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RESEARCH REPORT |
Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, MA 02118, USA;
* corresponding author, dgraves{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. We conducted in vivo experiments by subcutaneous inoculation of LPS in wild-type, TNFR1–/–R2–/–, TNFR1–/–, and TNFR2–/– mice to identify which TNF receptors are involved and the specific caspase pathway activated. LPS stimulated apoptosis through TNFR1 but not TNFR2, which was accompanied by the induced expression of 12 apoptotic genes. Fluorometric studies demonstrated that LPS in vivo significantly increased caspase-8 and caspase-3 activity, which was also dependent on TNF receptor signaling. By the use of specific caspase inhibitors, caspases-3 and -8 were shown to play an important role in LPS-induced apoptosis in vivo. Thus, LPS acts through TNFR1 to modulate the expression of apoptotic genes and activate caspases-3 and -8.
KEY WORDS: apoptosis • inflammation • caspases • cytokine • connective tissue • cell death
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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An important pro-inflammatory factor produced during infection is LPS, which is shed from the outer membrane of Gram-negative bacteria. In vitro, LPS directly promotes apoptosis in several cell types—including macrophages, hepatocytes, ventricular myocytes, and endothelial cells (Lakics and Vogel, 1998; Koteish et al., 2002; Li et al., 2002; Munshi et al., 2002)—but inhibits apoptosis of neutrophils (Hachiya et al., 1995). In vivo, LPS has been shown to have a pro-apoptotic effect on lymphocytes in Peyer’s patches and thymocytes, while it has anti-apoptotic effects in peritoneal neutrophils (Wang et al., 1994; Manhart et al., 2000; Feterowski et al., 2001). In addition to direct apoptotic effects, LPS can also stimulate recruitment of leukocytes and the production of pro-inflammatory cytokines, such as TNF- (Henderson et al., 1996; Mahalingam and Karupiah, 1999).
Like LPS, TNF- can induce apoptosis in different cell types (Laster et al., 1988; Fehsel et al., 1991). TNF- signals through 2 distinct cell-surface receptors, TNF receptor-1 (TNFR1) and TNF recptor-2 (TNFR2). The latter contains an intracellular ‘death domain’ (Orlinick and Chao, 1998; Singh et al., 1998). In most cases, it has been shown that TNFR1 mediates TNF--induced apoptosis by stimulating the activation of caspases (Hsu et al., 1995; Chen and Goeddel, 2002). Caspases are produced as pro-enzymes and become activated by proteolytic cleavage at internal aspartate residues upon apoptotic stimulation. These proteases can act as signaling molecules or participate in apotptosis (Thornberry et al., 1997; Budihardjo et al., 1999).
Previously, we demonstrated that LPS stimulates apoptosis in fibroblasts in vivo via TNF- but is not directly apoptotic for these cells in vitro (Alikhani et al., 2003). However, the role of specific TNF receptors and the apoptotic pathway through which they induce apoptosis remain to be established. To address this issue, we inoculated LPS into connective tissue of the scalp by subcutaneous injection. The results indicate that the apoptotic effect of LPS on fibroblasts is specifically mediated by TNFR1 signaling, with no contribution from TNFR2. LPS-stimulated fibroblast apoptosis via TNF- was dependent on caspases-3 and -8 activity and was accompanied by the expression of 12 pro-apoptotic genes.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Injection of LPS
Purified Escherichia coli serotype 0111:B4 LPS was purchased from List Biological (Campbell, CA, USA). Mice were anesthetized with an injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). LPS was inoculated adjacent to the periosteum at a point on the midline of the skull, between the ears. Injection at this anatomic site, which consists mainly of loose connective tissue, can be reproducibly achieved. This injection leads to a mild inflammatory response. For each data point, there were 6 mice (n = 6). Preliminary experiments established that 200 µg LPS in PBS (50 µL) induced a moderate number of apoptotic fibroblasts. Mice were killed 6 and 24 hrs following injection. Some animals were treated by intraperitoneal injection of caspase-8 or -3 inhibitor (1 mg/kg) 1 hr before LPS injection, which was supplemented by local application (1 mg/kg) at the time of LPS injection. The caspase-8 inhibitor (Z-IETD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) were purchased from R&D Systems (Minneapolis, MN, USA). Control mice received vehicle alone, sterile PBS containing 2% Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). In some mice, instead of LPS, recombinant murine TNF- (200 ng) (R&D Systems, Minneapolis, MN, USA) was inoculated adjacent to the periosteum at a point on the midline of the skull, between the ears.
