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(Journal of Leukocyte Biology. 2002;72:790-799.)
© 2002 by Society for Leukocyte Biology

Dissociated ROS production and ceramide generation in sulfasalazine-induced cell death in Raw 264.7 cells

B. Salh, K. Assi, S. Huang, L. O’Brien, U. Steinbrecher and A. Gómez-Muñoz

The Jack Bell Research Centre, Vancouver, British Columbia, Canada

Correspondence: Dr. B. Salh, Division of Gastroenterology, University of British Columbia, 100-2647 Willow Street, Vancouver, BC, V5Z 3P1, Canada. E-mail: bsalh{at}interchange.ubc.ca


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ABSTRACT
 
Sulfasalazine (SSZ) is a drug used in inflammatory bowel disease, whose precise mechanism of action remains to be clarified. Here, we report that incubation of Raw 264.7 cells with SSZ but not salicylates [acetylsalicylic acid (ASA), 4-aminosalicylic acid (4-ASA), and 5-ASA] causes a mixed apoptotic and necrotic form of cell death. In contrast to its metabolites, sulfapyridine and 5-ASA, SSZ exposure in Raw 264.7 cells resulted in a threefold increase in ceramide generation, as well as a robust production of reactive oxygen species (ROS). However, inhibition of ceramide production by fumonisin B1 failed to attenuate cell death. Preincubation with catalase, cyclosporin A (CsA), and bongkrekic acid attenuated ROS production. When dead cells were quantified for apoptotic versus necrotic cell death, catalase and N-acetylcysteine reproducibly attenuated apoptosis, whereas CsA, in addition to reducing apoptosis, was observed to dramatically enhance necrosis. In conclusion, the cell-death response induced by SSZ in Raw 264.7 cells involves ROS in the apoptotic limb but is independent of ceramide formation.

Key Words: macrophages • apoptosis • necrosis • MAPK • PLD


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INTRODUCTION
 
The pathogenesis of the two common forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis, involves inappropriately sustained activation of intestinal immune responses [1 , 2 ]. Macrophages play a critical role in such inflammatory responses, as they function as antigen presenting cells in addition to producing proinflammatory cytokines, which include tumor necrosis factor {alpha} (TNF-{alpha}), interferon-{gamma}, and interleukin-1ß (IL-1ß). These cytokines, which are present in intestinal lesions of patients with IBD [1 ], may stimulate the hydrolysis of sphingomyelin (a major phospholipid component of cell membranes), resulting in production of ceramide and phosphorylcholine [3 4 5 ]. Ceramides participate in signal transduction by activating specific serine/threonine kinases or by stimulating protein phosphatases that are involved in the regulation of cell differentiation, proliferation, apoptosis, and inflammatory responses [3 , 5 ].

The treatment of IBD includes sulfasalazine (SSZ), a drug that is formed by an azo-linkage of an antibiotic, sulfapyridine (SP), and an anti-inflammatory agent 5-aminosalicylic acid (5-ASA). SSZ has been demonstrated to inhibit the synthesis of IL-2 and lymphocyte proliferation as well as the production of IL-1 by monocytes [6 ]. It has been suggested that the active moiety of SSZ in the treatment of IBD is 5-ASA [7 ]. Consequently, over the last decade, 5-ASA has superceded SSZ as first-line treatment for mild to moderate, active IBD. 5-ASA has a number of inhibitory effects on the inflammatory cascades activated in IBD, including inhibition of cyclooxygenase and lipooxygenase activities, and it blocks the synthesis of platelet-activating factor [1 ]. More recently, 5-ASA has been shown to block TNF-{alpha}-induced growth inhibition and nuclear factor (NF)-{kappa}B activation in mouse colonocytes [8 ]. Another study has demonstrated an inhibition of extracellular release of proinflammatory secretory phospholipase A2 by SSZ [9 ]. Nevertheless, it should be pointed out that not all of the effects of SSZ can be mimicked by 5-ASA, as only the former was able to inhibit NF-{kappa}B activation by phorbol esters, TNF-{alpha}, or lipopolysaccharide (LPS) in the human colonic epithelial cell line SW620 [10 ]. Furthermore, only SSZ and SP, not 5-ASA, were able to inhibit fibroblast growth factor-mediated angiogenesis in human dermal microvascular endothelial cells [11 ].

