Tumor Necrosis Factor-α-Induced Apoptosis in Olfactory Epithelium in Vitro: Possible Roles of Caspase 1 (ICE), Caspase 2 (ICH-1), and Caspase 3 (CPP32)

Experimental Neurology 165, 35– 45 (2000) doi:10.1006/exnr.2000.7465, available online at http://www.idealibrary.com on

Tumor Necrosis Factor-␣-Induced Apoptosis in Olfactory Epithelium in Vitro: Possible Roles of Caspase 1 (ICE), Caspase 2 (ICH-1), and Caspase 3 (CPP32) 1 Yuko Suzuki 2 and Albert I. Farbman Department of Neurobiology and Physiology, Northwestern University, 2153 North Campus Drive, Evanston, Illinois 60208-3520 Received January 3, 2000; accepted April 19, 2000

INTRODUCTION We investigated the potential roles of three members of the interleukin-1␤-converting enzyme (ICE) protease family (caspases) in apoptosis in olfactory epithelium. By RT-PCR analysis, the mRNAs of caspase 1 (ICE), caspase 2 (ICH-1), and caspase 3 (CPP32) were detected in olfactory mucosa obtained from normal adults, E19 fetuses, and unilaterally bulbectomized rats. The transcript of caspase 2 disappeared in bulbectomized animals 3 and 5 days postoperatively, but reappeared 21 days postoperatively. This suggests that most of the caspase 2 transcript was in olfactory sensory neurons. We used TNF-␣ to induce cell death in organotypic cultures of E19 olfactory epithelium and assayed the ability of three caspase inhibitors to reverse the TNF-␣ effect. After 6 h of treatment with medium containing TNF-␣, a 2.5-fold increase in apoptotic body number was observed throughout the olfactory epithelium. Pretreatment of the cultures with either of two irreversible caspase inhibitors (Z-VADfmk, Ac-YVAD-cmk) for 4 h, followed by a 6-h treatment with TNF-␣ plus an inhibitor, blocked TNF-␣induced cell death completely. Pretreatment with a third caspase inhibitor (Z-DEVD-fmk) in the same treatment schedule reduced the numbers of apoptotic cells significantly but not to the same extent as Z-VADfmk or Ac-YVAD-cmk. Increasing the dose of any of the inhibitors reduced the numbers of apoptotic figures below those of control cultures, indicating that the inhibitory response is dose dependent. Taken together, the results suggest that caspases 1, 2, and 3, and perhaps others that are blocked by the inhibitors we used, participate in TNF-␣-induced cell death in vitro. © 2000 Academic Press Key Words: apoptosis; olfactory epithelium; TNF-␣; caspases; cell death.

Vertebrate olfactory neurons are unique because they are continually replaced throughout life. They die by programmed cell death under physiological conditions at all stages in their life cycle (8, 13, 19, 45). Dead sensory neurons are replaced by the progeny of dividing globose basal cells (25, 60, 62). Programmed cell death, also known as apoptosis, is a process by which cells within a multicellular organism are selectively eliminated in a controlled manner during normal differentiation and development (72). Apoptosis is characterized histologically by nuclear condensation, chromatin margination, cell shrinkage, plasma membrane blebbing, and fragmentation into dense bodies (apoptotic bodies) containing remnants of nucleus and cytoplasm (36). In the unperturbed olfactory epithelium apoptosis is presumably involved in tissue homeostasis and may be a direct or indirect trigger of neurogenesis (19). Little is known about the molecular mechanism involved in olfactory cell death. In a pilot study we showed that mRNAs of two receptors, Fas and tumor necrosis factor receptor 1 (TNFR1), and those of their respective ligands, Fas ligand and TNF-␣, are detectable in the olfactory mucosa (20). Moreover, both Fas and Fas ligand are detectable immunohistochemically in the epithelium. Both TNFR1 and Fas are transmembrane receptors that belong to a family of receptors including the low-affinity nerve growth factor receptor, TNFR2, CD40, CD27, and CD30 (50) and the family is still growing (49). Family members are defined by the presence of cysteine-rich repeats in their extracellular domains, but only Fas and TNFR1 contain, in their cytoplasmic portion, a 75- to 80-amino-acid “death domain” that appears to participate in apoptosis (65). Activation of these receptors by their cognate ligands, or by antibodies to the receptor, triggers an apoptotic cascade in various cell types, including T cells (5, 21, 50, 76), liver cells (55), neurons (14, 26, 53, 75), fibroblasts (34), and several cell lines (cf., 28, 42, 56, 59, 71).

