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Increased expression of soluble decoy receptor 3 in acutely inflamed intestinal epithelia
Clinical Immunology 115 (2005) 286 – 294 www.elsevier.com/locate/yclim
Increased expression of soluble decoy receptor 3 in acutely inflamed intestinal epithelia Sunghee Kima,b,T, Anastasia Fotiaduc, Vassiliki Kotoulac a
Department of Biological Sciences, University of Alabama, Rm 280 Nott Hall, Tuscaloosa, AL 35487, USA b BioPowerTech, 4734 Bluegrass Pkwy, Tuscaloosa, AL 35406, USA c Department of Pathology, School of Medicine, Aristotle University, 54006 Thessaloniki, Greece Received 14 January 2005; accepted with revision 22 February 2005
Abstract Decoy receptor 3 (DcR3), a soluble receptor in the tumor necrosis factor (TNF) receptor family, is known to inhibit apoptosis mediated by pro-apoptotic TNF family cytokines such as Fas ligand (FasL), TL1A, and LIGHT. Therefore, the regulation of DcR3 expression under certain pathophysiological conditions is of interest since the level of soluble DcR3 would most likely affect the homeostasis of cells and tissues. We found that human intestinal epithelial cell (IEC) lines (SW480, SW620, and HT29) could selectively increase DcR3 release in response to lipopolysaccharide (LPS) and that all the cells preferentially expressed Toll-like receptor 4 (TLR-4). LPS-induced DcR3 releases in IECs appeared to be via the activation of mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and c-Jun NH2-terminal protein kinase (JNK), and the transcription factor NF-nB. Moreover, the increased expression of DcR3 in appendix epithelia from patients with acute appendicitis was demonstrated. Taken together, the results indicated that DcR3 might play an important role in the human intestinal epithelium during acute inflammatory processes caused by endotoxin challenge. D 2005 Elsevier Inc. All rights reserved. Keywords: FasL; TL1A; LIGHT; Inflammation; Appendix
Introduction DcR3, a soluble decoy receptor in the TNF receptor (TNFR) family, is known to bind to ligands of the TNF family, namely, FasL, LIGHT, and TL1A [1–3]. As a result, DcR3 is believed to block cellular effects that are mediated by the interaction between these ligands and their memAbbreviations: DcR3, decoy receptor 3; TNF, tumor necrosis factor; TNFR, TNF receptor; TLR, Toll-like receptor; IEC, intestinal epithelial cell; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinase; LPS, lipopolysaccharide; FasL, fas ligand; LTA, lipoteichoic acid; Th, T helper; DC1, myeloid-derived dendritic cells; DC2, plasmacytoid-derived dendritic cells; MEK, MAP kinase activating kinase, MEK; MEKK, MEK activating kinase. T Corresponding author. Department of Biological Sciences, University of Alabama, Room 280 Nott Hall, Tuscaloosa, AL 35487, USA. Fax: +1 205 348 5976. E-mail address: [email protected] (S. Kim). 1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.02.014
brane-bound cognate receptors in the TNFR family: TL1A and FasL bind to death-domain containing receptors DR3 and Fas, respectively, and LIGHT engages with either herpes virus entry mediator (HVEM) or lymphotoxin beta receptor (LThR). One of the prominent biological functions of DcR3 is anti-apoptosis. A soluble recombinant DcR3 was reported to prevent apoptosis mediated by FasL [1,4,5] and LIGHT [2,6], and to inhibit TL1A-mediated caspase induction [3]. Moreover, DcR3 was shown to be effective at attenuating FasL-induced mortality [7] and acute pulmonary inflammation [8] in experimental animal models, thus demonstrating the therapeutic effects of soluble recombinant DcR3 in FasL-mediated tissue injury. Similarly, transgenic overexpression of DcR3 was shown to play a protective role in a rodent diabetic model [9]. DcR3 was also reported to modulate a variety of immune responses apparently via interaction with its ligands. Ectopic
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expression of DcR3 in rodent gliomas resulted in the inhibition of the infiltration of T cells and macrophages [5], and the pre-treatment of T cells with soluble DcR3 was shown to suppress T-cell chemotaxis [10]. Also, recombinant DcR3 was shown to downregulate cytotoxic T-cell activity against alloantigens [11], and transduce costimulatory signals in T cells resulting in proliferation of T helper [Th] 1 and 2 cells, and lymphokine production in Th 1 cells [12]. Additionally, exogenous DcR3 was reported to modulate dendritic cell maturation and differentiation [13]. It is apparent that the effects of recombinant DcR3 could be diverse because of its unique ability to interact with the three different ligands of type II transmembrane proteins that exist in both soluble and membrane-bound forms. The addition of excess exogenous soluble DcR3 in culture system would undoubtedly affect the behavior of the cells which express the ligands and their functional cognate receptors on the cell surface. Hence, the regulation of endogenous DcR3 expression and its tissue distribution under certain pathophysiological conditions might be important for our understanding of the role of DcR3 in the immune system. Overexpression of DcR3 has been reported in lung and gastrointestinal tract tumors, lymphomas, and gliomas [1,2,5,14]. At first, it was hypothesized that DcR3 might aid cancer cells to evade FasL-mediated cytotoxic attack by host immune cells [1]. In contrast, we have reported elevated levels of soluble DcR3 in sera of patients with bacterial infections and renal failure [15,16]. Additionally, we have demonstrated the selective induction of DcR3 release in human primary antigen-presenting cells such as monocytes and myeloidderived dendritic cells (DC1) but not plasmacytoid-derived dendritic cells (DC2) in response to both Gram-positive and Gram-negative bacterial antigens such as LPS and lipoteichoic acid (LTA). Thus, we have suggested that, in addition to its possible involvement in innate immune responses, DcR3 might preferentially play a role in cellmediated immune responses in bacterial pathogenesis [15]. However, little is known about the expression of DcR3 in acutely inflamed human organs. This prompted us to investigate DcR3 expression in acutely inflamed human appendix epithelium and the signaling mechanism involved in overexpression of DcR3 in human intestinal epithelial cells following LPS challenge.