Here, we show that SSZ causes cell death by a mechanism that involves oxidant stress. However, it is independent of ceramide generation. SSZ can stimulate the generation of phosphatidate (PA) via stimulation of phospholipase D (PLD), a major enzyme involved in the regulation of cell signaling [12 13 14 15 ]. Although ceramides and PA are antagonistic signals [16 17 18 ], we found that they are not generated simultaneously by SSZ and that both processes are independent of each other. Analysis of reactive oxygen species (ROS) production revealed that this was mechanistically dissociated from ceramide generation; however, using inhibitors of ROS production, we were able to uncover a significant role for them in the apoptotic limb of SSZ-induced cell death in Raw 264.7 cells.


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MATERIALS AND METHODS
 
Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were obtained from Life Technologies (Gaithersburg, MD). Fetal bovine serum (FBS) was from Hyclone Laboratories Inc. (Logan, UT). N-acetyl cysteine (NAC), z-DEVD-fmk, 2-LEHD-fmk, and bongkrekic acid were from Calbiochem (San Diego, CA). SP, 5-ASA, SSZ, bovine serum albumin (BSA), desipramine (DES), fumonisin B1 (FB1), phorbol 12-myristate 13-acetate (PMA), Hoechst 33342, propidium iodide (PI), superoxide dismutase (SOD), buthionine sulphoximine (BSO), butylated hydroxyanisole (BHA), catalase, and cyclosporin A (CsA) were from Sigma-Aldrich Canada (Oakville, Ontario). Caspase-3 antibody was from Stressgen Biotechnologies (Victoria, Canada); polyadenosine 5'-diphosphate-ribose polymerase (PARP) was from Oncogene Research Products (Cambridge, MA); dimethyl sulphoxide was from BDH Chemicals (Poole, Dorset, UK); and C2836 was from Molecular Probes (Portland, OR).

Cell culture
Raw 264.7 cells were grown in DMEM containing penicillin and streptomycin supplemented with 10% FBS. Then they were washed three times in DMEM, preincubated for 1 h in DMEM containing 1% FBS with or without inhibitors prior to addition of agonists and then incubated for the appropriate times before being harvested.

Bone marrow cells were isolated from the femurs of 6- to 8-week-old female CD-1 mice as described [19 ]. Cells were plated for 24 h in RPMI 1640 containing 10% FBS and 10% L-cell-conditioned medium, a crude source of macrophage-colony stimulating factor (M-CSF), kindly provided by Dr. Alice Mui (UBC, Vancouver). The nonadherent cells were removed and cultured in the above medium until confluence was reached (5–7 days). Thereafter, the cells were harvested using a Teflon cell lifter and seeded at 1 x 106 cells/well in six-well plates in RPMI 1640 with 10% FBS but without M-CSF for 24 h prior to use to render the cells quiescent. Thereafter, the medium was replaced with RPMI 1640 with or without 10% FBS.

Sample preparation
For caspase-3 immunoblotting, cells were scraped in medium using a rubber scraper, transferred into a 15-ml Falcon tube, and pelleted at 2000 rpm for 5 min, and the supernatant was aspirated and washed three times in ice-cold phosphate-buffered saline (PBS). The pellet was resuspended in homogenization buffer (20 mM Mops, 50 mM ß-glycerophosphate, 5 mM EGTA, 2 mM EDTA, 50 mM NaF, 2 mM sodium vanadate, 0.1% Nonidet P-40, pH 7.2, containing 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 40 µg/ml phenylmethylsulfonyl fluoride) and was sonicated for 20 s. The crude extracts were cleared by centrifugation, and protein determinations were carried out by Bradford’s method. For mitogen-activated protein kinase (MAPK) immunoblotting, the cell lysate was mixed with 5x sample buffer [2.5% sodium dodecyl sulfate (SDS), 1.25 M Tris-HCl, pH 6.8, 5 mM glycerol, 2.5 mM ß-mercaptoethanol, 2 mM 0.1% bromophenol blue] and was boiled for 10 min.

For PARP cleavage, scraped cells were resuspended in buffer (62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 5% ß-mercaptoethanol), sonicated for 15 s, and incubated at 65°C for 15 min.

Western immunoblotting
Aliquots of sample were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane using a Biorad transblot apparatus at 300 mA for 1.5 h. The membrane was blocked with 4% skimmed milk in Tris-buffered saline/Tween 20 (TBST; 20 mM Tris-HCl, pH 7.4, 250 mM NaCl, 0.05% Tween 20) for 1.5 h. The primary antibody was applied for 2 h (dilution according to manufacturer’s recommendations) and washed in TBST, and the secondary antibody was conjugated to horseradish peroxidase, added at a dilution of 1:5000 for 1 h, washed 3x in TBST, and finally washed for 10 min in TBS. The blots were visualized using enhanced chemiluminescence.

