Introduction

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Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant, which is an active anti-inflammatory component of propolis (honeybee resin) (Mirzoeva and Calder, 1996; Michaulart et al., 1999). Various investigators have demonstrated that CAPE has anti-inflammatory properties both in vitro and in vivo (Huang et al., 1996; Mirzoeva and Calder, 1996; Michaulart et al., 1999; Orban et al., 2000). CAPE was also proposed to be a specific inhibitor of the transcription factor nuclear factor-B (NF-B), which may account for its anti-inflammatory actions (Natarajan et al., 1996). Specifically, in a human histiocytic cell line, CAPE only inhibited NF-B, as opposed to other transcription factors such as AP1, TFIID, and Oct-1. In this regard, it was proposed that CAPE directly inhibited the interaction of NF-B with DNA (Natarajan et al., 1996). CAPE also clearly induces apoptosis in various cell types (Chiao et al., 1995; Orban et al., 2000; Chen et al., 2001).

An initial goal of our study was to assess the anti-inflammatory and apoptosis-inducing effects of CAPE in macrophage and colonic epithelial cell lines. We utilized these cells because they clearly have relevance to the pathogenesis of inflammatory bowel disease (IBD) (Rogler et al., 1998). In this regard, we were specifically interested in evaluating the effects of CAPE on the activation of NF-B in these cell types. Although CAPE has demonstrated efficacy in various animal models of inflammation, it has apparently not been tested previously in an animal model of IBD. Therefore, another important goal of this study was to evaluate the efficacy of CAPE in a relevant animal model of human Crohn's Disease [bacterial peptidoglycan polysaccharide (PG-PS)-induced colitis in Lewis rats] (Yamada et al., 1993; Sartor et al., 1996).

	Materials and Methods

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A rat macrophage cell line (NR8383) and a human colonic epithelial cell line (SW620) were obtained from the American Type Culture Collection (Manassas, VA). NR8383 cells were grown in F-12K culture medium, whereas SW620 cells were grown in RPMI-1640 culture medium. PG-PS (product 10S from Streptococcus group A bacteria) was purchased from Lee Laboratories (Grayson, GA). CAPE was obtained from Sigma Chemical Co. (St. Louis, MO). Female Lewis rats (weighing 125-150 g) were purchased from Charles River Laboratories (Southbridge, MA). Animals were housed one to a cage. In the animal housing area, rats were maintained on a 12-h light/dark cycle. All animals were provided with a standard rodent diet and water ad libitum. Rats were acclimated to these environmental conditions for at least 5 days prior to their use in colitis studies.

Evaluation of NF-B Binding from Cell Lines. NF-B was detected in nuclear protein extracts by an electrophoretic mobility shift assay (EMSA). The EMSA was performed using a specific ³²P-oligonucleotide sequence for NF-B, which was obtained from Promega (Madison, WI). This EMSA technique was described previously (Fitzpatrick et al., 2000).

Evaluation of Cytokine Production from Cell Lines. NR8383 cells (0.5 million/ml) were exposed to various concentrations of CAPE (3-30 µg/ml) or vehicle (0.2% DMSO) for 1 h. PG-PS (100 µg/ml) was then added to stimulate TNF- production. TNF- was measured 6 h after PG-PS stimulation with an ELISA kit (Biosource, Camarillo, CA). SW620 cells (1 million/ml) were exposed to CAPE (3-30 µg/ml) or vehicle (0.2% DMSO) for 1 h. TNF- (20 ng/ml) was then used to stimulate IL-8 production. IL-8 was measured by ELISA (Biosource) 24 h after TNF- stimulation (Fitzpatrick et al., 2000).

Evaluation of Apoptosis. For the apoptosis assay, NR8383 cells (100,000/ml), or SW-620 cells (150,000/ml) were exposed to CAPE (3-30 µg/ml) or vehicle (0.2% DMSO) for 24 h. The Cell Death Detection Elisa Plus assay (Roche Molecular Biochemicals, Mannheim, Germany) was used to measure DNA fragmentation. Data are expressed as the fold change relative to parallel vehicle-treated cells.