1 Supported by NIH Grants P01 DC 00347 and P60 DC 02764. We gratefully acknowledge the assistance of Mrs. Judith A. Buchholz. 2 Present address: Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, 061-0293, Japan.

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0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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The death domains of the receptors are thought to be linked by intermediate adaptor proteins to effector enzymes belonging to the interleukin-1␤-converting enzyme (ICE) protease family, a family of cysteine proteases now known as caspases (3). The identification of the product of the Caenorhabditis elegans cell death gene, ced-3, as a homologue of mammalian ICE (caspase 1) has led to extensive evidence implicating the caspases in apoptosis (11, 12, 17, 37). This has been established by the use of specific enzyme inhibitors such as the cowpox virus CrmA protein, baculovirus p35 protein, certain peptide methyl ketones, and specific peptide aldehydes (10, 22, 47). These inhibitors reversibly or irreversibly prevent cleavage of known substrates of the various caspases. The mammalian ICE/ced-3 family comprises 14 known caspases (2, 31, 68). Cells that undergo apoptosis, either physiological or induced, apparently do not contain all caspases or, at least, not all are activated under specified experimental conditions (reviewed by 11, 12, 66). Activated caspases cleave proteins at Asp-X peptide bonds (33, 73, 74). Thus, although the apoptotic pathways in various cells share many features, the cumulative evidence indicates there is no single universal molecular cascade for cells undergoing apoptosis nor, at least in some cases, is there a single cascade for a given cell type. The fact that a cell expresses a particular caspase does not necessarily mean that it will be activated and will participate in cell death. The aim of the present study was to examine some of the molecular aspects of TNF-␣-induced apoptosis in olfactory epithelium in vitro. In preliminary studies we had shown that when TNF-␣ was added to medium of organotypic olfactory cultures the number of apoptotic cells was increased 2.5-fold (20). In the present study we test the hypothesis that TNF-␣-induced apoptosis in this tissue is mediated by caspases. We showed that the mRNAs of three caspases were present in fetal and adult rat olfactory mucosa. In addition, we found that addition of certain caspase inhibitors reversed the apoptotic effect of TNF-␣. MATERIALS AND METHODS

Adult Sprague–Dawley rats (200 –250 g; Harlan Inc., Indianapolis, IN) were sacrificed by an overdose of CO 2; the olfactory mucosa, spleen, and liver were dissected out and stored at ⫺80°C. In addition, whole brains removed from E16 rat fetuses, olfactory mucosa from E19 fetuses, and olfactory mucosa from 3- to 4-month-old animals, in which one olfactory bulb had been ablated, were used. For the surgery animals were anesthetized with ketamine (9 mg/100 g body wt) and xylazine (1 mg/100 g body wt). The skin above the right olfactory bulb was incised, a small hole was made in the bone covering the olfactory bulb with a dental drill, and the bulb was removed by aspiration. Gelfoam (Up-

john, Kalamazoo, MI) was placed in the wound to control bleeding. The skin wound was sutured and animals were allowed to recover. Gentamicin sulfate (2.5 mg) and buprenorphine HCl (0.02 mg/100 g body wt) were given intramuscularly after surgery. Animals were killed with an overdose of CO 2 3, 5, and 21 days after surgery. Olfactory mucosa from both ipsilateral and contralateral sides to the bulbectomy were taken for analysis. All animal procedures were done according to guidelines established by the National Institutes of Health and approved by the Northwestern University Animal Care and Use Committee. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total cellular RNA was extracted by a modification of the acid guanidinium thiocyanate–phenol– chloroform method (9) using Trizol reagent (Gibco BRL, Life Technologies, Rockville, MD). RNA concentration was determined spectrophotometrically. Using random hexamers and reverse transcriptase (Gibco BRL, Life Technologies), cDNA was synthesized from total RNA (5 ␮g). PCR was performed using 2 ␮l of the cDNA, 0.5 ␮l Taq polymerase (Perkin Elmer, Foster City, CA), 2 ␮l each NTP, and 50 pmol of the following oligonucleotide primers (all synthesized at the Biotechnology Facility, Northwestern University): 5⬘-CCA CTC CTT GTT TCT CTC-3⬘ (caspase 1 antisense primer; Accession No. U14647); 5⬘-CCT TCC TTG TAT TCA TGT C-3⬘ (caspase 1 sense primer), expected product length 189; 5⬘-ATC TCC ACG ACA TGC TTG GAT GAA-3⬘ (caspase 2, antisense primer; Accession No., U77933); 5⬘-TAG ATA ATG GTG ATG GTC CTC CCT-3⬘ (caspase 2 sense primer), expected product length 475; 5⬘-TGA GCA TTG ACA CAA TAC AC-3⬘ (caspase 3 antisense primer; Accession No. U84410); and 5⬘-AAG CCG AAA CTC TTC ATC-3⬘ (caspase 3 sense primer), expected product length 349. PCRs for all caspases were carried out for 35 cycles using the following program for each cycle: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the 35 cycles there was an extension step at 72°C for 5 min. Control tubes in which water was substituted for the reverse transcriptase product were used. The PCR products were electrophoresed in 2% agarose gels buffered with Tris– borate EDTA (TBE) and visualized under UV illumination by ethidium bromide staining. Organotypic Cultures Pregnant Sprague–Dawley rats were sacrificed by an overdose of CO 2 in the 19th day of gestation (E19). Fetuses were removed and decapitated, and the olfactory mucosa was peeled off from both sides of the nasal septum. The sheets of olfactory mucosa were explanted onto collagen-coated Millipore substrates resting on