Measurement of ceramide production, sphingomyelin levels, and PLD activity
[3H]Ceramides were determined by labeling the cells with 5 µCi [3H]palmitate/ml for 24 h. The radioactive medium was aspirated, and the cells were washed twice with nonradioactive DMEM containing 0.1% BSA. The macrophages were incubated for a further 2.5 h in BSA- or serum-free DMEM. No intermediate washes were carried out along the procedure to prevent the burst of sphingolipids and diacylglycerol, which occurs after changing the medium [17 ]. The cells were washed once with ice-cold, calcium-free PBS and extracted with chloroform/methanol as follows: Cells were scraped into 0.5 ml methanol and further washed with 0.5 ml methanol. The two aliquots were combined and mixed with 1 ml chloroform and 0.9 ml 2 M KCl + 0.2 M H3PO4 to separate phases. Chloroform phases were dried under nitrogen, and ceramides were separated by thin-layer chromatography (TLC) using Silica Gel 60-coated glass plates. TLC plates were developed for 50% of their length with chloroform/methanol/acetic acid (9:1:1, v/v/v) and then dried. The plates were redeveloped for their full length with petroleum ether/diethyether/acetic acid (60:40:1, v/v/v). The identity of the ceramides was confirmed by cochromatography with authentic ceramide standards after staining with iodine vapor. Radioactive ceramides were quantitated after scraping from the TLC plates followed by liquid scintillation counting. The levels of [3H]sphingomyelin were also determined from [3H]palmitate-labeled cells by developing the TLC plates in chloroform/methanol/acetic acid/formic acid/water (35:15:6:2:1 by vol). Radioactive sphingomyelin was quantified by liquid scintillation counting. PLD was measured on the basis of its transphosphatidylation activity, which leads to the production of [3H]phosphatidylethanol when cells containing [3H]phosphatidylcholine are incubated in the presence of ethanol [18 ].

Fluorescein-activated cell sorter (FACS) analysis for PI staining
Cells were seeded in 12-well plates and grown to 80% confluence. After incubation in 1% FBS containing media for 1 h, cells were exposed to SSZ for 6 h. Cells were mechanically lifted off by repeated pipetting, centrifuged, and resuspended in hypotonic lysis buffer, 25 µg/ml RNAse (Qiagen, Toronto, Ontario), 0.1% sodium citrate, 50 µg/ml PI (Sigma-Aldrich Canada), and 0.1% Triton X-100. Fluorescence was measured using a FACS (Epics XL-MCL, Beckman Coulter, Fullerton, CA). At least 104 cellular events were counted on FL3.

ROS determination
This was performed essentially as previously described [20 ] using the C2938 fluorescent probe. Raw 264.7 cells were labeled with 1 µM C2938 (2', 7'-dichlorofluorescein diacetate) for 1 h and then incubated with various concentrations of SSZ. The cells were then washed in PBS three times, lifted by repeated pipetting, and resuspended in PBS. Cell fluorescence was measured using flow cytometry (FL3). Inhibitor experiments using ROS scavengers were performed by preincubating the cells in the respective compound for 1 h prior to the assay.

Simultaneous measurement of apoptosis and necrosis
This technique relies on the principle that apoptotic cells take up the vital dye Hoechst 33342 compared with live cells. PI is added to distinguish from late apoptotic or necrotic cells, which have lost membrane integrity [21 , 22 ]. Cells were incubated for 4 h with SSZ, with or without inhibitors. Dual fluorescence with Hoechst 33342 (1 µg added to 106 cells followed by incubation for 7 min at 37°C, FL1) and PI (1 µg added and incubated on ice for 10 min, FL3) was measured. The live cells were gated out, and the relative proportions of apoptotic and necrotic cells were estimated by flow cytometry.


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RESULTS
 
Induction of cell death by SSZ in murine macrophages
We initially observed that when macrophages were exposed to SSZ overnight, they underwent cell death. This was seen in primary murine peritoneal macrophages (data not shown) and in Raw 264.7 cells (see Fig. 1A and 1B ). Although some of the cells are contracted and show membrane blebbing (arrowhead, Fig. 1B ), it is clear that others are obviously undergoing necrosis, as shown by the cellular swelling and intracellular vacuolization (arrow, Fig. 1B ). Apoptosis is occurring, as the representative Western immunoblot (Fig. 1C) demonstrates PARP cleavage, indicating a caspase-dependent mechanism. FACS analysis for subdiploid DNA (Fig. 1D) indicates that apoptosis is a prominent mechanism with over 50% of the cell population exhibiting this parameter after 24 h exposure to SSZ.