Materials and methods Reagents, cell lines, antibody, and human tissue samples Inhibitors, SB203580, SB202190, Ro31-8220, Wortmannin, and pyrrolidine dithiocarbamate (PDTC), and bacterial antigens, LPS (E. coli serotype 0111:B4) and LTAs (Bacillus subtilis and Staphylococcus aureus) were purchased from Sigma (St. Louis, MO). Kinase inhibitors,
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PD98059, U0126, and SP600125 were obtained from LC Laboratories (Woburn, MA). DMEM was obtained from Cellgro (Herndon, VA) and heat-inactivated FBS was from Invitrogen (Carlsbad, CA). Cell lines, SW480, SW620, HT29, and U937 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). We have previously reported the development of monoclonal antibodies (MAbs) against human soluble DcR3 [16]. After testing a dozen MAbs, we selected MD3F4 based on its highest sensitivity and specificity to react with DcR3 in human tissue samples. The specificity of MD3F4 was extensively tested with a vast array of normal and diseased human tissue samples in comparison with other isotype matching anti-DcR3 MAbs. Also, a lack of MD3F4 reactivity was confirmed in the presence of excess soluble DcR3 during incubation of tissue slides with MD3F4. Cell culture and ELISA Intestinal epithelial cell lines that were originally derived from colon adenocarcinomas, SW480, SW620, and HT29, were cultured in DMEM supplemented with 10% FBS. One day before inhibitor treatment, cells were plated at 2 105 cells/well in 24-well tissue culture plates. After aspirating the medium, fresh culture medium with or without inhibitors was added to the wells (0.5 ml/well). The culture plates were then incubated for 24 h at a 378C, 5% CO2 culture incubator. The levels of DcR3 in culture supernatants were measured by the human DcR3 specific quantitative ELISA as described in [16]. Briefly, 100 Al of culture supernatants was added to wells of 96-well ELISA plates that were coated with the capture antibody (MD3E2) and blocked with 3% BSA. The standard DcR3 protein and test samples were added to the wells and the plates were incubated overnight at 48C. After washing, a biotinylated detection antibody (MD3B1) was incubated to wells for 2 h at room temperature. After washing, a peroxidase-conjugated streptavidin (Vector Lab., Burlingame, CA) was added to the wells. Color was developed using TMB substrate solution (KPL, Gaithersburg, MD). After stopping the color reaction with 1 N H2SO4, plates were read at absorbance 450 nm in an Emax ELISA plate reader (Molecular Device Co., Sunnyvale, CA). Real-time quantitative PCR (TaqMan) and reverse transcription (RT)-PCR analyses Total RNAs from SW480, SW620, HT29, and U937 were harvested by using Trizol RNA extraction solution (Invitrogen). RT-PCR was performed using SuperScript One-Step RT-PCR System according to the manufacturer’s recommendation (Invitrogen). A total of 50 Al reaction mixture contained 100 ng RNA, 0.2 AM each of forward and reverse primers specific for human TLR2 (5Vcccatttccgtctttttgaa-3V and 5V-cgcagctctcagatttaccc-3V), TLR4 (5V-tgagcagtcgtgctggtatc-3V and 5V-cagggcttttctgagtcgtc-3V), TLR5 (5V-ggaaccagctcctagctcct-3V, 5V-aagagg-
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gaaaccccagagaa-3V), or TLR9 (5V-cagcagctctgcagtacgtc-3V and 5V-aaggccaggtaattgtcacg-3V). The reaction was carried out at 458C for 30 min and 948C for 2 min followed by 40 cycles of 948C 15 s, 558C 30 s, 688C 15 s. The primers were designed using the web-accessible Primer 3 software [17] and the primers were purchased from Integrated DNA Technologies (Coralville, IA). For TaqMan analysis, the reaction was carried out in a 25-Al reaction mixture containing 25 ng of RNA, 0.6 AM each primers specific for human DcR3 (forward: 5Vctgatcctggccccctctta-3V; reverse: 5V-ttcttctatttaaaaaaaagcctctttca-3V; probe:5-tctacatccttggcaccccacttgca-3V). Probes labeled with 6-FAM and TAMRA were purchased from Biosource International (Camarillo, CA, USA). PCR products were measured with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). To ensure no contamination from genomic DNA, parallel reactions were conducted without reverse transcriptase. The level of DcR3 mRNA was normalized to the mRNA level of 18S rRNA in the same sample. Immunohistochemistry (IHC) Archival paraffin blocks from ten routine surgical pathology cases, previously diagnosed with acute appendicitis upon histological examination, were used. Patient age ranged 13–50 years (median: 22). Each block contained sections from the apical and the basal part of the appendectomy specimen. Apical and basal sections of the appendix from each case were sliced at 2 Am thickness and mounted on positively charged slides. Paraffin slides were then incubated overnight at 558C. After dewaxing in multiple fresh xylenes, the slides were rehydrated in descending ethanol changes. Endogenous peroxidase was blocked for 20 min with 3% H2O2 in methanol and, subsequently, slides were rinsed in distilled water. No epitope unmasking was used since this step was determined to be unnecessary for DcR3 reactivity with MD3F4. To abrogate non-specific binding, sections were incubated with a commercially available blocking solution, Powerblock (Innogenex, San Ramon, CA), at 378C for 30 min. Slides
were incubated with MD3F4 (2.5 Ag/ml) diluted in Powerblock at 378C for 1 h. After washing in PBS, a secondary biotinylated anti-mouse antibody (DAKO, Glostroup, Denmark), diluted at 1:200 in Powerblock, was applied for 1 h at room temperature. Color development was achieved with streptavidin–biotin complex and liquid diaminobenzidine (DAB, both from DAKO). No counterstaining was used for the appendicitis specimens. Qualitative evaluation of DcR3 localization was accomplished by comparison to H&E stained serial sections for each case.
Results Increases of DcR3 protein in colon epithelial cells following LPS stimulation By utilizing quantitative ELISA that was validated to measure soluble DcR3 in human cell culture supernatants and sera samples [16], the regulation of soluble DcR3 expression was investigated in the human IEC lines SW480, SW620, and HT29 that are derived from colorectal carcinomas. Previously, we had reported the purification of native soluble DcR3 secreted in SW480 culture [16]. Also, we showed that SW480 expressed one order of magnitude higher soluble DcR3 compared to SW620 in culture supernatants [16]. Regardless of their constitutive DcR3 expression, following LPS treatment, both SW480 and SW620 released 3- to 4-fold increased DcR3 compared with the cells with no LPS stimulation (Fig. 1). Additionally, HT29 which expressed no detectable DcR3 (apparently lower than the detection limit of ELISA, 36 pg/ml) could also release detectable amounts of DcR3 after LPS treatment. All three cell lines appeared to be highly responsive to LPS to release DcR3: LPS concentration as low as 0.1 ng/ml could induce protein release and 10 ng/ml was sufficient to maximize the release of soluble DcR3 from the cells, and there was no further induction of DcR3 at higher LPS concentrations. Unlike DC1 that responded to both Gram-positive and Gram-negative bacterial antigens, LPS and LTA, respectively [15], these IEC cell lines only responded to LPS and there was
Fig. 1. Selective induction of DcR3 in IECs, SW480, SW620, and HT29, following LPS challenge. Cells seeded in 24-well culture plates were incubated in the presence of LPS at the indicated concentrations for 24 h. The levels of soluble DcR3 in culture supernatants were measured by ELISA. Experiments were conducted at least three independent times with triplicate samples. Data are mean F SD and statistical analysis was performed using unpaired t test. All doses of LPS (0.1, 1, 10 ng/ml) increased DcR3 release in all the cell lines compared to the cells without LPS. P values between untreated and LPS (grouped all doses) treated SW480 and SW620 were less than 0.001.