Preparation of Histologic Sections
Animals were killed by decapitation, and their heads were fixed for 72 hrs in cold 4% paraformaldehyde. The soft and hard tissues were kept intact so that tissue architecture would be preserved. Specimens were decalcified by incubation in cold Immunocal (Decal Corporation, Congers, NY, USA) for approximately 12 days, with solution changed daily. Paraffin-embedded sagittal sections were prepared at a thickness of 5 microns.
Detection of TUNEL-positive/Vimentin-positive Apoptotic Fibroblasts
Apoptotic cells were detected by an in situ TUNEL assay by means of a TACS 2 TdT-Blue Label kit purchased from Trevigen (Gaithersburg, MD, USA), following the manufacturer’s instructions. Sections were then incubated with polyclonal goat anti-vimentin (Cortex Biochem, San Leandro, CA, USA). Primary antibody was localized by the avidin-biotin immunoperoxidase method, with the use of a kit from Vector Laboratories (Burlingame, CA, USA). The signal was enhanced by tyramide signal amplification with the use of a kit from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). At high magnification (1000x), the number of apoptotic fibroblasts was counted in the loose connective tissue between the coronal and occipital sutures. This area typically consisted of approximately 50 fields per specimen. The number was normalized per area of connective tissue (mm2). Counts and measurements were confirmed by re-analysis of the specimens by one other independent examiner. Student’s t test was used to determine significant differences between the experimental and control groups.
RNase Protection Assay
Following the death of the mice at the indicated time points, their scalps were immediately dissected from the calvaria and frozen in liquid nitrogen. Total RNA was extracted with Trizol (Life Technologies, Rockville, MD, USA) from pulverized frozen tissue, following the manufacturer’s instructions. P32-labeled riboprobes were incubated with 10 µg of total RNA and then subjected to RNase digestion with the use of a kit from Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA), following the manufacturer’s instructions. Following electrophoresis on a 6% polyacrylamide gel, radiolabeled bands were visualized with a PhosphoImager (Bio-Rad Laboratories, Hercules, CA, USA). The optical density of each band was normalized by the value of GAPDH in the same lane. Each value represents the mean of 3 separate RNase protection assays ± SEM. Statistical difference between samples was determined by one-way analysis of variance, followed by Tukey’s multiple-comparison test.
Caspases’ Activities
Caspases’ activities were assayed by a fluorometric kit purchased from R&D Systems. Briefly, following the animals’ death at the indicated time points, murine scalps were immediately dissected from the calvaria and frozen in liquid nitrogen. Frozen tissues were pulverized and lysates prepared with the use of cell lysis buffer provided by R&D Systems. Caspases-3, -8, and -9 activities were detected with specific fluorogenic substrates with the use of excitation (400 nm) and detection (505 nm) filters. In some assays, recombinant caspase-3 enzyme (R&D Systems) was used as a positive control. Buffers without cell lysate and cell lysate without substrate were used as negative controls. Statistical difference between samples was determined by one-way analysis of variance, followed by Tukey’s multiple-comparison test.
Cell Culture
Human adult dermal fibroblasts were purchased from Cambrex (Walkersville, MD, USA). Cells were propagated and maintained in Dulbecco’s Modified Eagle’s Medium (Cambrex) supplemented with 10% fetal bovine serum, gentamycin (100 µg/mL), and amphotericin B (100 ng/mL). TNF- was purchased from R&D and tested in culture medium supplemented with 0.5% fetal bovine serum. Assays were performed when the cultures reached 75% to 85% confluence. Apoptosis of fibroblasts was determined by the measurement of histone-associated DNA fragments (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, respectively, increased apoptosis of fibroblasts 666% and 734%, compared with vehicle alone (P < 0.05) (Table
). However, in the absence of TNF receptor signaling, LPS induced very little fibroblast apoptosis.
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activity in the host response to LPS, we studied the expression of apoptotic genes by RNase protection assay in TNFR1–/–R2–/– compared with matched wild-type mice (Fig. 1). At the zero time point, the expression of pro-apoptotic genes could be detected at relatively low levels. LPS increased mRNA levels of apoptosis-inducing ligands TNF-, FasL (Fas Ligand), and TRAIL (TNF-related apoptosis-inducing ligand) in wild-type mice from 220% to 300%, while in the TNF-receptor-ablated mice, the increase was considerably less. The expression of the apoptotic TNF receptor family member Fas in wild-type mice increased by 160% at 24 hrs, while in TNF-receptor-ablated mice, this increase was 50%. The expression of initiator caspases (caspases-2 and -8) increased approximately 300% at 6 hrs and slightly less at 24 hrs. In the TNF-receptor-ablated group, induction was from 35% to 85%. The expression of effector caspases (caspases-3, -6, and -7) in wild-type mice increased from 190% to 265% at 24 hrs in comparison with an 11% to 71% increase in TNFR1–/–R2–/– mice. The expression of the death domain family of proteins—FADD (Fas-associated protein with death domain), CRADD (Caspase and RIP adaptor with death domain), and TRADD (TNFR1-associated death domain protein)—increased 200% to 250%, while in the TNF-receptor-ablated group, induction was 10% to 30%. The difference between wild-type mice and TNFR1–/–R2–/– mice for all genes at both 6 and 24 hrs was statistically significant (P < 0.05). Thus, LPS, largely through TNF-, induced the expression of several different pro-apoptotic factors in fibroblasts.