PG-PS-Induced Colitis in Rats. Female Lewis rats (fasted for 24 h) were anesthetized with a Ketamine:Rompun (xylazine) combination. The descending colon was exposed using aseptic techniques. Subsequently, nine intramural (subserosal) injections of PG-PS were made into a 5-cm segment of distal colon. Some animals received saline injections as a study control. After the injections were made, the colon was placed back into the abdominal cavity, and the muscle wall was closed using sterile, absorbable 3-0 black silk suture. Next, sterile surgical staples were used to close the skin incision. Some rats were euthanized on day 14 (using CO₂ euthanasia) to obtain baseline measurements. Other groups of rats (n = 6-13 per group) were treated by i.p. injection with vehicle (polyethylene glycol 400/saline, 1:2) or CAPE (30 mg/kg) for a 7-day period, starting on day 14. Injections were given once daily at 8 AM. The dose volume was 4 ml/kg. Animals were euthanized on day 21. Various indices were used to assess the degree of colitis at this time point (Fitzpatrick et al., 2000). This protocol was reviewed/approved by the Otsuka Maryland Research Institute Animal Care and Use Committee.

Evaluation of PG-PS-Induced Colitis. During the 7-day study period, the body weight was measured on a daily basis. A gross colonic injury score (0-5 scale) was calculated according to the criteria set forth previously (Yamada et al., 1993). The slightly modified scoring system is: 0, normal appearance; 1, serosal adhesions to other organs or mesentery, with no mucosal injury; 2, serosal nodules, with no mucosal injury; 3, hyperemic mucosal lesions/inflammation; 4, single mucosal erosion and/or ulcer; 5, multiple mucosal erosions and/or ulcers. This macroscopic evaluation was done with the aid of 2-fold magnification. All of these measurements were done on a blinded basis (i.e., the evaluator was unaware of the prior treatment). Utilizing half of the 5-cm segment of colon, mucosal myeloperoxidase activity was determined by the tetramethylbenzidine method (Fitzpatrick et al., 2000). Colonic mucosal cytokines (IL-1 and TNF-) were also measured by commercially available ELISA kits (Fitzpatrick et al., 2000).

Evaluation of Associated Arthritis. Any associated arthritis was evaluated by measuring the rear ankle joint diameters of Lewis rats with an electronic calipers. Since arthritis could be unilateral or bilateral in nature, measurements were made on both ankle joints. The values obtained from both joints were averaged, and expressed as the rear ankle joint diameter (in mm).

Statistical Analysis. Data are represented as the mean ± standard error. IC₅₀ values (in vitro studies) were calculated via a Graph-Pad Prism software package (i.e., by regression analysis). Statistical analyses between or among treatment groups were done in conjunction with a Sigma-Stat statistical software program. Drug (CAPE) treatment data was usually compared with data from a parallel vehicle-treated condition, using a two-tailed unpaired t test analysis. If a normal data distribution was not present, the Mann-Whitney test was used. A difference of p < 0.05 was considered significant for all statistical analyses.