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Fig. 2. Increases of DcR3 mRNAs in SW480 cells following LPS stimulation. Cells seeded in 6-well culture plates were incubated in the presence or absence of LPS or LTA for 8 h. Total RNAs were harvested from the cells and then subjected to TaqMan. The primers were expected to amplify 81 base pairs (1000~1080) of the DcR3 nucleotide sequence obtained from Genbank accession number AF1044419. Experiments were independently conducted two times with triplicate samples and data are mean F SD. LTA (B) is an LTA source from B. subtilus, and LTA (S) is an LTA source from S. aureus. No significant increases in DcR3 were observed by the two LTAs. The increase of DcR3 mRNA by LPS was highly significant with P value less than 0.0001 according to unpaired t test.
no noticeable increase in DcR3 in these three cells following the treatment with LTA at various concentrations. In accordance with protein expression, the treatment of SW480 with two bacterial LTA sources did not increase the expression of DcR3 mRNA while LPS stimulation resulted in up to a 4-fold increase in the message (Fig. 2). The selective responsiveness to LPS might be in part due to the preferential expression of Toll-like receptor 4 (TLR-4) in IECs. While the U937 monocytic cell line that was employed as a control cell line to show integrity of the primer sets expressed detectable mRNA levels of TLR-2, TLR-4, TLR-5, and TLR-9, the three IEC lines appeared to predominantly express TLR-4 mRNA (Fig. 3). It has been established that LTA and LPS mediate signaling preferentially via TLR-2 and TLR-4, respectively [18].
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which is a potent selective inhibitor of MAP kinase activating kinase (MEK). PD98059 inhibits phosphorylation of MEK by the upstream kinases, MEK activating kinase 1 (MEKK-1) and Raf-1 [20]. Also, U0126 was utilized since this compound selectively blocks MEK1/2, the kinases upstream of ERK1/2 [21,22]. This compound was also shown to inhibit AP-1 transactivation by suppressing synthesis of c-jun and cfos mRNA and protein by MAP kinases [21,22]. A specific inhibitor of JNK, SP600125, was also employed. This relatively new synthetic inhibitor selectively inhibits JNK1, 2, 3 thus resulting in the inhibition of AP-1 phosphorylation [23]. The upstream kinase of MAPK pathway, MEKK-1, is required for JNK activation by LPS [24]. MEK7, a downstream kinase of MEKK, activates only JNK while MEK4 activates both JNK and p38 [25]. Once activated, JNK phosphorylates c-Jun which dimerizes with c-Fos to activate AP-1. The treatment of cells with ERK and JNK inhibitors (U0126, PD98059, and SP600125) in the presence of 10 ng/ ml LPS resulted in significant inhibition of DcR3 release in SW480 (Fig. 4). However, potent pyridinyl imidazole inhibitors of p38 MAP kinase, SB202190 and SB203580, had no inhibitory effects on the cells. Instead, these inhibitors, at concentrations at 0.5–1 AM, resulted in DcR3 increases up to 30% in the cells (Fig. 4). There was no significant DcR3 inhibition by both inhibitors, but rather slightly deteriorated cell viability above 2 AM of the inhibitors. The increases in DcR3 release by these inhibitors might be due to the activation of Raf-1, an upstream kinase of MEK, by the inhibitors. Previously, SB203580 was
Involvement of MAP kinase pathways in signaling of DcR3 release The signaling mechanisms involved in DcR3 release was studied utilizing SW480 cells since this cell line expresses a relatively high level of DcR3. Specific inhibitors of intracellular kinases in signaling pathways were chosen based on their well-recognized specificity and no apparent cytotoxic effect on the cells even at ranges well above IC50 which was previously reported in the literature. The cells were treated with inhibitor concentrations around IC50 and the cell viability was always confirmed at the end of inhibitor treatment using MTT assay. The activation of MAP kinases, ERK1/2, JNK and p38, in the TLR-mediated signal transduction pathway has been well established (reviewed in [19]). Therefore, we employed specific inhibitors of MAPK pathways such as PD98059
Fig. 3. Preferential expression of TLR-4 in IECs. Total RNAs from IEC lines, SW480, SW620, and HT29, were subjected to RT-PCR. The U937 cell line was included as a control to show the specificity of primer sets. A master reaction mixture was made for RNA from each of the cell lines and then primers for each TLR were added to aliquots. Each RT-PCR reaction tube contained 100 ng of RNA. One fifth of the RT-PCR reaction volume (10 Al) was analyzed on 1 or 1.2% agarose gels. A 2-kb DNA ladder marker was obtained from Invitrogen. Expected PCR fragments for TLR-2, -4, -5, and -9 are 185 (2288~2472), 167 (3709~3875), 196 (2373~2568), 224 (1921~2144) base pairs (bp), respectively. Complete cDNA sequences for TLR-2, -4, -5, and -9 were obtained from Genbank accession numbers U88878, AF172169, AB060695, and AF245704, respectively.
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Fig. 4. LPS-induced DcR3 release in SW480 cells was inhibited by inhibitors of MAPK (ERK1/2 and JNK) and NF-nB. Cells seeded in 24-well tissue culture plates were treated with both LPS (10 ng/ml) and the indicated inhibitors for 24 h. DcR3 release in culture supernatants of LPS-stimulated cells with or without inhibitor treatments were quantified by ELISA. Experiments were conducted two independent times with triplicate samples and data are mean F SD. PDTC (50 AM, 100 AM), PD98059 (5, 10, 20 AM), U0126 (100, 200, 400 nM), and SP600125 (20, 40 AM) significantly inhibited DcR3 release in the cells compared to the cells with no inhibitor treatment. P values between untreated and each inhibitor (grouped according to the indicated doses) were less than 0.001. SB202190 and SB203580 significantly increased DcR3 release with P values less than 0.05 between untreated and inhibitor (combined 0.5 and 1 AM) according to unpaired t test.
reported to activate Raf-1 [26]. It is known that LPS signaling involves Raf-1 activation [27]. The inhibition of DcR3 releases by ERK and JNK inhibitors was more prominent in LPS-treated cells since the inhibition by the same inhibitors in the cells treated with PMA, a potent activator of protein kinase C (PKC), was at most 15% to 20% by U0126 and PD98059, respectively. We found that PMA could also increase DcR3 release up to one order of magnitude higher in the three IEC lines, and, as expected, a PKC inhibitor Ro31-8220 (inhibits conventional PKC isoforms, a, h-I, h-II, and g) could effectively inhibit PMA activated DcR3 release in the IEC cell lines. In contrast, Ro31-8220 had no inhibitory effects on the IEC cells following LPS treatment; therefore, MAP kinases but not conventional PKC isoforms are involved in LPS-inducible DcR3 release in IECs (Fig. 4). Also, a PI3-K (phosphatidylinositol 3kinase) inhibitor, Wortmannin, had no inhibitory effect on the cells (Fig. 4). It is widely known that LPS activated downstream transcription factor NF-nB [19]. As expected, a specific inhibitor of NF-nB activation, PDTC, selectively inhibited LPS-induced DcR3 release about 70% (Fig. 4). In addition, we observed that the constitutive expression of soluble DcR3 in SW480 cells could also be inhibited by the same inhibitors of ERK and JNK that showed the inhibitory effects on LPS-inducible DcR3 release (the inhibition profile was similar to Fig. 4) but the two p38 inhibitors had no inhibitory effects in the protein expression (data not presented). Also, PDTC did not inhibit any constitutive DcR3 release in SW480 cells probably due to the lack of activated NF-nB activity in the resting cells. Based on the results from the inhibitor studies, we have deduced the signaling pathways that are most likely involved in LPS-inducible DcR3 release in the cells as shown (Fig. 5).