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bated for 6 h in TNF-␣. The control solution for this set of cultures was the basic medium containing 0.5% BSA. In the inhibitor experiments cultures were preincubated for 4 h in basic medium containing one of the following inhibitors (all purchased from Calbiochem, San Diego, CA): 100 ␮M Z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), 150 ␮M AcYVAD-cmk (Acetyl-Tyr-Val-Ala-Asp-chloromethylketone), 150 ␮M Z-DEVD-fmk (N-benzyloxycarbonylAsp-Glu-Val-Asp-fluoromethyketone), or 100 ␮M Z-FA-fmk (N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone). After the preincubation explants were incubated for 6 h with medium containing TNF-␣ (and BSA) plus the same inhibitor. Ac-YVAD-cmk was designed to be a potent, cell permeable irreversible caspase 1 inhibitor at the dose used (41), but other work showed clearly that it also inhibits the activity of caspase 6 (42). Z-DEVD-fmk was presumed to be an

FIG. 1. RT-PCR for caspases 1, 2, and 3 mRNAs. All three transcripts are detectable in the control adult rat olfactory mucosa (Cont. Olf., lane 2), E16 brain (lane 6), and liver (lane 8). MRNAs of caspases 1 and 3 were detectable in adult spleen (lane 7) but not that of caspase 2. Where caspase 2 was present it showed as a doublet, probably representing the long and truncated transcripts. The truncated form was brighter in the gels. In the bulbectomized animals there was no apparent change in the transcripts of caspase 1 and 3, but the signal for caspase 2 in the 3 (D3 obx) and 5 day (D5 obx) postbulbectomy animals was significantly reduced and returned to control values 21 days (D21 obx) after surgery. The expected size for these PCR products are 189 bp for caspase 1, 475 bp for caspase 2, and 349 bp for caspase 3. The estimated sizes for these PCR amplimers match their expected sizes. The first lane shows 100-bp molecular weight ladder and the last lane contained no RT product.

stainless-steel grid platforms in humidified organ culture dishes (18). Serum-free Waymouth’s MB/752 medium with supplements (7) was used as the basic medium for all the organotypic culture studies and the cultures were grown at 35°C in an atmosphere of 5% CO 2 in air. For each experimental group a minimum of 10 organotypic cultures were grown. For quantification 7–15 pregnant rats were used and usually 9 –15 embryos were obtained from each rat. Cultures were grown for 4 h in the basic medium followed by 6 h in basic medium containing TNF-␣ (40 ng/ml medium) and 0.5% bovine serum albumin (BSA, added to help keep TNF-␣ in solution). In our first experiments we also incubated for 20 h, but found the effect was easy to see at 6 h, so all of the experimental results reported in this paper were from cultures incu-

FIG. 2. TUNEL responses in cultured olfactory epithelium. (A) E19 olfactory mucosa was cultured for 4 h in the basic medium and followed by TNF-␣ for 6h. Many TUNEL-positive (dark brown-colored) nuclei are seen throughout the epithelium (OE). (B) Control. E19 olfactory mucosa was cultured for 4 h in the basic medium, followed by the basic medium plus 0.5% BSA for 6 h. A few TUNELpositive nuclei are seen in the olfactory epithelium and the lamina propria. bl, basal lamina. Sections were counterstained with methyl green. Bar, 20 ␮m.