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  		Figure 1. Induction of apoptosis by SSZ in Raw 264.7 cells. (A, B) Morphological changes with or without exposure to 10 mM of SSZ. Visible changes indicative of membrane disruption (arrow) or membrane blebbing (arrowhead) appear as early as 1 h after treatment with SSZ. (C) Western blot analysis of PARP cleavage in Raw 264.7 cells following treatment with 10 mM SSZ for 6 h. Samples were harvested and subjected to immunoblotting as described in Materials and Methods. (D) Increased subdiploid population following exposure of Raw 264.7 cells to SSZ. Cells were seeded in 12-well plates, were grown to 80% confluence, and then were exposed to SSZ for 6 h. Cells were mechanically lifted off and resuspended in hypotonic lysis buffer as described in Materials and Methods. Fluorescence was measured using a FACS (Epics XL-MCL, Beckman Coulter). At least 104 cellular events were counted on FL3.

Collectively, these results indicate that SSZ is capable of inducing a mixed form of apoptotic and necrotic cell death in Raw 264.7 cells and that this response is characterized by PARP cleavage, DNA fragmentation, as well as loss of membrane integrity.

Temporal and concentration characteristics of SSZ-induced apoptosis
The time course of caspase-3/CPP32 cleavage was next investigated using 10 mM SSZ. The data in Figure 2A and 2B show that there is a reduction in the intensity of the CPP32 band after 4 h, which coincides with the appearance of the cleaved product of PARP. Using the disappearance of the CPP32 signal as a surrogate marker of apoptosis, we explored the kinetics of this response with a range of concentrations and the cells harvested after 12, 24, and 48 h of exposure to SSZ. The data indicate that concentrations of SSZ as low as 0.5 mM were able to activate the caspase system. In this case, there is a significant reduction in the intensity of the CPP32 band at 48 h (Fig. 2C) . This observation indicates that the apoptosis-inducing property of SSZ may well be a relevant phenomenon in vivo.



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  		Figure 2. (A, B) Time course and concentration curve of SSZ-induced apoptosis. Raw 264.7 cells were exposed to SSZ (10 mM) for up to 6 h, and samples were harvested and subjected to immunoblotting for PARP and CPP32 as for Figure 1A . Notable loss of the intensity of the CPP32 band occurs between 3 and 4 h. (B) PARP cleavage is detectable as early as 4 h, which temporally coincides with the loss of CPP32 signal. (C) Raw 264.7 cells were cultured in six-well plates with the indicated concentrations of SSZ, and the cells were harvested at the times indicated. The results show apoptosis occurring at doses as low as 0.5 mM after a 48 h exposure.

Generation of ceramides by SSZ in Raw 264.7 cells
Several reports have shown that caspase-3 activation can be mediated by ceramides, which are proapoptotic in a variety of cell types [23 24 25 ]. Therefore, we investigated the generation of ceramides in response to exposure to SSZ. Strikingly, in addition to stimulating caspase-3, SSZ also induced the generation of ceramides in a manner that was concentration- and time-dependent (Fig. 3A and 3B ). Maximal ceramide generation occurred using 5 mM SSZ. Above 10 mM SSZ, there was actually a slight reduction in ceramide generation. Using 5 mM SSZ, it can be seen (Fig. 3B) that ceramide formation can be detected after 1 h of incubation with the drug, reaching maximal accumulation of ceramides by about 8–12 h. Furthermore, only SSZ (Fig. 3C) and neither of its constituent parts 5-ASA or SP, alone or in combination, leads to any significant increase in ceramide generation.



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  		Figure 3. Ceramide generation by SSZ. (A) Concentration curve showing that 5 mM SSZ or above is required for maximum generation of ceramide. Raw 264.7 cells were grown to a confluency of 60–70% and were labeled with [3H]palmitic acid (5 µCi/ml) for 24 h in DMEM containing 0.1% BSA. The cells were washed three times with this same medium and incubated further for 2.5 h in serum- or BSA-free DMEM. Macrophages were then treated with 0–10 mM SSZ for 8 h. [3H]Ceramides were analyzed by TLC using authentic standards, as indicated in Materials and Methods. The results were calculated as the percentage of the total lipid radioactivity that was present in ceramide and are expressed as the fold-increase relative to control incubations in the absence of SSZ. The results are the means ± SEM of three independent experiments performed in duplicate. (B) Time course of ceramide formation using 5 mM SSZ. Raw 264.7 cells were labeled as in (A) and exposed to SSZ for various times as indicated, without changing the medium. Results are the means ± range of two independent experiments, except for the time point at 8 h, which is the mean ± SEM of four experiments performed in duplicate. (C) Ceramide generation after exposure of Raw 264.7 cells to SSZ, 5-ASA, and SP. Using 5 mM treatment of each of these drugs, SSZ causes an increase of approximately threefold in ceramide generation compared with each of the SSZ metabolites or their combination. Results are calculated and expressed as in (A), and they are the means ± range of two independent experiments performed in duplicate.