Immunolocalization of DcR3 in acutely inflamed appendix epithelium We then sought to investigate the possible changes in DcR3 expressions in human intestinal epithelia with acute
Fig. 5. A schematic presentation of signaling mechanisms involved in DcR3 releases after LPS challenge. LPS stimulates cells via TLR-4 on the cell surface that leads to the activation of MEKK-1 and Raf-1 followed by downstream kinases, MEKs. MEK1/2 activates ERK1/2 while MEK4/7 activates JNK. The activation of ERK and JNK will result in the downstream transcription factors including AP-1. The activation of NF-nB will be likely achieved via the pathway involving NF-nB inducing kinase (NIK) pathways (not shown in the diagram). The binding of transcription factors including NF-nB and AP-1 to the promoter of DcR3 gene will eventually cause release of soluble DcR3 in intestinal epithelial cells. Inhibitors are indicated in parentheses.
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inflammation. A common example of acute inflammation whereupon surgical specimens for histological evaluation are obtained is spontaneous acute appendicitis. As shown in Fig. 6, non-inflamed areas in the basal part of the organ were used as a normal control to evaluate the inflammatory response usually localized in the apical part of the organ. In fact, absence of inflammation was noticed in 7 out of 10 cases in the basal section of the removed appendix, minimal to mild inflammation in 2 cases and severe ulcerative one in 1 case. Various degrees of inflammation were observed in all 10 cases in the apical part of the appendix, justifying the clinical diagnosis and removal of the organ. In the non-inflammatory (normal) mucosa, submucosa, and muscular wall components (epithelial cells, local lymphocytic aggregates and follicles, smooth muscle cells, as well as capillary, small and larger vessel endothelia), DcR3 was either absent or barely detectable. Only mast cells, often quite numerous in the submucosa, as well as few monocytes, were found positive with the DcR3 antibody in these histologically non-inflammatory areas (Fig. 6, panels A–D). The expression of DcR3 in mast cells is of interest. A previously unrecognized protective role of mast cells in acute bacterial peritonitis was reported, and
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its protective role against enterobacterial infection was reported to be mediated by TLR-4 [28,29]. By contrast, most of the above mentioned tissue components were found positive in areas with histological signs of inflammation in all ten cases examined (Fig. 6, panels E–L). DcR3 immunoreactivity was observed in the surface epithelial cells and in the glandular structures; in the inflammatory cell infiltrates, inside and diffusely around the cells; in the endothelial cells of sizes of vessels ranging from capillaries up to the larger vessels located in the submucosa; in the smooth muscle cells of the vessel wall and in the muscularis externa; and, in fibroblasts in the lamina propria. Practically, DcR3 was localized in all inflamed tissue components and layers, according to the extent of inflammatory infiltration, with one exception: the germinal centers of reactive follicles remained negative (Fig. 6, panels J and K), as in the non-inflamed tissue (Fig. 6, panel B). In regions with inflammatory infiltrates, diffuse DcR3 positivity was also observed around the cells, in line with the soluble nature of this protein. In the same areas, DcR3 positive lymphocytes were also present in vascular lumina (Fig. 6, panel L). As a control for antibody specificity, erythrocytes and necrotic debris did not stain for DcR3, as expected.
Fig. 6. Representative examples of DcR3 localization in tissue components of normal and acutely inflamed vermiform appendix. (A~D) Tissue sections with no detectable inflammation in the basal part of the appendix. None or very weak DcR3 expression is shown in the epithelial and endothelial cells, vessel walls, as well as in most stromal components (B, C). Mast cells (m) and scarce monocytes are positive for DcR3 (C). (E ~L) Tissue sections from acute appendicitis specimens. (E~H) Severe inflammation of the appendiceal wall up to the serosa spanning the whole organ. (I~L) Mild inflammation of the mucosa in the apical part of the appendix, without involving the submucosa. In the inflammatory tissue, epithelial cells, lymphocytic infiltrates, endothelia, and smooth muscle vascular walls are positive for DcR3. Germinal centers of the lymphatic follicles (J, K, compared with B) remain largely negative. In L, DcR3 positive lymphocytes can be recognized in vascular lumina. e = Epithelial cells; li = lymphocytic infiltrate; lf = lymphatic follicle; v = vessel; arrows = endothelial cells; * = erythrocytes, negative for DcR3, as expected. Stains: A, E, G, I, H, and E; all other, DcR3 IHC. Original magnifications: A, B, E, I, J, 40; F, K, 100; C, D, G, H, L, 400.
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Discussion In spite of their basal levels of DcR3, all three colonic epithelial cell lines significantly increased DcR3 secretion in response to LPS that is widely known to activate NF-nB, a central regulator of immune and inflammation responses. The activation of NF-nB has been detected in epithelial cells of inflamed intestinal mucosa as well as in intestinal mucosa during endotoxemia [30,31]. Therefore, our data seemed to indicate that the induction of DcR3 would most likely occur in most parts of the human intestinal epithelium in response to LPS challenge. The pathogenesis of acute appendicitis is believed to involve bacterial infection due to the blockage of appendix lumen. Recently, it has been suggested that bacterial LPS may play a central role in the development of appendicitis and in the consequently developing generalized systemic anti-inflammatory response, which is observed in the majority of patients with clinically characterized disease [32]. The extensive DcR3 staining in many different cell types in inflamed appendix appeared to be in line with our findings that LPS treatment results in increases of DcR3 in a variety of human primary cells including monocytes and DC1 [15], and dermal microvascular endothelial cells (HMVEC) and keratinocytes (unpublished data). It is also likely that the cytokines that are released in the course of acute inflammatory reaction are a contributing factor to the extensive DcR3 expression in acute appendicitis. Although not to the extent of LPS stimulation, we found that proinflammatory cytokines such as TNF and IL-1 can also induce soluble DcR3 release in HMVEC (unpublished data). In sharp contrast to intensive DcR3 staining in surrounding epithelial cells, infiltrating inflammatory and endothelial cells, lymphoid follicles appeared to be devoid of DcR3 immunoreactivity. Similarly, we had reported that human primary B and T cells in culture did not express any significant level of DcR3 even in the presence of activators: LPS and S. aureus Cowan I (SAC) for B cells and phytohemagglutinin (PHA) and anti-CD3/CD28 for T cells [15]. The intestinal mucosa is regarded as a first line of defense against invading pathogens in the intestine. The overexpresion of DcR3 in acutely inflamed appendix indicates that DcR3 might play a potentially important role in the innate immune response in the intestine. Similarly to the biological effects that were observed with recombinant soluble DcR3 in culture systems, the endogenously secreted DcR3 might also exert its anti-apoptotic activity via binding to its pro-apoptotic ligands expressed in the course of acute inflammatory responses in the intestine. On the other hand, overly elevated DcR3 in the intestine under certain pathological conditions might also contribute to uncontrolled cell growth resulting in colorectal neoplasia. This might be one of the reasons that DcR3 overexpression was detected in certain colorectal carcinomas.