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irreversible inhibitor for caspase 3 (52), but, again, later work showed it inhibited the activity of caspases 6, 7, and 10. Z-FA-fmk (granzyme B inhibitor), which lacks Asp, was used as a negative control for the inhibitors. The stock solutions of inhibitors were dissolved in dimethyl sulfoxide (DMSO). The final concentrations of DMSO in cultures were 0.04 – 0.07%. In each experiment, i.e., each time fetal tissue was harvested from a pregnant rat, at least three specimens of olfactory mucosa were used for each of five experimental conditions, i.e., medium containing TNF-␣, medium containing TNF-␣ plus inhibitor, and three controls. The controls were (i) medium with BSA, (ii) medium with BSA plus inhibitor, or (iii) medium with BSA plus Z-FA-fmk. A different set of cultures was grown to test the effect of inhibitors on apoptosis occurring in the cultures grown in basic medium alone, not treated with TNF-␣. In other words, because some apoptosis occurred in all of the control cultures described above, we did this second set of experiments to determine whether the inhibitors would reduce the numbers of apoptotic cells below the levels occurring in the controls. These cultures were incubated with medium containing one of the inhibitors at higher doses than those used above in combination with TNF-␣. We did four groups of cultures: (i) a control, incubated with basic medium alone; (ii) basic medium plus 200 ␮M Z-DEVD-fmk; (iii) basic medium plus 200 ␮M Ac-YVAD-fmk; and (iv) basic medium plus 150 ␮M Z-VAD-fmk. All cultures were incubated for 7 h. Light and Electron Microscopy For the TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling) method (24) the cultured olfactory mucosa was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C and processed for embedding in paraffin. Sagittal sections were cut on a microtome at a thickness of 10 ␮m. The slides were deparaffinized to water and treated with 5 ␮g/ml proteinase K (Sigma) for 4 min at room temperature. The reaction for TUNEL labeling was carried out by using an Apop Tag in situ apoptosis detection kit (Onchor, Gaithersburg, MD). The sections were immersed in equilibration buffer for 15 min and incubated in a solution of TdT and digoxigenin– dUTP at 37°C for 1 h in a humidified chamber. The reaction was terminated by incubating in stop/wash buffer for 30 min at 37°C. Then sections were incubated in antidigoxigenin peroxidase at room temperature for 30 min. After washing in phosphate-buffered saline, the sections were colored by incubation with hydrogen peroxide (0.3%) in the presence of 50 ␮g/ml diaminobenzidine. For direct staining of apoptotic cells the sections were treated by the Feulgen procedure which specifically stains DNA

(cf., 8). The nuclei of the apoptotic cells are visualized as intensely stained structures. The Feulgen-stained sections were counterstained with Fast Green FCF. For quantitative studies, the number of pyknotic cells along a 450 ␮m length of olfactory epithelium was counted under a light microscope by using an ocular micrometer. Counts were made every fifth section (40 ␮m between sections). To determine whether the groups were different from one another, the analysis of variance (ANOVA) and post hoc test between means (Fisher’s PLSD) were used. For electron microscopic observations, the cultured olfactory mucosa was fixed in a phosphate-buffered (pH 7.4) solution containing 2% glutaraldehyde and 1.6% paraformaldehyde. They were postfixed in phosphate-buffered 1% OsO 4, dehydrated, and embedded in Epon 812. Ultrathin sections were cut, stained with uranyl acetate followed by lead citrate, and examined under a Hitachi H7000 electron microscope. RESULTS

Caspase Transcripts in Olfactory Mucosa RT-PCR was used to determine whether the mRNAs of caspases 1, 2, and 3 were expressed in the rat olfactory mucosa. The transcripts of all three caspases were present in olfactory mucosa from normal adult rats, in fetal rat brains (at E16), in adult rat liver, and in olfactory mucosa from rats that had been unilaterally bulbectomized 21 days following surgery (Fig. 1). The caspase 1 and 3 transcripts were present in all of the other tissues including the three time points after unilateral bulbectomy (Fig. 1) and in E19 olfactory mucosa (not shown). Caspase 2 was revealed as a doublet, probably showing the long and truncated forms; the latter showed a much brighter band on the gel. However the mRNA of caspase 2 was not demonstrable in the olfactory mucosa 3 days following bulbectomy, was very faint at 5 days, and was virtually restored to control levels 21 days after surgery. Caspase 2 transcripts were apparently absent in spleen. Induction of Apoptosis in Organotypic Cultures Olfactory mucosa stripped from the nasal septum of E19 rat fetuses was cultured for 4 h in the basic medium and for 6 h in the presence of 40 ng/ml TNF-␣. As a control the mucosa was cultured in the basic medium alone for 6 h. Apoptotic cell death was observed both in the olfactory and in the respiratory mucosa in both controls and TNF-␣-exposed explants. The TUNEL method, which detects apoptotic bodies, revealed many TUNEL-positive nuclei in the epithelium of the olfactory mucosa treated with TNF-␣ for 6 h (Fig. 2A), thus confirming our earlier results (20). In the lamina propria a few TUNEL-positive cells were also observed. In