Collectively, these data unequivocally indicate that ceramide generation occurs prior to the activation of caspase-3 and depend on SSZ and not on either of its constituents.

SSZ induces ceramide synthesis via the de novo pathway
Ceramides can be produced through the stimulation of sphingomyelinase (Smase) activity or by de novo synthesis [4 , 15 , 16 ]. To investigate these pathways in SSZ-induced apoptosis, the levels of sphingomyelin (SM) were measured at 2 or 8 h with increasing concentrations of SSZ (0.5–10 mM). Under these conditions, the levels of SM were unchanged, suggesting that ceramide formation by SSZ was independent of SMase activation. In addition, pretreatment of macrophages with the SMase inhibitor DES (5–25 µM), prior to stimulation of the macrophages with SSZ, did not cause any significant change in ceramide levels (data not shown). Finally, the activities of the neutral and acidic SMases were determined. We found that more than 98% of the sphingomyelinase activity in Raw 264.7 cells corresponded to the acidic form of the enzyme. Acidic sphingomyelinase activity was 91 ± 11 nmoles/h/mg protein (mean±range of two independent experiments performed in triplicate), and this was not significantly altered by incubation of the macrophages with 5 mM SSZ for 4 h. To investigate whether the production of ceramides by SSZ was caused by de novo synthesis, macrophages were preincubated with FB1, which is a selective inhibitor of ceramide synthase [26 , 27 ]. Figure 4A shows that ceramide production was completely inhibited by 75 µM FB1, which suggests that ceramide formation by SSZ occurs through activation of the de novo pathway.



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  		Figure 4. (A) Inhibition of de novo biosynthesis of ceramides. Raw 264.7 cells preincubated for 1 h with varying concentrations of FB1 were exposed to 5 mM SSZ for 2 h, as indicated. [3H]Ceramides were analyzed by TLC using authentic standards, as indicated in Materials and Methods. The results were calculated and expressed as in Figure 3 , and they are the means ± range of two independent experiments. (B) Effect of FB1 on SSZ-induced CPP32 activation. Raw 264.7 cells were preincubated in DMEM containing 1% FBS with or without 75 µM FB1 prior to addition of 2.5 mM SSZ and were incubated for 24 h. Western immunoblot showing the intensity of the CPP32 protein is shown. (C) FB1 (also FUM B1) does not influence apoptotic or necrotic SSZ-induced cell death. Raw 264.7 cells were preincubated in FB1 and then exposed to SSZ. Relative proportions of apoptotic (solid bars) and necrotic (shaded bars) cells were determined by flow cytometry as described in Materials and Methods.

The next objective was to determine whether ceramides were involved in the induction of apoptosis by SSZ. Therefore, we investigated whether FB1 was capable of blocking SSZ-induced caspase-3 activation. Preincubation of macrophages with this inhibitor did not alter SSZ-induced caspase-3 activation (Fig. 4B) , indicating that the production of ceramide and caspase-3 activation (and thus apoptosis) by SSZ were dissociated events. Examination of cell morphology confirmed that there was no reversal of cell death. However, to establish that FB1 did not influence cell death, we simultaneously measured apoptotic and necrotic fractions of macrophages sequentially exposed to FB1 and SSZ [21 , 22 ]. The data indeed confirm a lack of a role for FB1 in SSZ-induced cell death (Fig. 4C) . These observations are in agreement with those of other workers [28 ] who showed that Fas or TNF-{alpha}-induced apoptosis was mediated by caspase-3, independently of ceramide signaling.