Previously, IEC lines including HT29 were shown to be unresponsive to Gram-positive bacterial antigens primarily due to the lack of TLR-2 protein [33]. Our result also seemed to indicate that IECs selectively induce DcR3 in response to LPS but not to LTA in part due to their preferential expression of TLR-4 transcripts. Unlike monocytes and DC1 that responded to both LPS and LTA to induce DcR3, we had reported that DC2 which was known to express neither TLR-2 nor TLR-4 did not induce DcR3 after LPS or LTA stimulation [15]. DC2, although it was known to express TLR-7 and TLR-9, did not induce DcR3 release in response to single-stranded RNA virus infection or bacterial DNA sequence CpG-oligodeoxynucleotides while it released IFN-a against both agents in the same culture supernatant [15]. In addition, we found that human primary keratinocytes could induce DcR3 in response to both LPS and LTA while human primary HMVEC only responded to LPS (unpublished data). It has been reported that human keratinocytes express both functional TLR-4 and TLR-2 [34] while HMVEC expresses functional TLR-4 but not TLR-2 [35]. Therefore, the regulation of DcR3 in response to LPS and LTA appeared to be critically dependent on the presence of functional TLR-4 and TLR-2 in different human cell types. As it was seen with DC1, the increase in DcR3 release in IECs following LPS was significantly inhibited by the inhibitors of ERK1/2 (p44/p42) MAP kinase. However, inhibitors of p38 MAPK again showed no effects on DcR3 release in IECs with or without LPS stimulation. We had already shown that the p38 inhibitor SB203580 did not inhibit DcR3 release in human primary DC1 after LPS challenge [15]. The MAPK p38 is well known to play a critical role in induction of the proinflammatory cytokines TNF and IL-1 in monocytes, and we had also demonstrated that TNF release in DC1 after LPS stimulation could be significantly inhibited by the p38 MAPK inhibitor [15]. Hence, the lack of p38 involvement might be a characteristic feature in DcR3 regulation, compared to proinflammatory cytokines such as TNF and IL-1. If DcR3 turns out to play a protective role in acute inflammatory conditions induced by LPS, the inhibition of p38 might prove to be more effective in controlling inflammation than the inhibition of upstream MAPK pathway kinases or the downstream NF-nB that indiscriminately control expression of both survival and death molecules that are activated in the course of inflammatory responses. An inhibitor of p38 MAPK, FR167653, which is being actively evaluated for its clinical use, was shown to be highly effective to ameliorate numerous inflammatory symptoms including experimentally induced acute colitis by suppressing the production of TNF and IL-1 [36]. Also, SB203580 was found to reduce mortality in a murine model of LPSinduced septic shock, through its inhibitory effects on LPSinduced TNF increases in mice [37]. LPS is widely known to trigger sepsis, a spectrum of clinical conditions characterized by systemic inflammation
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and coagulation that result from the host immune response to infection. Increasing evidence indicates that apoptosis may be responsible for sepsis-related mortality. Increased apoptosis in columnar epithelial cells in colon and ileum were detected in patients who died from sepsis [38]. It has been reported that patients with severe sepsis and septic shock have elevated plasma levels of nucleosomes, specific markers released by cells during the later stages of apoptosis [39], and elevated serum levels of epithelial cell apoptosis specific markers were also detected in critically ill septic patients [40]. Furthermore, increased serum levels of soluble Fas and FasL were demonstrated in patients with sepsis [41,42] and increased lymphoid tissue apoptosis in experimental baboons with Gram-negative bacteremic shock resulted from multiple cell death pathways including FasL-mediated signaling [43]. Moreover, inhibition of Fas/ FasL signaling was shown to improve septic survival in experimental mice [44]. Therefore, soluble DcR3, having been demonstrated for its therapeutic effects on many FasLmediated pathological conditions, may represent a potential therapeutic candidate in sepsis. The increases in soluble DcR3 release in many different cell types after endotoxin challenge and its overexpression in the acutely inflamed organ suggests that DcR3 may affect the outcome of cellular behavior in the course of acute inflammatory responses presumably via interaction with its ligands, FasL, LIGHT, and TL1A. Therefore, our findings might also aid the understanding of certain pathophysiological conditions mediated by these ligands of DcR3.
Acknowledgment
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The authors thank Dr. Prodromos Hytiroglou for his expert advice in the histological studies in the paper.
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Increased expression of soluble decoy receptor 3 in acutely inflamed intestinal epithelia Sunghee Kima,b,T, Anastasia Fotiaduc, Vassiliki Kotoulac a
Department of Biological Sciences, University of Alabama, Rm 280 Nott Hall, Tuscaloosa, AL 35487, USA b BioPowerTech, 4734 Bluegrass Pkwy, Tuscaloosa, AL 35406, USA c Department of Pathology, School of Medicine, Aristotle University, 54006 Thessaloniki, Greece Received 14 January 2005; accepted with revision 22 February 2005
Abstract Decoy receptor 3 (DcR3), a soluble receptor in the tumor necrosis factor (TNF) receptor family, is known to inhibit apoptosis mediated by pro-apoptotic TNF family cytokines such as Fas ligand (FasL), TL1A, and LIGHT. Therefore, the regulation of DcR3 expression under certain pathophysiological conditions is of interest since the level of soluble DcR3 would most likely affect the homeostasis of cells and tissues. We found that human intestinal epithelial cell (IEC) lines (SW480, SW620, and HT29) could selectively increase DcR3 release in response to lipopolysaccharide (LPS) and that all the cells preferentially expressed Toll-like receptor 4 (TLR-4). LPS-induced DcR3 releases in IECs appeared to be via the activation of mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and c-Jun NH2-terminal protein kinase (JNK), and the transcription factor NF-nB. Moreover, the increased expression of DcR3 in appendix epithelia from patients with acute appendicitis was demonstrated. Taken together, the results indicated that DcR3 might play an important role in the human intestinal epithelium during acute inflammatory processes caused by endotoxin challenge. D 2005 Elsevier Inc. All rights reserved. Keywords: FasL; TL1A; LIGHT; Inflammation; Appendix
Introduction DcR3, a soluble decoy receptor in the TNF receptor (TNFR) family, is known to bind to ligands of the TNF family, namely, FasL, LIGHT, and TL1A [1–3]. As a result, DcR3 is believed to block cellular effects that are mediated by the interaction between these ligands and their memAbbreviations: DcR3, decoy receptor 3; TNF, tumor necrosis factor; TNFR, TNF receptor; TLR, Toll-like receptor; IEC, intestinal epithelial cell; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinase; LPS, lipopolysaccharide; FasL, fas ligand; LTA, lipoteichoic acid; Th, T helper; DC1, myeloid-derived dendritic cells; DC2, plasmacytoid-derived dendritic cells; MEK, MAP kinase activating kinase, MEK; MEKK, MEK activating kinase. T Corresponding author. Department of Biological Sciences, University of Alabama, Room 280 Nott Hall, Tuscaloosa, AL 35487, USA. Fax: +1 205 348 5976. E-mail address: [email protected] (S. Kim). 1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.02.014
brane-bound cognate receptors in the TNFR family: TL1A and FasL bind to death-domain containing receptors DR3 and Fas, respectively, and LIGHT engages with either herpes virus entry mediator (HVEM) or lymphotoxin beta receptor (LThR). One of the prominent biological functions of DcR3 is anti-apoptosis. A soluble recombinant DcR3 was reported to prevent apoptosis mediated by FasL [1,4,5] and LIGHT [2,6], and to inhibit TL1A-mediated caspase induction [3]. Moreover, DcR3 was shown to be effective at attenuating FasL-induced mortality [7] and acute pulmonary inflammation [8] in experimental animal models, thus demonstrating the therapeutic effects of soluble recombinant DcR3 in FasL-mediated tissue injury. Similarly, transgenic overexpression of DcR3 was shown to play a protective role in a rodent diabetic model [9]. DcR3 was also reported to modulate a variety of immune responses apparently via interaction with its ligands. Ectopic