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Evidence for Caspase Activation in Explants of Olfactory Epithelium during TNF-␣-Induced Apoptosis

FIG. 3. Electron micrograph of cultured olfactory epithelium treated with basic medium for 4 h and TNF-␣ for 6 h. (A) Apical region. A few olfactory knobs (O) are seen between supporting cells. Arrows, apoptotic bodies. (B) Middle to basal region. Supporting cells (S), which are characterized by the presence of tonofilaments (T), contain many apoptotic bodies (arrows). O, olfactory cells. Bar, 1 ␮m.

the control specimen many fewer TUNEL-positive nuclei were observed in the epithelium and lamina propria (Fig. 2B). In electron micrographs of the specimens treated with TNF-␣ for 6 h most of the apoptotic bodies were located in the middle to basal region of the olfactory epithelium (Figs. 3A and 3B). In the apical region a few dendritic knobs of normal appearing olfactory neurons were observed, suggesting that some sensory neurons survived the treatment with TNF-␣. In the middle and basal regions most of the apoptotic cells had been phagocytized by supporting cells (Fig. 3B) as has been reported to occur in vivo (63).

To determine whether caspases play a role in TNF␣-induced apoptosis, known irreversible inhibitors of caspases, Z-VAD-fmk, Ac-YVAD-cmk, and Z-DEVDfmk, were used before and during TNF-␣ treatment of the explants. For quantification the Feulgen reaction, which specifically stains DNA, was used. With the Feulgen reaction apoptotic cells were detected as condensed or pyknotic cell nuclei in the olfactory epithelium (Fig. 4). We determined that with either the Feulgen method or the TUNEL method the number of apoptotic bodies was approximately 2.5-fold higher in TNF-␣-treated cultures than in controls (Figs. 4A, 4B, and 5). The Feulgen method was used for the remaining studies because it was more convenient and costeffective, given the large number of sections examined in this study. In cultures that had been pretreated with Z-VADfmk or Ac-YVAD-cmk for 4 h, followed by 6 h of treatment with TNF-␣ plus inhibitors, the number of apoptotic nuclei was essentially the same as that seen in untreated cultures or cultures treated only with the inhibitor. Moreover this number was significantly different from that in specimens treated only with TNF-␣ (Figs. 4C, 4D, and 5; post hoc tests, P ⬍ 0.0001). Thus, in the doses used, Z-VAD-fmk and Ac-YVAD-cmk completely inhibited TNF-␣-induced apoptosis. The numbers of apoptotic nuclei in specimens treated with the two inhibitors were not significantly different from one another or from the numbers in the control cultures. Treatment with the inhibitor Z-DEVD-fmk resulted in a reduction in the number of apoptotic cells over that treated with TNF-␣ alone (Figs. 4E and 5, post hoc tests, P ⬍ 0.0001). However, the potency of this inhibitor was significantly less than that of the other two caspase inhibitors (P ⫽ 0.0002). Treatment with ZFA-fmk (lacking aspartate) had no effect in preventing TNF-␣-induced apoptosis (Figs. 4F and 5), suggesting that the aspartate-containing tripeptide or tetrapeptide components of the specific caspase inhibitors were the active agents in this system. Treatment of the cultures with inhibitors alone at the initial doses had no effect when compared with the cultures treated with the basic medium. In other words, there appeared to be a “basal” level of cell death in the cultures. However, when the doses of the inhibitors were increased the basal levels were significantly reduced (Fig. 6; post hoc tests, P ⫽ 0.0001 for Z-DEVD-fmk and Ac-YVAD, P ⬍ 0.0001 for Z-VAD-fmk), suggesting that the basal level of apoptosis level was at least partly dependent on the caspases.