We have recently shown that 5-ASA and SSZ can activate PLD in primary murine macrophages [29 ]. A similar effect could be detected as early as 5 min in Raw 264.7 cells, which peaked by 15–30 min (data not shown). As SSZ induced ceramide formation, the activation of PLD by this drug was unexpected, as PA, the product of PLD activity, and ceramides are antagonistic signals [30 ], and ceramides block PLD activation [13 , 14 ]. However, the generation of ceramide by SSZ followed a different kinetic profile, and PLD was activated well before ceramide accumulation or caspase-3 activation. Although in a number of instances PLD has been associated with the induction of cell proliferation [15 , 31 ], there is also evidence for an involvement of PLD in the regulation of apoptosis. In this regard, Kasai et al. [32 ] showed that the activity of oleate-dependent PLD was increased during actinomycin D-induced apoptosis in Jurkat T cells, and Gilbert et al. [33 ] reported that phosphatidylcholine-PLD mediates antiproliferative signals in murine B lymphocytes. These observations led us to investigate whether PLD activation by SSZ was associated with caspase-3 activation. Lacking specific inhibitors of PLD, this assumption was tested by treating the macrophages with prolonged incubation (20 h) with 100 nM PMA to down-regulate protein kinase C (PKC), as this enzyme activity is involved in the regulation of PLD [34 ]. This experimental condition prevented the stimulation of PLD by SSZ. The fold-increase in PLD activity was decreased from 3.3 ± 0.3 to 1.1 ± 0.2 (mean±range of two independent experiments performed in duplicate). However, down-regulation of PKC did not alter caspase-3 activation (data not shown). These observations suggest that activation of PLD and caspase-3 are independent and unrelated events. SSZ-induced caspase-3 stimulation was also independent of Ca2+-dependent or Ca2+-independent cytosolic phospholipase A2 activation, as preincubation of macrophages with the specific inhibitors arachidonoyl trifluoromethyl ketone or palmitoyl trifluoromethyl ketone, respectively, failed to alter caspase-3 activation (data not shown).

Based on the ability of protein phosphorylation events to influence apoptosis [35 ], we investigated whether activation of any of the MAPKs was associated with the induction of apoptosis. MAPK activation was assessed by comparing the activation induced by different doses of SSZ with that induced by LPS. It was found that SSZ does not activate any of these kinases (data not shown), thereby excluding a direct role for them in the cell-death response induced by SSZ.

Involvement of ROS in SSZ-induced cell death
Several observations support the formation of ROS as being integrally involved in apoptosis [36 , 37 ]. To test this possibility in Raw 264.7 cells, macrophages were exposed to 2.5 mM SSZ, 5-ASA, and SP (alone or in combination) after loading the cells with C2938 and were then analyzed by flow cytometry. The data indicate that only SSZ is able to affect the generation of ROS (Fig. 5A and 5B ). Using this concentration, the time course characteristics were determined, and it is apparent that this effect occurs within 1 h and plateaus after 6 h (Fig. 5C) .



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  		Figure 5. (A) SSZ causes increased ROS generation in Raw 264.7 cells. Cells were labeled with 1 µM C2938 (2', 7'-dichlorofluorescein diacetate; DCF–DA) for 1 h and were then incubated with 5 mM SSZ, 5-ASA, SP, or the latter two in combination for 4 h. The cells were then washed in PBS three times, lifted by repeated pipetting, and resuspended in PBS. Cell fluorescence was measured using flow cytometry (FL3). (B) Representative histogram of data shown in (A). (C) Time-course of ROS generation by SSZ. Cells were exposed to SSZ for the indicated times, and ROS was determined as described above.

The next objective was to determine the origin of the ROS. It is known that there are several sources of intracellular ROS, the most prominent sites being the mitochondria and the endoplasmic reticulum. Other sources include the cyclooxygenase, lipooxygenase, and hypoxanthine/xanthine oxidase systems. Therefore, cells were pretreated with ROS scavengers, acting at enzymatic level (e.g., catalase and SOD) and post-generation (e.g., BHA), an agent depleting glutathione, BSO, and two agents that are known to inhibit the mitochondrial permeability transition in mitochondria, namely CsA and bongkrekic acid [38 ]. Additionally, as we had evidence of caspase activation, inhibitors of nonspecific/broad-based (z-DEVD-fmk) as well as a specific inhibitor of caspase-9 (LEHD-CHO) were included in the analysis. The data (Fig. 6 ) indicate that the most effective inhibitors within this system are catalase, CsA, and bongkrekic acid. Less dramatic inhibition was observed with BSO, BHA, NAC, and SOD. In contrast, no change was seen with either of the caspase inhibitors, indicating that although caspase activation occurred within this model system, it was not upstream of ROS generation. By using FB1, we were able to rule-out ceramide generation upstream of ROS production. Furthermore, using catalase, we were also able to rule-out the converse (i.e., ROS generation upstream of ceramide generation; data not shown). This experiment confirmed that the most likely source of ROS in Raw 264.7 cells exposed to SSZ was the mitochondria.



