG (Southern Biotechnology Associates, Birmingham, Alabama) and enhanced chemoluminescence (Pierce).

Figure 2

Analysis of FasL expression in 293 T cells transfected with a FLAG-FasL expression vector. (Left) analysis of untransfected cells; (right) analysis of transfected cells. Total cell lysates were incubated with antibodies as indicated and analyzed with a protein immunoblot. Arrows show FasL-specific signals. Signal detected by mAb33 is not found with any of the other FasL-specific antibodies.

Figure 3

Total cell lysates from FLAG-FasL–transfected 293 T cells were incubated with the FLAG- specific mAb M2 coupled to agarose beads (Kodak). FasL was immunoprecipitated and subjected to Western blotting. Nitrocellulose filters were developed with G247-4, or with mAb33 and HRPO-coupled goat–anti-mouse IgG. Mab33 detects a 37-kD signal present in the supernatant, but not immunoprecipitated FasL.

We therefore examined further the FasL specificity of mAb33. We transiently transfected human 293T embryonic kidney carcinoma cells with a FasL expression vector that encodes human FasL that has an NH2-terminal FLAG tag, and we performed a protein immunoblot, with the use of the FasL-specific antibodies mAb33, C-20, G247-4 (Pharmingen), and P62 (2), as well as M2 (Eastman Kodak, New Haven, Connecticut), which is specific for the NH2-terminal FLAG tag (Fig.11B).

With the use of mAbs C247-4 and M2, the FasL-transfected 293T cells showed FasL-specific signals that were absent in the untransfected control cells. Although the polyclonal rabbit antibodies C-20 and PE62 showed some background staining in untransfected 293T cells, they also revealed CD95L-specific signals in the lysates of the transfectants, as would be expected from earlier reports (2-4). In contrast, mAb33 detected a 37-kD signal (similar to the band in Fig. 11A) in both transfected and untransfected cells. To further characterize the specificity of mAb33, we immunoprecipitated FLAG-tagged FasL from lysates of transfected 293T cells with the use of the FLAG-specific antibody M2. The immunoprecipitates were tested by protein immunoblotting with G247-4 and mAb33. FasL expression was detected with G247-4, but not with mAb33. However, mA33 (but not G247-4) produced a strong 37-kD signal in the supernatant of the immunoprecipitate (Fig.11C).

Thus, C-20, PE62, and G247-4—but not mAb33—seem to be suited for the analysis of CD95L expression by protein immunoblotting. However, mAb33 does not stain human FasL, but a different protein expressed in many cell types. It is therefore questionable whether the signals shown in figure 4 of the report by Giordano et al. correspond to FasL expressed by thyroid cells.

With the use of 293T cells transfected with FLAG-tagged FasL, we then analysed several antibodies (NOK-1, NOK-2 (5), and G247-4 (PharmIngen, San Diego, California), mAb33, C-20, and MIKE 2 (Alexis, San Diego, California) for FasL specific staining with the use of flow cytometry and immunoflorescence. Untransfected cells served as specificity controls. With the use of a fluorescent activated cell sorter (FACS), only NOK-1, NOK-2, and MIKE-2 detected FasL expressed on the 293T cell transfectants, whereas C-20, mAb33, and G247-4 did not show any specific signals (6). G247-4 is known to work only in Western blots, but C-20 was also reported by Giordano et al. to stain FasL-expressing thyrocytes with the use of flow cytometrical and immunohistochemical analyses. In the immunoflurescence studies, FasL-specific signals were easily detected with NOK-1, G247-4, and with the FLAG-specific antibody M2. The peptide-specific rabbit polyclonal antibody C-20, however, gave a high background staining already with untransfected cells. FasL transfectants showed stronger signals, but also a similar background staining similar to the controls. Both stainings were not seen when the blocking peptide was added (6).

On the basis of these results, we conclude that C-20 is suited for analyzing CD95L expression by protein immunoblotting, but not by flow cytrometry, immunofluorescence, or immunohistochemistry. Because C-20 detects in such blots a signal of about 65 kD, which does not correspond to FasL (Fig. 11B), it may also stain by immunofluorescence antigens unrelated to FasL that may bear epitopes similar to the peptide used to generate the antibody. Flow cytometrical experiments using C-20 may also reveal cells expressing such epitopes that may be absent in 293 T cells but not in thyroid cells. We therefore cannot recommend the application of C-20 in flow cytrometrical and immunofluorescence studies designed to show FasL expression.

In comparison to the other FasL-specific antibodies, mAb33 detects a different intracellular protein that seems to be expressed ubiquitously. Previous studies that used this antibody should be interpreted with caution. In conclusion, our studies describe a panel of antibodies that are specific for FasL and that may be used either in flow cytrometrical, immunofluorescence, or Western blot analysis. They also show that the two antibodies C-20 and mAb33 may bear additional specificities or might not be specific for FasL. Because both antibodies are commercially available, they seem to be used frequently by many investigators. We suggest that results from such studies (1, 7) should be reinterpreted keeping in mind the specificity of these antibodies, or repeated with the use of reagents that are known to be specific for FasL.

REFERENCES

Response: We appreciate the comments by Stokes et al. and Fiedler et al. about our earlier report (1). With regard to detecting FasL in primary thyrocyte cultures with RNA protection assay and PCR, such detection would be difficult if the thyrocytes were not freshly excised and immediately analyzed, because FasL expression is labile ex vivo (mRNA or protein).

Concerning Fas expression, Stokes et al. used thyrocytes from controlateral lobes of thyroid cancers as their “normal” controls (2). We also observed variable Fas expression in uninvolved thyrocytes from thyroids with cancer, but we did not use such samples for normal controls. We obtained more normal samples from thyroid sections from patients undergoing laringectomy for laringeal cancer. These thyrocytes consistently expressed very low amounts of Fas, similar to thyrocytes from patients with nontoxic goiters (NTG), which we used in our report (1).