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FIG. 4. Feulgen reactions in the cultured olfactory epithelium after treatment with inhibitors of caspase 1 and caspase 3. (A) Control. The olfactory mucosa was cultured in the basic medium for 4 h and followed by the medium containing 0.5% BSA for 6 h. Arrowheads indicate apoptotic cells. (B) TNF-␣ treatment. The olfactory mucosa was cultured in the basic medium for 4 h, followed by the medium containing TNF-␣ and BSA for 6 h. (C) Preincubation with Z-VAD-fmk (100 ␮M, 4 h) before treatment with TNF-␣ ⫹ BSA in the presence of Z-VAD-fmk (100 ␮M, 6 h). (D) Preincubation with Ac-YVAD-cmk (150 ␮M, 4 h) before treatment with TNF-␣ ⫹ BSA in the presence of Ac-YVAD-cmk (150 ␮M, 6 h). (E) Preincubation with Z-DEVD-fmk (150 ␮M, 4 h) before treatment with TNF-␣ ⫹ BSA in the presence of Z-DEVD-fmk (150 ␮M, 6 h). (F) Preincubation with Z-FA-fmk (100 ␮M, 4 h) before treatment with TNF-␣ ⫹ BSA in the presence of Z-FA-fmk (100 ␮M, 6 h). Counter staining, Fast Green. Bar, 20 ␮m.

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The Roles of Caspases in Cell Death

FIG. 5. The number of apoptotic cells per 450 ␮m length of olfactory epithelium. The olfactory mucosa was treated for 4 h with each inhibitor (Z-DEVD-fmk, Ac-VAD-cmk, Z-VAD-fmk, Z-FA-fmk) or not treated (⫺) and subsequently cultured for 6 h in the presence (⫹) or absence (⫺) of TNF-␣ together with each inhibitor. The symbol (⫺) on the TNF-␣ and inhibitor lines indicate controls. The bars show the mean and the vertical line on each bar represents the standard error. The numbers of cultures are as follows: n ⫽ 15 for control and TNF-␣ alone (TNF-␣ ⫹, inhibitor⫺); n ⫽ 10 for Z-DEVD-fmk, AcYVAD-cmk, and Z-VAD-fmk; n ⫽ 7 for Z-FA-fmk.

DISCUSSION

The present results show that TNF-␣ rapidly and potently induces apoptosis in the organotypic cultures of olfactory epithelium, as shown by both TUNEL and Feulgen staining. Adding caspase inhibitors to the culture medium significantly reduced the number of apoptotic bodies in the cultures to levels below those seen in cultures grown in control medium. The effect was dose-dependent. These data implicate several caspases as possible participants in the apoptotic cascade. The fact that transcripts of three caspases are expressed in olfactory mucosa of untreated adult and fetal rats is consistent with the notion that they may be participants in apoptosis in vivo as well. Moreover, the reduced expression of caspase 2 transcripts within 3 and 5 days following bulbectomy and its restoration 21 days after bulbectomy suggest that the transcripts for this caspase are present primarily in the neuronal population and that the truncated form was apparently prevalent. The truncated form is associated more with inhibition of cell death whereas the long form is associated with apoptosis (40). Our studies suggest that the three caspases may participate in olfactory cell death; further, they provide data that permit us to formulate testable hypotheses and a system on which these hypotheses can be analyzed.

There is evidence suggesting that diverse stimuli can trigger cell death and the activation of the caspase family enzymes. Moreover, it is possible that separate pathways may be responsible for activating the three caspases we studied. In the Fas ligand/Fas mediated cell death of mouse T lymphocytes apoptosis was completely inhibited by Z-VAD-fmk, one of the inhibitors we used, and BD-fmk, a caspase 3 inhibitor (57). On the other hand, apoptosis induced by dexamethasone, etoposide, or irradiation was more sensitive to inhibition by BD-fmk. Moreover, in murine T lymphocytes, cell death induced by withdrawal of interleukin-2 was not inhibited by Z-VAD-fmk, but was inhibited by BDfmk (57). In death of rat hippocampal neurons induced by traumatic brain injury, caspase 3 was activated but caspase 1 was apparently not involved (75). In contrast, caspase 1 was activated in the death of neurons induced by ␤-amyloid (35). In the present study, we have focused on one pathway of cell death in the olfactory epithelium in vitro, that induced by TNF-␣, but by no means have we ruled out the possibility that other molecular cascades may be involved in this tissue, as is the case in other tissues. The coupling of caspase 2, one of the presumed effector caspases acting via the TNF-␣/TNFR signaling pathway, apparently occurs by binding to an adaptor molecule known as RAIDD (RIP-associated ICH-1/ CED-3-homologous protein with a death domain; 15) or CRADD (caspase and RIP adaptor with death domain; 1). The carboxy terminal of RAIDD/CRADD binds to

FIG. 6. The number of apoptotic cells per 450-␮m length of olfactory epithelium. The olfactory mucosa was treated with each inhibitor (200 ␮M Z-DEVD-fmk, 200 ␮M Ac-YVAD-cmk, 150 ␮M Z-VAD-fmk) or without inhibitor (medium) for 7 h. The bars show the mean and the vertical line on each bar represents SE. n ⫽ 7 cultures for each treatment.