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  		Figure 6. Attenuation of SSZ-induced ROS production by catalase, bongkrekic acid, CsA, and free radical scavengers. Cells were prepared as described in the legend to Figure 7 and incubated in the presence of the indicated inhibitor for 1 h prior to exposure to SSZ for 4 h. ROS production was determined as described above.

Inhibition of ROS generation reduces the apoptotic component of SSZ-induced cell death
Having found that ROS generation could be modulated by using specific inhibitors working at the level of mitochondrial permeability transition, we investigated whether we could attenuate cell death using these agents and compare them with caspase inhibitors and the other ROS antagonists. Using flow cytometry to examine the subdiploid population at 24 h post-incubation, we were initially unable to detect any difference in the magnitude of cell death (data not shown). Subsequently, we modified the procedure to facilitate distinction between cells undergoing apoptosis or necrosis in the early stages, as previously described [22 , 23 ]. This was obviously important, as our morphological findings indicated that SSZ induced a mixed form of cell death. The data (Fig. 7A ) shows that we were indeed able to reverse apoptosis using catalase, NAC, and CsA. Surprisingly, we observed a dramatic increase in the proportion of CsA-treated cells that underwent necrosis. Again, we were unable to uncover a significant role for caspase-3 or -9 in this process.



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  		Figure 7. Attenuation of ROS production decreases apoptotic cell-death fraction in response to SSZ. Raw 264.7 cells (A) or BMDMs (B) were incubated for 4 h with SSZ, with or without inhibitors. Dual fluorescence with Hoechst 33342 (1 µg added to 106 cells followed by incubation for 7 min at 37°C, FL1) and PI (1 µg added and incubated on ice for 10 min, FL3) was measured. The live cells were gated out, and the relative proportions of apoptotic (solid bars) and necrotic (open bars) cells were estimated by flow cytometry. (*, P<0.05 using a two-tailed Student’s t-test.) The experiment was performed on three separate occasions in the Raw 264.7 cells and confirmed on two occasions in the primary BMDMs.

As most of the work described in this report was carried out in a cell line, it was elected to determine whether the latter effects could be duplicated in primary macrophages. We obtained bone marrow-derived macrophages (BMDMs) as previously described [19 ] and initially found that they were more sensitive to SSZ with similar effects occurring at 2.5 mM. When we preincubated these cells with the ROS inhibitors and then exposed them to SSZ and performed flow cytometry, it was apparent that CsA, NAC, and catalase were capable of attenating the apoptotic limb of cell death (Fig. 7B) . There was a reduction from 7.3% apoptosis down to 2.3%, 3.2%, and 4.1% for the three agents, respectively. This confirmed our findings in the Raw 264.7 cells.


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DISCUSSION
 
In this work, we have demonstrated for the first time that SSZ-induced apoptosis in Raw 264.7 murine macrophages is preceded by the formation of ceramides, which were generated by de novo synthesis. Additionally, we have observed that SSZ-induced apoptosis involves mitochondrial generation of ROS. However, ceramides were not mechanistically involved in SSZ-induced apoptosis in this system. Furthermore, we have demonstrated a role for ROS in the apoptotic component of the cell death induced by SSZ in Raw 264.7 cells and primary bone marrow-derived macrophages. Finally, we have shown that there is no accompanying activation of any members of the MAPK family.

Several recent publications have addressed the effects of SSZ on leukocyte function and/or viability [39 40 41 ]. Akahoshi et al. [39 ] reported that at concentrations of 100 µM SSZ, apoptosis was readily induced in neutrophils, which underwent extensive apoptosis spontaneously over the study period (24 h). The effect could be shown to involve the generation of ROS, and in addition, apoptosis could be attenuated with inhibitors of protein kinase A and tyrosine kinases. No effect was seen with inhibition of PKC in accordance with our findings. Subsequently, it was shown by Liptay et al. [40 ] that SSZ inhibited NF-{kappa}B and induced apoptosis in T-lymphocytes. A 50% apoptotic effect was observed at concentrations of 2.5 mM SSZ within 4 h, with an ED50 for 24 h of 0.625 mM. It should be noted that this concentration (0.625 mM) is not too dissimilar from our findings of significant CPP32 activation at 24 h with 1 mM SSZ (see Fig. 2 ). A definitive role for caspases in the apoptotic response induced by SSZ was first documented by Rodenburg et al. [41 ] while investigating the expression of TNF-{alpha} by LPS in macrophages. As in our study, apoptosis occurred after 4–6 h, depending on the cell line used. A specific effect was seen using inhibitors of caspase-3 and -8 but not for inhibitors of caspase-1 and -9. The mechanism involved in the apoptotic response was not specifically addressed in this work. The minor disparity between their and our observations may be a result of differences between the cell lines used. Collectively, all of these studies indicate that the SSZ-associated effects reported probably occur in vivo, as concentrations of SSZ in the intestine may reach millimolar levels [42 ].