Prompted by concerns raised about the specificity of the polyclonal antibody to FasL C20 (Santa Cruz, Biotechnology, Santa Cruz, California) and mAb 33 (Transduction Laboratories, Lexington, Kentucky) (see below), we repeated an immunohistochemical study with the NOK-2 antibody (PharMingen, San Diego, California) on thyroid sections from laringectomy patients (above). Most thyrocytes from normal thyroids showed detectable FasL expression (3) (Fig. 21). In situ hybridization would further address this issue. Moreover, FACS analysis of ex vivo thyrocytes from NTG patients confirmed the constitutive expression of FasL on most thyrocytes, with the use of both the NOK-2 antibody and the H11 (Alexis, San Diego, California) antibody (Fig. 22A).

Figure 1

Immunoistolocalization of FasL in thyroid tissue from a laringectomized patient. (A) control mAb, (B) NOK-2 mAb.

Figure 2

(A) Surface FasL expression of ex vivo NTG thyrocytes detected by NOK-2 and H11 antibodies. Species/isotype-matched antibodies were used as controls. (B) Protein immunoblot analysis of FasL expression in thyrocytes as detected by mAbs 33 and G247-4. Lane 1: thyroid tissue from a NTG patient. Lane 2: thyroid tissue from a laringectomized patient. Lane 3: thyrocytes from a laringectomized patient, immunodepleted from CD45+ cells. Lane 4: 8 hours PMA-activated Jurkat J77 cells. Lane 5: untreated Jurkat J77 cells.

Finally, (i) thyroid tissue from patients with a laringectomy or NTG and (ii) hematopoietic cell-depleted thyrocytes from patients with NTG expressed FasL, as detected by Western blot analysis with 33 mAb (Transduction Laboratories) or with G247-4 mAb (PharMingen) (Fig. 22B). We detect a single band in our blots, similar to some investigators (4), while others detect multiple bands, which might represent glycosylated and unglycosylated forms (5).

Although we agree that C20 antibody may give a relatively high background signal as compared with other commercially available reagents, these new data support our earlier conclusion that normal thyrocytes express substantial amounts of FasL in vivo.

Fiedler et al. conclude that mAb 33 recognizes a protein different from FasL. We performed similar experiments with the use of three different cellular systems: (i) 293T cells transiently transfected with human FasL, (ii) COS-7 cells transiently transfected with human FasL (6), and (iii) NIH 3T3 stably transfected with murine FasL (7). To detect the expression of FasL by FACS analysis, we used NOK-1 mAb (PharMingen), C20, G247-4 mAb, and 33 mAb. Species and isotype-matched antibodies were used as control primary reagents. All of these antibodies gave a specific staining only in transfected cells (Fig. 23; only data on NIH 3T3 are shown), although with different distributions.

Figure 3

FACS analysis of NIH 3T3 stably transfected with murine FasL, with the use of NOK-1, C20, G247-4, and 33 antibodies. Antibodies appear to cross-react between human and mouse FasL.

It is not clear why Fiedler et al. did not detect FasL expression by FACS analysis (with the use of the 33, C20, and G247-4 antibodies), or why the FLAG-FasL immunoprecipitate reacts with G247-4, but not with the mAb 33. We used a human FasL construct; they used a human FasL-NH2-terminal FLAG construct. Perhaps the FLAG is not unharmful to some FasL epitopes.

Fielder et al. used untransfected cells as specificity controls for their FACS analysis. This procedure may lead to an underestimation of specific signals, as our data show that all the antibodies tested are crossreactive between human and murine FasL. Because many cell lines express low levels of FasL constitutively, or under certain culture conditions, specificity controls should be performed with isotype-matched antibodies and mock-transfected cells, as opposed to untransfected cells.

We also analyzed protein immunoblots of 293T cells transiently transfected with human FasL, COS-7 cells transiently transfected with human FasL, and NIH 3T3 stably transfected with murine FasL. To reveal FasL, we used mAb 33 and mAb G247-4. Both mAbs detected an ∼37 kD band only in transfected cells (Fig. 24) and in PMA-treated, but not in PMA-untreated, Jurkat cells (8).

Figure 4

Protein immunoblot analysis of FasL transfectants, with the use of both 33 and G247-4 antibodies. Lane 1: 293T cells, pcDNA3 empty-transfected. Lane 2: 293T cells, pcDNA3 hFasL-transfected. Lane 3: COS-7 cells pcDNA3empty-transfected. Lane 4: COS-7 cells pcDNA3hFasL-transfected. Lane 5: NIH-3T3 pSRa-72(N.1)empty-transfected. Lane 6: NIH-3T3 pSRa-72(N.1)mFas-L transfected. Lane 7: untreated Jurkat J77 cells. Lane 8: 8 hours PMA-activated Jurkat J77 cells.

Fielder et al. find a uniform strong band reactive with the mAb 33 by protein immunoblot analysis in untransfected murine 293T cells, as well as in other human cell lines. As we detect mAb 33-reactive signals in transfected cells or in Jurkat only after PMA exposure, their finding is intriguing, and we have no explanation for it.

In conclusion, we find evidence that mAb 33 does recognize FasL in three different cell types, transiently or stably transfected with human or murine FasL, both by FACS and protein immunoblot analysis. Nevertheless, because of possible differences in specificity among the various available antibodies, we recommend the simultaneous use of several anti-FasL reagents. Constitutive FasL expression on in vivo and ex vivo normal thyrocytes has now been described by five different antibodies against FasL.

REFERENCES AND NOTES

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