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the homologous domain in RIP, a serine/threonine kinase component of the death pathway. RIP binds, via adaptor proteins, to the activated TNF receptor. However, although caspase 2 is activated in this pathway the substrate on which it effects its action is, so far as we know, unknown. The evidence favoring the interpretation that caspase 2 is important in the apoptosis pathway is circumstantial and largely based on the familial relationship to CED-3 and other caspases. Overexpression of caspase 2 (Nedd2) in cultured fibroblast and neuroblastoma cells resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene (39). Moreover, during embryonic development this caspase is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death, such as central nervous system and kidney; it is also expressed in adult neurons (39). Caspase 2 is thought to be activated early during the apoptotic process in the human monocytic tumor cell line, THP.1, but it was not possible to discern whether its activation preceded that of other caspases (42). Our evidence showing it is selectively expressed, both in long and in truncated forms, in olfactory neurons suggests that it may play a role in apoptosis in the olfactory system. Consistent with this hypothesis is the evidence that one of the inhibitors we used, Z-VAD-fmk, does block activation of caspase 2, but this inhibitor also blocks activities of caspases 1, 3, 6, and 7 (23, 42) and possibly others. In caspase 2-deficient mutant mice excess numbers of germ cells were present in the ovaries and the oocytes were resistant to cell death following exposure to chemotherapeutic drugs, suggesting that caspase 2 is required for oocyte cell death (6). However, these mutant mice reached adulthood with no gross abnormality. Although caspase 2 is highly expressed in the central nervous system it does not seem to be activated in the ischemic model of neuronal death in the central nervous system or in the rate of facial motor neuron death after axotomy (6). Thus, although our data indicate caspase 2 is expressed in olfactory neurons further studies are required to determine whether it is activated in our organotypic model system or in vivo when cell death is induced experimentally. Mouse mutants deficient in caspase 1 develop normally, appear healthy, are fertile, and do not exhibit a prominent cell death-defective phenotype (37). The observations in these mutants suggest either that there are no gross defects in normal physiological processes involving apoptosis or that in the caspase 1-deficient mice one or more other caspases easily substitute for it. There is evidence that caspase 1 does participate in Fas-induced cell death (17, 29). Our evidence that it may be a participant is based on the presence of caspase 1 transcripts in olfactory mucosa, although it is not entirely clear from the RT-PCR data that this enzyme is specifically in neurons. Our in vitro data

using peptides designed to inhibit caspase 1, although suggestive, do not rule out the possibility that the inhibitors could be acting on other members of the caspase family. The only known substrate for caspase 1 is interleukin-1␤ (reviewed by 11), and it is not known how cleavage of this substrate might fit into the apoptotic cascade. On the other hand mouse mutants deficient in caspase 3 are smaller than their littermates and die at 1–3 weeks after birth (38). Brain development in these deficient mice is severely affected with hyperplasias in several brain locales. Pyknotic cells, usually observed at sites of major morphogenetic change in normal brain development, are absent in caspase 3-deficient animals (38). These data suggest that caspase 3 plays a critical role during morphogenetic cell death in the mammalian brain (11, 38, 51). Our data are consistent with this interpretation. Caspase 3 has many known intracellular substrates, including poly(ADP-ribose) polymerase (a DNA repair enzyme), U1-70 kDa (a 70-kDa protein component of the U1 small nuclear ribonucleoprotein), protein kinase C ␦, huntingtin, gelsolin, and others (reviewed by 11). Cell Death in Olfactory Epithelium It is well known that even under unperturbed conditions the olfactory sensory neurons continuously die and are replaced by progeny of precursor cells in the basal layer (e.g., 8, 13, 27, 32, 43– 45, 63, 69). Supporting cells, on the other hand, are thought to be much longer lived and do not die in significant numbers (70). The morphology of apoptotic cell death in our organotypic cultures mimics that seen in vivo, i.e., most dead cells were neurons and basal cells found in the middle and basal regions of the epithelium. Further, the disposition of apoptotic bodies in our cultures resembled that occurring in vivo, as supporting cells phagocytized the apoptotic bodies (63). The rate of apoptotic cell death in olfactory epithelium can be increased in several ways in vivo. For example, surgical interventions, such as bulbectomy or axotomy (e.g., 8, 30, 48, 63) or administration of colchicine (64) increase apoptosis. Moreover, in vitro, treatment of olfactory explants by addition of Fas ligand or TNF-␣ (20) to culture medium can significantly increase the number of apoptotic bodies. Inhibitors of Cell Death Z-VAD-fmk and Ac-YVAD-cmk were both considered to be caspase 1 inhibitors and were designed as such but further experimentation has shown that they are less than absolutely specific; i.e., they block other caspases as well as caspase 1 (e.g., 11, 42). Thus, studies based on the assumption that these inhibitors are specific for caspase 1 must be reevaluated.