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expression of DcR3 in rodent gliomas resulted in the inhibition of the infiltration of T cells and macrophages [5], and the pre-treatment of T cells with soluble DcR3 was shown to suppress T-cell chemotaxis [10]. Also, recombinant DcR3 was shown to downregulate cytotoxic T-cell activity against alloantigens [11], and transduce costimulatory signals in T cells resulting in proliferation of T helper [Th] 1 and 2 cells, and lymphokine production in Th 1 cells [12]. Additionally, exogenous DcR3 was reported to modulate dendritic cell maturation and differentiation [13]. It is apparent that the effects of recombinant DcR3 could be diverse because of its unique ability to interact with the three different ligands of type II transmembrane proteins that exist in both soluble and membrane-bound forms. The addition of excess exogenous soluble DcR3 in culture system would undoubtedly affect the behavior of the cells which express the ligands and their functional cognate receptors on the cell surface. Hence, the regulation of endogenous DcR3 expression and its tissue distribution under certain pathophysiological conditions might be important for our understanding of the role of DcR3 in the immune system. Overexpression of DcR3 has been reported in lung and gastrointestinal tract tumors, lymphomas, and gliomas [1,2,5,14]. At first, it was hypothesized that DcR3 might aid cancer cells to evade FasL-mediated cytotoxic attack by host immune cells [1]. In contrast, we have reported elevated levels of soluble DcR3 in sera of patients with bacterial infections and renal failure [15,16]. Additionally, we have demonstrated the selective induction of DcR3 release in human primary antigen-presenting cells such as monocytes and myeloidderived dendritic cells (DC1) but not plasmacytoid-derived dendritic cells (DC2) in response to both Gram-positive and Gram-negative bacterial antigens such as LPS and lipoteichoic acid (LTA). Thus, we have suggested that, in addition to its possible involvement in innate immune responses, DcR3 might preferentially play a role in cellmediated immune responses in bacterial pathogenesis [15]. However, little is known about the expression of DcR3 in acutely inflamed human organs. This prompted us to investigate DcR3 expression in acutely inflamed human appendix epithelium and the signaling mechanism involved in overexpression of DcR3 in human intestinal epithelial cells following LPS challenge.
Materials and methods Reagents, cell lines, antibody, and human tissue samples Inhibitors, SB203580, SB202190, Ro31-8220, Wortmannin, and pyrrolidine dithiocarbamate (PDTC), and bacterial antigens, LPS (E. coli serotype 0111:B4) and LTAs (Bacillus subtilis and Staphylococcus aureus) were purchased from Sigma (St. Louis, MO). Kinase inhibitors,
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PD98059, U0126, and SP600125 were obtained from LC Laboratories (Woburn, MA). DMEM was obtained from Cellgro (Herndon, VA) and heat-inactivated FBS was from Invitrogen (Carlsbad, CA). Cell lines, SW480, SW620, HT29, and U937 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). We have previously reported the development of monoclonal antibodies (MAbs) against human soluble DcR3 [16]. After testing a dozen MAbs, we selected MD3F4 based on its highest sensitivity and specificity to react with DcR3 in human tissue samples. The specificity of MD3F4 was extensively tested with a vast array of normal and diseased human tissue samples in comparison with other isotype matching anti-DcR3 MAbs. Also, a lack of MD3F4 reactivity was confirmed in the presence of excess soluble DcR3 during incubation of tissue slides with MD3F4. Cell culture and ELISA Intestinal epithelial cell lines that were originally derived from colon adenocarcinomas, SW480, SW620, and HT29, were cultured in DMEM supplemented with 10% FBS. One day before inhibitor treatment, cells were plated at 2 105 cells/well in 24-well tissue culture plates. After aspirating the medium, fresh culture medium with or without inhibitors was added to the wells (0.5 ml/well). The culture plates were then incubated for 24 h at a 378C, 5% CO2 culture incubator. The levels of DcR3 in culture supernatants were measured by the human DcR3 specific quantitative ELISA as described in [16]. Briefly, 100 Al of culture supernatants was added to wells of 96-well ELISA plates that were coated with the capture antibody (MD3E2) and blocked with 3% BSA. The standard DcR3 protein and test samples were added to the wells and the plates were incubated overnight at 48C. After washing, a biotinylated detection antibody (MD3B1) was incubated to wells for 2 h at room temperature. After washing, a peroxidase-conjugated streptavidin (Vector Lab., Burlingame, CA) was added to the wells. Color was developed using TMB substrate solution (KPL, Gaithersburg, MD). After stopping the color reaction with 1 N H2SO4, plates were read at absorbance 450 nm in an Emax ELISA plate reader (Molecular Device Co., Sunnyvale, CA). Real-time quantitative PCR (TaqMan) and reverse transcription (RT)-PCR analyses Total RNAs from SW480, SW620, HT29, and U937 were harvested by using Trizol RNA extraction solution (Invitrogen). RT-PCR was performed using SuperScript One-Step RT-PCR System according to the manufacturer’s recommendation (Invitrogen). A total of 50 Al reaction mixture contained 100 ng RNA, 0.2 AM each of forward and reverse primers specific for human TLR2 (5Vcccatttccgtctttttgaa-3V and 5V-cgcagctctcagatttaccc-3V), TLR4 (5V-tgagcagtcgtgctggtatc-3V and 5V-cagggcttttctgagtcgtc-3V), TLR5 (5V-ggaaccagctcctagctcct-3V, 5V-aagagg-
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gaaaccccagagaa-3V), or TLR9 (5V-cagcagctctgcagtacgtc-3V and 5V-aaggccaggtaattgtcacg-3V). The reaction was carried out at 458C for 30 min and 948C for 2 min followed by 40 cycles of 948C 15 s, 558C 30 s, 688C 15 s. The primers were designed using the web-accessible Primer 3 software [17] and the primers were purchased from Integrated DNA Technologies (Coralville, IA). For TaqMan analysis, the reaction was carried out in a 25-Al reaction mixture containing 25 ng of RNA, 0.6 AM each primers specific for human DcR3 (forward: 5Vctgatcctggccccctctta-3V; reverse: 5V-ttcttctatttaaaaaaaagcctctttca-3V; probe:5-tctacatccttggcaccccacttgca-3V). Probes labeled with 6-FAM and TAMRA were purchased from Biosource International (Camarillo, CA, USA). PCR products were measured with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). To ensure no contamination from genomic DNA, parallel reactions were conducted without reverse transcriptase. The level of DcR3 mRNA was normalized to the mRNA level of 18S rRNA in the same sample. Immunohistochemistry (IHC) Archival paraffin blocks from ten routine surgical pathology cases, previously diagnosed with acute appendicitis upon histological examination, were used. Patient age ranged 13–50 years (median: 22). Each block contained sections from the apical and the basal part of the appendectomy specimen. Apical and basal sections of the appendix from each case were sliced at 2 Am thickness and mounted on positively charged slides. Paraffin slides were then incubated overnight at 558C. After dewaxing in multiple fresh xylenes, the slides were rehydrated in descending ethanol changes. Endogenous peroxidase was blocked for 20 min with 3% H2O2 in methanol and, subsequently, slides were rinsed in distilled water. No epitope unmasking was used since this step was determined to be unnecessary for DcR3 reactivity with MD3F4. To abrogate non-specific binding, sections were incubated with a commercially available blocking solution, Powerblock (Innogenex, San Ramon, CA), at 378C for 30 min. Slides
were incubated with MD3F4 (2.5 Ag/ml) diluted in Powerblock at 378C for 1 h. After washing in PBS, a secondary biotinylated anti-mouse antibody (DAKO, Glostroup, Denmark), diluted at 1:200 in Powerblock, was applied for 1 h at room temperature. Color development was achieved with streptavidin–biotin complex and liquid diaminobenzidine (DAB, both from DAKO). No counterstaining was used for the appendicitis specimens. Qualitative evaluation of DcR3 localization was accomplished by comparison to H&E stained serial sections for each case.