	Results

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CAPE Inhibits NF-B in Two Cell Lines. Supershift experiments (data not shown), which were conducted in both cell lines, demonstrated that the NF-B DNA binding complex consisted of p50 and p65 subunits. Similar results have been reported previously by several other investigators (e.g., Wahl et al., 1998). As shown in Fig. 1A, CAPE dose-dependently inhibited the PG-PS-stimulated nuclear binding of NF-B in a rat macrophage (NR8383) cell line. The mean percent inhibitions obtained with CAPE treatment were: 31.2 ± 6.8 (3 µg/ml), 41.5 ± 14.0 (10 µg/ml), and 62.3 ± 11.0 (30 µg/ml). All these mean reductions in the nuclear binding of NF-B were statistically significant (p < 0.05 versus vehicle treatment). The calculated IC₅₀ value (mean of two representative studies) was 14 µg/ml. CAPE also inhibited NF-B in the colonic epithelial (SW-620) cell line (Fig. 1B). The mean percentage inhibitions obtained with CAPE treatment were: 40.5 ± 1.3 (3 µg/ml), 43.7 ± 1.2 (10 µg/ml), and 72.3 ± 7.8 (30 µg/ml). All these mean reductions in the nuclear binding of NF-B were also statistically significant (p < 0.05 versus vehicle treatment). The calculated IC₅₀ value was 8 µg/ml. A larger concentration of CAPE (100 µg/ml) was clearly toxic (i.e., by trypan blue exclusion) in both cell types.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   A, CAPE inhibits the nuclear binding of NF-B in the rat macrophage cell line. Representative EMSA results showing NF-B binding activity under various conditions. Lanes 1 and 2, CAPE (3 µg/ml) + PG-PS; lanes 3 and 4, CAPE (10 µg/ml) + PG-PS; lanes 5 and 6, CAPE (30 µg/ml) + PG-PS; lanes 7 and 8, vehicle + PG-PS; and lane 9, vehicle + no PG-PS. Based on a relative densitometry analysis, IC50 of CAPE for inhibiting the nuclear binding of NF-B was calculated to be 14 µg/ml. B, CAPE inhibits the nuclear binding of NF-B in the colonic epithelial cell line. Representative EMSA results showing NF-B binding activity under various conditions. Lanes 1 and 2, CAPE (3 µg/ml) + TNF- (20 ng/ml); lanes 3 and 4, CAPE (10 µg/ml) + TNF-; lanes 5 and 6, CAPE (30 µg/ml) + TNF-; lanes 7 and 8, vehicle + TNF-; and lane 9, vehicle + no TNF- (background level in this picture). Based on a relative densitometry analysis, IC50 of CAPE for inhibiting the nuclear binding of NF-B was calculated to be 8 µg/ml.

CAPE Inhibits Cytokine Production in Two Cell Lines. CAPE also inhibited PG-PS-stimulated TNF- production from the rat macrophage cell line. However, a strict dose-related effect was not observed. Moreover, all utilized concentrations of CAPE inhibited TNF- production by >50%. At CAPE concentrations of 10 µg/ml, TNF- was inhibited by 79% (Fig. 2A). CAPE (30 µg/ml) also inhibited TNF--stimulated IL-8 production (by 60%) in the SW-620 colonic epithelial cell line (Fig. 2B). However, lower concentrations of CAPE (3 or 10 µg/ml) were ineffective in this cell line.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A, CAPE inhibits PG-PS (100 µg/ml)-stimulated TNF- production from a rat macrophage (NR8383) cell line. Cells were exposed to CAPE (3-30 µg/ml) or vehicle for 1 h prior to PG-PS stimulation. TNF- in the supernatant was measured 6 h after PG-PS exposure. n = 2 to 4 per group. , p < 0.05 versus no PG-PS; or +, p < 0.05 versus vehicle/PG-PS. B, CAPE inhibits TNF- (20 ng/ml)-stimulated IL-8 production from a colonic epithelial (SW620) cell line. Cells were exposed to CAPE (3-30 µg/ml) or vehicle for 1 h prior to TNF- stimulation. IL-8 in the supernatant was measured 24 h after TNF- exposure. n = 2 (no TNF) or 3 other treatment groups. , p < 0.05 versus no TNF-; or +, p < 0.05 versus vehicle/TNF-.

CAPE Induces Apoptosis in Two Cell Lines. As illustrated in Fig. 3, CAPE induced apoptosis (increased DNA fragmentation) in both macrophages and colonic epithelial cells. However, CAPE appeared to be a more effective inducer of apoptosis in the rat macrophage cell line.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A, CAPE induces apoptosis (DNA-fragmentation) in a rat macrophage (NR8383) cell line. Cells were exposed to culture medium (no treatment), vehicle (0.2% DMSO), or CAPE (3-30 µg/ml) for 24 h. DNA fragmentation was evaluated by ELISA. n = 8 to 9 per treatment group. , p < 0.05 versus vehicle treatment. B, CAPE induces apoptosis (DNA fragmentation) in a colonic epithelial (SW620) cell line. Cells were exposed to culture medium (no treatment), vehicle (0.2% DMSO), or CAPE (3-30 µg/ml) for 24 h. DNA fragmentation was evaluated by ELISA. n = 7 to 8 per treatment group. , p < 0.05 versus vehicle treatment.