As no effect was seen for the component parts of SSZ, it is likely that other pathways are involved in mediating the anti-inflammatory actions of 5-ASA. There appears to be some controversy as to whether 5-ASA targets activation of NF-{kappa}B. One report indicated an inhibitory effect upon the phosphorylation of p65 [43 ], and others have implicated an effect on I kappa B kinase (IKK) [8 , 44 ]. In contrast to this, Weber et al. [45 ] were unable to show any effect on NF-{kappa}B activation, using up to 5 mM 5-ASA in their studies.

One of the most frequently cited reports linking drug-induced cell death to ceramide generation comes from the observations of Bose et al. [46 ]. The latter authors demonstrated that daunorubicin-induced apoptosis is dependent on an increase in ceramide synthesis through the de novo pathway using FB1, a selective inhibitor of dihydroceramide synthase, to attenuate this response. By investigating etoposide-induced apoptosis in Molt-4 human leukemia cells, Perry et al. [47 ] have observed that the rate-limiting step for ceramide generation was not the dihydroceramide synthase but serine palmitoyltransferase. Furthermore, it was demonstrated that ceramide generated through the de novo pathway contributed to membrane disruption but was not involved in activation of caspases leading to PARP cleavage. These observations indicate that standard chemotherapeutics may impact differently on these lipid messengers and that much still remains to be learned about the precise role of ceramide generation under these circumstances.

Another possible means by which SSZ could be inducing apoptosis is through competition for adenosine 5'-triphosphate (ATP) binding. In this regard, Weber et al. [45 ] could counteract the effects of SSZ on the activities of IKK{alpha} and IKKß, simply by increasing the concentration of ATP in the assay mixture. Furthermore, Cronstein et al. [48 ] have reported that SSZ (like salicylates) mediates its anti-inflammatory activity through the release of adenosine, consequent on uncoupling oxidative phosphorylation, thus leading to a depletion of intracellular ATP.

Our findings are clearly in accord with others who have shown that SSZ-induced apoptosis in neutrophils could be attenuated by NAC [39 ]. However, to our knowledge, this issue has not been addressed in macrophages. Thus, we have significantly extended the findings of Rodenberg et al. [41 ] by demonstrating that ROS production and ceramide generation via the de novo pathway accompany SSZ-induced cell death. There have been interesting developments in other systems that have examined this phenomenon. NAC has been shown to inhibit ceramide formation and cell death in TNF-{alpha}-treated rat primary astrocytes [49 ] as well as daunorubicin-treated human leukemic cells [50 ]. Similarly, an association has been uncovered in response to various physical treatments such as UV or ionizing radiation, and photodynamic therapy [51 52 53 ]. A direct link between ceramide generation and apoptosis has been demonstrated by the ability of ceramide to induce release of cytochrome C from mitochondria [54 ]. Furthermore, bacterial SMase was able to induce ROS formation and caspase-3 activation in cultured rat hepatocytes [55 ]. Although a proapoptotic effect has been suggested by most studies, other work may have direct relevance to our findings in that ceramide has been shown to increase the activity of Mn-SOD in astrocytes and neurons [56 , 57 ].

In conclusion, we have demonstrated in this work that SSZ induces cell death as a result of apoptosis and necrosis in Raw 264.7 cells and BMDMs. This event is associated with caspase-3 activation and PARP cleavage. SSZ stimulated ceramide formation through the de novo pathway and PLD activity, but neither of these effects was associated with the induction of cell death. In addition, SSZ did not cause any significant changes on either of the stress-activated protein kinases (SAPKs) (p54/45 JNK or p38 HOG). Therefore, the apoptotic component of the cell-death response induced by SSZ involves a ROS-dependent mechanism that is dissociated from ceramide formation or PLD activation.


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ACKNOWLEDGEMENTS
 
This study was supported by in part by grants from Crohn’s and Colitis Foundation of Canada to B. S., the Northwestern Society for Intestinal Research to A. G-M. and B. S., and in part by funds from the Geraldine Dow Foundation. B. S. and A. G-M. contributed equally to this manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We are grateful to Dr. J. Kong for her invaluable assistance with some of the FACS analyses.


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FOOTNOTES
 
Current address of A. Gómez-Muñoz: Department of Biochemistry and Molecular Biology, Faculty of Science, University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain. Back

Received January 22, 2002; revised June 7, 2002; accepted June 27, 2002.


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