Results Increases of DcR3 protein in colon epithelial cells following LPS stimulation By utilizing quantitative ELISA that was validated to measure soluble DcR3 in human cell culture supernatants and sera samples [16], the regulation of soluble DcR3 expression was investigated in the human IEC lines SW480, SW620, and HT29 that are derived from colorectal carcinomas. Previously, we had reported the purification of native soluble DcR3 secreted in SW480 culture [16]. Also, we showed that SW480 expressed one order of magnitude higher soluble DcR3 compared to SW620 in culture supernatants [16]. Regardless of their constitutive DcR3 expression, following LPS treatment, both SW480 and SW620 released 3- to 4-fold increased DcR3 compared with the cells with no LPS stimulation (Fig. 1). Additionally, HT29 which expressed no detectable DcR3 (apparently lower than the detection limit of ELISA, 36 pg/ml) could also release detectable amounts of DcR3 after LPS treatment. All three cell lines appeared to be highly responsive to LPS to release DcR3: LPS concentration as low as 0.1 ng/ml could induce protein release and 10 ng/ml was sufficient to maximize the release of soluble DcR3 from the cells, and there was no further induction of DcR3 at higher LPS concentrations. Unlike DC1 that responded to both Gram-positive and Gram-negative bacterial antigens, LPS and LTA, respectively [15], these IEC cell lines only responded to LPS and there was
Fig. 1. Selective induction of DcR3 in IECs, SW480, SW620, and HT29, following LPS challenge. Cells seeded in 24-well culture plates were incubated in the presence of LPS at the indicated concentrations for 24 h. The levels of soluble DcR3 in culture supernatants were measured by ELISA. Experiments were conducted at least three independent times with triplicate samples. Data are mean F SD and statistical analysis was performed using unpaired t test. All doses of LPS (0.1, 1, 10 ng/ml) increased DcR3 release in all the cell lines compared to the cells without LPS. P values between untreated and LPS (grouped all doses) treated SW480 and SW620 were less than 0.001.
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Fig. 2. Increases of DcR3 mRNAs in SW480 cells following LPS stimulation. Cells seeded in 6-well culture plates were incubated in the presence or absence of LPS or LTA for 8 h. Total RNAs were harvested from the cells and then subjected to TaqMan. The primers were expected to amplify 81 base pairs (1000~1080) of the DcR3 nucleotide sequence obtained from Genbank accession number AF1044419. Experiments were independently conducted two times with triplicate samples and data are mean F SD. LTA (B) is an LTA source from B. subtilus, and LTA (S) is an LTA source from S. aureus. No significant increases in DcR3 were observed by the two LTAs. The increase of DcR3 mRNA by LPS was highly significant with P value less than 0.0001 according to unpaired t test.
no noticeable increase in DcR3 in these three cells following the treatment with LTA at various concentrations. In accordance with protein expression, the treatment of SW480 with two bacterial LTA sources did not increase the expression of DcR3 mRNA while LPS stimulation resulted in up to a 4-fold increase in the message (Fig. 2). The selective responsiveness to LPS might be in part due to the preferential expression of Toll-like receptor 4 (TLR-4) in IECs. While the U937 monocytic cell line that was employed as a control cell line to show integrity of the primer sets expressed detectable mRNA levels of TLR-2, TLR-4, TLR-5, and TLR-9, the three IEC lines appeared to predominantly express TLR-4 mRNA (Fig. 3). It has been established that LTA and LPS mediate signaling preferentially via TLR-2 and TLR-4, respectively [18].
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which is a potent selective inhibitor of MAP kinase activating kinase (MEK). PD98059 inhibits phosphorylation of MEK by the upstream kinases, MEK activating kinase 1 (MEKK-1) and Raf-1 [20]. Also, U0126 was utilized since this compound selectively blocks MEK1/2, the kinases upstream of ERK1/2 [21,22]. This compound was also shown to inhibit AP-1 transactivation by suppressing synthesis of c-jun and cfos mRNA and protein by MAP kinases [21,22]. A specific inhibitor of JNK, SP600125, was also employed. This relatively new synthetic inhibitor selectively inhibits JNK1, 2, 3 thus resulting in the inhibition of AP-1 phosphorylation [23]. The upstream kinase of MAPK pathway, MEKK-1, is required for JNK activation by LPS [24]. MEK7, a downstream kinase of MEKK, activates only JNK while MEK4 activates both JNK and p38 [25]. Once activated, JNK phosphorylates c-Jun which dimerizes with c-Fos to activate AP-1. The treatment of cells with ERK and JNK inhibitors (U0126, PD98059, and SP600125) in the presence of 10 ng/ ml LPS resulted in significant inhibition of DcR3 release in SW480 (Fig. 4). However, potent pyridinyl imidazole inhibitors of p38 MAP kinase, SB202190 and SB203580, had no inhibitory effects on the cells. Instead, these inhibitors, at concentrations at 0.5–1 AM, resulted in DcR3 increases up to 30% in the cells (Fig. 4). There was no significant DcR3 inhibition by both inhibitors, but rather slightly deteriorated cell viability above 2 AM of the inhibitors. The increases in DcR3 release by these inhibitors might be due to the activation of Raf-1, an upstream kinase of MEK, by the inhibitors. Previously, SB203580 was
Involvement of MAP kinase pathways in signaling of DcR3 release The signaling mechanisms involved in DcR3 release was studied utilizing SW480 cells since this cell line expresses a relatively high level of DcR3. Specific inhibitors of intracellular kinases in signaling pathways were chosen based on their well-recognized specificity and no apparent cytotoxic effect on the cells even at ranges well above IC50 which was previously reported in the literature. The cells were treated with inhibitor concentrations around IC50 and the cell viability was always confirmed at the end of inhibitor treatment using MTT assay. The activation of MAP kinases, ERK1/2, JNK and p38, in the TLR-mediated signal transduction pathway has been well established (reviewed in [19]). Therefore, we employed specific inhibitors of MAPK pathways such as PD98059
Fig. 3. Preferential expression of TLR-4 in IECs. Total RNAs from IEC lines, SW480, SW620, and HT29, were subjected to RT-PCR. The U937 cell line was included as a control to show the specificity of primer sets. A master reaction mixture was made for RNA from each of the cell lines and then primers for each TLR were added to aliquots. Each RT-PCR reaction tube contained 100 ng of RNA. One fifth of the RT-PCR reaction volume (10 Al) was analyzed on 1 or 1.2% agarose gels. A 2-kb DNA ladder marker was obtained from Invitrogen. Expected PCR fragments for TLR-2, -4, -5, and -9 are 185 (2288~2472), 167 (3709~3875), 196 (2373~2568), 224 (1921~2144) base pairs (bp), respectively. Complete cDNA sequences for TLR-2, -4, -5, and -9 were obtained from Genbank accession numbers U88878, AF172169, AB060695, and AF245704, respectively.