CAPE Attenuates Colonic NF-B and PG-PS-Induced Colitis in Rats. Treatment of Lewis rats with CAPE for a 1-week period resulted in a significant reduction in the gross colonic injury of Lewis rats (Fig. 4). Specifically, on day 21 after the induction of PG-PS-induced colitis, the gross colonic injury scores were 0.5 ± 0.3 (vehicle/saline), 4.7 ± 0.2 (vehicle/PG-PS), and 3.5 ± 0.4 (CAPE/PG-PS). CAPE treatment resulted in a reduction of gross colonic injury to approximately 50% of the level found in PG-PS-treated rats on day 14 (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   CAPE treatment, for a 1-week period, reduced the gross colonic injury (adhesions, nodules, and mucosal damage) associated with the chronic reactivation phase (days 14-21) of PG-PS-induced colitis in rats. The gross colonic injury score was calculated as described under Materials and Methods. n = 5 to 12 per time point. , p < 0.05 versus vehicle + saline treatment; and +, p < 0.05 versus vehicle + PG-PS treatment.

View this table: [in this window] [in a new window]   TABLE 1 Effect of CAPE (30 mg/kg) on PG-PS-induced colitis in rats (day 21)data summary

View larger version (20K): [in this window] [in a new window]   Fig. 5.   There is a significant association between the colonic levels of NF-B and gross colonic injury (PG-PS colitis model). NF-B was determined by EMSA and relative densitometry analysis. (n = 29 rats, r = 0.574, p < 0.01 by linear regression analysis).

CAPE Slightly Reduces the Arthritis Associated with PG-PS-Induced Colitis in Rats. As indicated in Table 1, CAPE treatment resulted in a slight reduction in the arthritis associated with PG-PS administration to Lewis rats. Specifically, the mean ankle joint diameters (in mm) on day 21 were 5.44 ± 0.04 (vehicle/saline), 7.28 ± 0.39 (vehicle/PG-PS), and 6.85 ± 0.29 (CAPE/PG-PS).

	Discussion

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Our study demonstrates that CAPE is an effective in vitro inhibitor of NF-B and relevant cytokines in two different cell lines. Similar to our previous results with gliotoxin (another NF-B inhibitor), we found that CAPE was also an effective inhibitor of NF-B and cytokines in both our rat macrophage and colonic epithelial cell lines (Fitzpatrick et al., 2000). Interestingly, CAPE appeared to be a more effective inducer of apoptosis in macrophages, as opposed to colonic epithelial cells. In this regard, other investigators have also reported that CAPE can preferentially induce apoptosis, depending on the cell type treated with this compound (Chiao et al., 1995). The reason(s) for a selective effect on apoptosis are not fully understood but may be related to the inherent redox state of a particular cell type (Chiao et al., 1995; Chen et al., 2001; Orban et al., 2000).

NF-B is up-regulated in macrophages and epithelial cells of patients with IBD (Ellis et al., 1998; Rogler et al., 1998; Bantel et al., 2000). Moreover, macrophages have been reported to play an important role in the pathogenesis of PG-PS-induced intestinal injury in Lewis rats (Yamada et al., 1993; Sartor et al., 1996). In our study, the mean colonic NF-B level was only slightly attenuated in CAPE-treated Lewis rats. Nevertheless, there was a significant correlation between the colonic NF-B level and gross colonic injury in rats. Specifically, clear evidence of a reduced colonic NF-B level and associated gross colonic injury was present in three CAPE-treated animals (Fig. 5). In these rats, the colonic NF-B levels were similar to those found in vehicle/saline-treated animals. These rats also had gross colonic injury scores of <4. It is possible that the inherent pharmacokinetic properties of CAPE may have resulted in the lack of more prominent reductions in all treated animals. Specifically, it is possible that relevant pharmacological concentrations of CAPE may not have been consistently achieved in the rat colon. A similar suggestion was put forth previously by other investigators, regarding the in vivo anti-inflammatory profile of CAPE (Orban et al., 2000). The dose of CAPE used in this study was sufficient to induce in vivo biological effects in other rat inflammatory models (Michaulart et al., 1999; Orban et al., 2000). Larger doses, which might lead to higher in vivo drug levels, were not pursued because of concerns with possible toxicity. In this regard, some weight loss was observed in our CAPE-treated animals.