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). RNase protection assays showed that LPS did not increase the expression of apoptotic genes in TNFR1–/– mice. In TNFR2–/– mice, LPS increased the expression of TNF-, FasL, TRAIL, Fas, Caspase-3, Caspase-7, Caspase-8, TRADD, CRADD, and FADD from 120 to 275%. There was no difference between TNFR2–/– mice and wild-type mice (P > 0.05). The difference between TNFR1–/– mice and TNFR2–/– mice was statistically significant (P < 0.05). Thus, all of the LPS-induced expression of pro-apoptotic molecules is mediated through TNFR1 receptor signaling.
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). In TNFR1–/–R2–/– mice, LPS increased caspase-3 activity 31%, caspase-8 activity 42%, and caspase-9 activity 6.3%.
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). Based on quantitative analysis of double-stained histologic sections, caspases-3 and -8 inhibitors significantly reduced apoptosis induced by LPS by 93% and 87%, respectively, which was statistically significant (P < 0.05) (Fig. 3C). These results indicate that in vivo caspase-8 serves as the main initiator caspase, while caspase-3 serves as the main executioner caspase stimulated by LPS. As shown above, this occurs via TNF stimulation. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, we did not explore the role of different TNF receptors in this process. Studies presented here demonstrate that LPS inoculation induced apoptotic gene expression through TNFR1. This is based on results showing greatly reduced up-regulation of apoptotic genes in TNFR1–/– mice compared with wild-type mice. It is further supported by findings that the reduced up-regulation in TNFR1–/– mice was equal to that of TNFR1–/–R2–/–, and that ablation of TNFR2–/– had no impact. These data are consistent with the structure of the TNF receptors (Peschon et al., 1998; Idriss and Naismith, 2000; Chen and Goeddel, 2002). TNFR1 is a member of the TNF receptor superfamily that contains a ‘death domain’, which TNFR2 lacks. The physiologic significance of this effect is due to the dependence of apoptosis on the balance of pro- and anti-apoptotic factors within a cell. Thus, LPS, through induction of TNF-, can predispose a cell toward apoptosis by stimulating the expression of pro-apoptotic factors and potentially renders the cell more sensitive to apoptotic signals.
Whether a cell becomes apoptotic depends upon the activation of caspases. Different pathways have been described for LPS-stimulated apoptosis. It has been shown that, in different cell types, LPS may modulate caspase-3 through either the cytoplasmic (caspase-8-dependent) or mitochondrial (caspase-9-dependent) pathway, or both (Kawahara et al., 2001; Munshi et al., 2002; Koizumi et al., 2003; Okuyama et al., 2003). The specific inhibition of caspase-8 prevented the apoptotic effect of LPS, indicating that the cytoplasmic pathway is the principal pathway through which LPS induces apoptosis in connective tissue. We also demonstrated that activation of caspases occurs through TNF receptor signaling. This agrees with results from previous studies showing that binding of TNF- to TNFR1 can activate caspase-8 through adaptor proteins such as TRADD and FADD (Hsu et al., 1995; Chen and Goeddel, 2002).
In connective tissue, TNF- injection was able to produce apoptotic effects simlar to those produced by LPS injection. These data—in addition to our other observation that LPS was unable to induce apoptosis in TNF-receptor-ablated mice—are consistent with findings that the apoptotic effect of P. gingivalis is mediated through TNF- (Graves et al., 2001). Thus, bacterial LPS—through interaction with LPS receptors such as LPS-binding protein, CD14, and Toll-like receptors—may significantly contribute to tissue damage associated with infection by inducing TNF- expression, thereby stimulating expression of pro-apoptotic genes and inducing the cytoplasmic apoptotic pathway.
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ACKNOWLEDGMENTS |
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Received December 13, 2003; Last revision May 31, 2004; Accepted July 1, 2004
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