POSSIBLE ROLE OF CASPASES IN OLFACTORY CELL DEATH

In our first set of in vitro experiments both inhibitors blocked TNF-␣-induced cell death completely; i.e., the numbers of apoptotic figures in the TNF-␣-treated cultures was not significantly different from those seen in untreated cultures or those treated with inhibitor alone. However, when the dose of either inhibitor was increased the inhibitory effect was even more marked (Fig. 6). For example, addition of 100 ␮M of Z-VAD-fmk to the medium reduced the numbers of TNF-␣-induced apoptotic bodies to the level of control cultures, but 150 ␮M this tripeptide inhibitor reduced the numbers to about half those seen in the controls. Increasing the doses of the two tetrapeptide inhibitors had a similar effect, though not quite so large. To achieve an effect with the tetrapeptide compounds, Ac-YVAD-cmk and Z-DEVD-fmk, a higher dose was required probably because the explants are less permeable to the modified tetrapeptide than to the modified tripeptide (23, 67). The doses we used in both culture experiments were consistent with doses used in cell cultures and not considered deleterious (cf., 16). Moreover, the total incubation time in media containing inhibitors was no longer than 10 h, well below the 48 h used in experiments using cerebellar granule cells, and the 48-h exposure was not considered toxic (cf., 16). In the first set of experiments we chose the doses on the basis of the observation that there were no differences between the numbers of apoptotic cells in control cultures, containing neither inhibitor nor TNF-␣, and cultures containing the inhibitor but no TNF-␣ (cf., 16). From these first experiments we concluded that the inhibitors were not themselves deleterious to the explants. Our observations in the second set of culture experiments indicate that the inhibitors reduced the numbers of apoptotic cells that were not induced by exogenous TNF-␣. The dose-dependent response to the inhibitors supports the notion that the caspases participate in apoptosis occurring under control conditions in the olfactory epithelium. However, although the use of peptidebased caspase inhibitors may be used to implicate caspases in apoptosis, these inhibitors cannot be used to implicate a particular caspase in whole cell models of apoptosis or in animal models of disease, given the current state of the art (23). Whole cell models differ in many significant ways from analytical kinetic studies of the caspases done in cell free systems, in which the role of inhibitors can be directly tested on individual caspases (e.g., 11, 41, 46). In whole cell models, extensive analysis of individual caspase activation must be done, and in those cases in which the substrate is known it must be demonstrated that the activated enzyme cleaves its substrate (cf., 42). In future experiments on TNF-␣induced apoptosis in the olfactory model system we will perform these analyses.

43

CONCLUSIONS

In summary, our data indicate that TNF-␣ is a potent inducer of apoptosis in organotypic cultures of olfactory epithelium and that both the morphological distribution of apoptotic figures and the phagocytosis of apoptotic bodies resemble the in vivo findings. Preincubation of the explants with caspase inhibitors inhibits cell death in these cultures of olfactory epithelium. The organotypic system offers advantages for further testing of the hypotheses developed in this study, because it enables the investigator to monitor the specific cell types involved in the apoptotic response in a tissue while (a) retaining relationships of cells to each other and (b) using doses and exposure times essentially similar to those used in monolayer cultures of various cell types (4, 16, 54, 58, 61, 75). Some questions that can be approached include the following. Which agents other than TNF-␣ and Fas Ligand can induce olfactory neuron apoptosis? Do caspases participate in olfactory neuron apoptosis, and if so, which ones? Is olfactory neuron apoptosis linked to olfactory neurogenesis, and if so, what is the molecular basis of the linkage? Why are olfactory supporting cells so much more resistant to apoptosis than olfactory neurons? REFERENCES 1.

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