In this study, CAPE significantly reduced the gross colonic injury associated with the chronic reactivation phase (days 14-21) of PG-PS-induced colitis. However, CAPE exhibited only partial inhibition of this damage to approximately 50% of the level initially found in PG-PS-treated rats on day 14. CAPE also significantly, but partially, reduced the increased colonic cytokine levels (IL-1 and TNF-) associated with chronic PG-PS-induced colitis. Such attenuations in these pro-inflammatory cytokines may have contributed to the attenuation of gross colonic injury in rats. This statement is supported by results from a previous study, which suggested that IL-1 plays an important pathogenic role in PG-PS-induced intestinal damage (McCall et al., 1994). In our studies, CAPE (at 30 µg/ml) effectively reduced chemokine (i.e., IL-8) production in vitro, but had only a slight effect on colonic neutrophil infiltration (myeloperoxidase activity) in vivo. In this regard, it is known that PG-PS-induced colitis has a multi-factorial pathogenesis (Sartor et al., 1996). Therefore, conceivably other/additional chemotactic factors may be involved in regulating neutrophil influx into the colon. As mentioned above, it is also possible that a sufficient level of CAPE (e.g., 30 µg/ml) was not consistently maintained in the rat colon. Therefore, based on our in vitro findings (Fig. 2B), chemokine production may not have been dramatically altered in all CAPE-treated rats. It is somewhat difficult to gauge the "clinical" significance of these reductions with CAPE treatment. Particularly, because the PG-PS model is not well-suited for examining symptoms of colitis (e.g., diarrhea, rectal bleeding). Nevertheless, the overall results obtained with CAPE on gross colonic injury and measured biochemical parameters were better than those that we obtained previously in this rat colitis model with olsalazine (L. R. Fitzpatrick and T. Le, unpublished data).

Theoretically, it would seem desirable to develop an agent for the treatment of IBD that could induce apoptosis in relevant inflammatory cells, but not in colonic epithelial cells. In this regard, reduced apoptosis has been described in inflammatory cells (including macrophages) of IBD patients, whereas apoptosis in colonic epithelial cells is apparently increased. This pattern of apoptosis may contribute to the initiation and perpetuation of IBD (Strater et al., 1997; Abreu et al., 2000; Brannigan et al., 2000; Suzuki et al., 2000; Bu et al., 2001). In this regard, our in vitro results indicate that CAPE could possibly induce more enhanced apoptosis of macrophages, as opposed to colonic epithelial cells. However, future in vivo studies are still needed to confirm this initial in vitro observation. Of note, it was recently reported that a similar selective apoptosis profile was responsible for the efficacy of anti-TNF therapy in a SAMP1/Yit mouse IBD model (Marini et al., 2001).

As shown in Table 1, CAPE treatment resulted in a slight anti-arthritis effect in PG-PS-treated rats. The magnitude/significance of this effect is hard to gauge because only 73% of the vehicle/PG-PS-treated rats developed significant arthritis (Yamada et al., 1993; Sartor et al., 1996). Nevertheless, our results may indeed have some relevance/importance because another inhibitor of NF-B (PS-341) has been shown to reduce the joint swelling associated with PG-PS-induced arthritis in rats (Palombella et al., 1998). Moreover, an extract of propolis has been shown previously to significantly reduce adjuvant-induced arthritis in rats (Park and Kahn, 1999).

In summary, we found that CAPE inhibits NF-B and cytokine production in two relevant cell types for IBD. In terms of in vivo relevance, CAPE reduced these parameters in conjunction with stimulation of rat macrophages by PG-PS. Moreover, CAPE induced apoptosis in macrophages. There was also in vivo evidence that CAPE reduced colonic levels of NF-B, as well as relevant pro-inflammatory cytokines. The aforementioned pharmacological profile of CAPE likely contributed to the observed attenuation of PG-PS-induced colitis by this compound.