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Fig. 4. LPS-induced DcR3 release in SW480 cells was inhibited by inhibitors of MAPK (ERK1/2 and JNK) and NF-nB. Cells seeded in 24-well tissue culture plates were treated with both LPS (10 ng/ml) and the indicated inhibitors for 24 h. DcR3 release in culture supernatants of LPS-stimulated cells with or without inhibitor treatments were quantified by ELISA. Experiments were conducted two independent times with triplicate samples and data are mean F SD. PDTC (50 AM, 100 AM), PD98059 (5, 10, 20 AM), U0126 (100, 200, 400 nM), and SP600125 (20, 40 AM) significantly inhibited DcR3 release in the cells compared to the cells with no inhibitor treatment. P values between untreated and each inhibitor (grouped according to the indicated doses) were less than 0.001. SB202190 and SB203580 significantly increased DcR3 release with P values less than 0.05 between untreated and inhibitor (combined 0.5 and 1 AM) according to unpaired t test.
reported to activate Raf-1 [26]. It is known that LPS signaling involves Raf-1 activation [27]. The inhibition of DcR3 releases by ERK and JNK inhibitors was more prominent in LPS-treated cells since the inhibition by the same inhibitors in the cells treated with PMA, a potent activator of protein kinase C (PKC), was at most 15% to 20% by U0126 and PD98059, respectively. We found that PMA could also increase DcR3 release up to one order of magnitude higher in the three IEC lines, and, as expected, a PKC inhibitor Ro31-8220 (inhibits conventional PKC isoforms, a, h-I, h-II, and g) could effectively inhibit PMA activated DcR3 release in the IEC cell lines. In contrast, Ro31-8220 had no inhibitory effects on the IEC cells following LPS treatment; therefore, MAP kinases but not conventional PKC isoforms are involved in LPS-inducible DcR3 release in IECs (Fig. 4). Also, a PI3-K (phosphatidylinositol 3kinase) inhibitor, Wortmannin, had no inhibitory effect on the cells (Fig. 4). It is widely known that LPS activated downstream transcription factor NF-nB [19]. As expected, a specific inhibitor of NF-nB activation, PDTC, selectively inhibited LPS-induced DcR3 release about 70% (Fig. 4). In addition, we observed that the constitutive expression of soluble DcR3 in SW480 cells could also be inhibited by the same inhibitors of ERK and JNK that showed the inhibitory effects on LPS-inducible DcR3 release (the inhibition profile was similar to Fig. 4) but the two p38 inhibitors had no inhibitory effects in the protein expression (data not presented). Also, PDTC did not inhibit any constitutive DcR3 release in SW480 cells probably due to the lack of activated NF-nB activity in the resting cells. Based on the results from the inhibitor studies, we have deduced the signaling pathways that are most likely involved in LPS-inducible DcR3 release in the cells as shown (Fig. 5).
Immunolocalization of DcR3 in acutely inflamed appendix epithelium We then sought to investigate the possible changes in DcR3 expressions in human intestinal epithelia with acute
Fig. 5. A schematic presentation of signaling mechanisms involved in DcR3 releases after LPS challenge. LPS stimulates cells via TLR-4 on the cell surface that leads to the activation of MEKK-1 and Raf-1 followed by downstream kinases, MEKs. MEK1/2 activates ERK1/2 while MEK4/7 activates JNK. The activation of ERK and JNK will result in the downstream transcription factors including AP-1. The activation of NF-nB will be likely achieved via the pathway involving NF-nB inducing kinase (NIK) pathways (not shown in the diagram). The binding of transcription factors including NF-nB and AP-1 to the promoter of DcR3 gene will eventually cause release of soluble DcR3 in intestinal epithelial cells. Inhibitors are indicated in parentheses.
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inflammation. A common example of acute inflammation whereupon surgical specimens for histological evaluation are obtained is spontaneous acute appendicitis. As shown in Fig. 6, non-inflamed areas in the basal part of the organ were used as a normal control to evaluate the inflammatory response usually localized in the apical part of the organ. In fact, absence of inflammation was noticed in 7 out of 10 cases in the basal section of the removed appendix, minimal to mild inflammation in 2 cases and severe ulcerative one in 1 case. Various degrees of inflammation were observed in all 10 cases in the apical part of the appendix, justifying the clinical diagnosis and removal of the organ. In the non-inflammatory (normal) mucosa, submucosa, and muscular wall components (epithelial cells, local lymphocytic aggregates and follicles, smooth muscle cells, as well as capillary, small and larger vessel endothelia), DcR3 was either absent or barely detectable. Only mast cells, often quite numerous in the submucosa, as well as few monocytes, were found positive with the DcR3 antibody in these histologically non-inflammatory areas (Fig. 6, panels A–D). The expression of DcR3 in mast cells is of interest. A previously unrecognized protective role of mast cells in acute bacterial peritonitis was reported, and
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its protective role against enterobacterial infection was reported to be mediated by TLR-4 [28,29]. By contrast, most of the above mentioned tissue components were found positive in areas with histological signs of inflammation in all ten cases examined (Fig. 6, panels E–L). DcR3 immunoreactivity was observed in the surface epithelial cells and in the glandular structures; in the inflammatory cell infiltrates, inside and diffusely around the cells; in the endothelial cells of sizes of vessels ranging from capillaries up to the larger vessels located in the submucosa; in the smooth muscle cells of the vessel wall and in the muscularis externa; and, in fibroblasts in the lamina propria. Practically, DcR3 was localized in all inflamed tissue components and layers, according to the extent of inflammatory infiltration, with one exception: the germinal centers of reactive follicles remained negative (Fig. 6, panels J and K), as in the non-inflamed tissue (Fig. 6, panel B). In regions with inflammatory infiltrates, diffuse DcR3 positivity was also observed around the cells, in line with the soluble nature of this protein. In the same areas, DcR3 positive lymphocytes were also present in vascular lumina (Fig. 6, panel L). As a control for antibody specificity, erythrocytes and necrotic debris did not stain for DcR3, as expected.
Fig. 6. Representative examples of DcR3 localization in tissue components of normal and acutely inflamed vermiform appendix. (A~D) Tissue sections with no detectable inflammation in the basal part of the appendix. None or very weak DcR3 expression is shown in the epithelial and endothelial cells, vessel walls, as well as in most stromal components (B, C). Mast cells (m) and scarce monocytes are positive for DcR3 (C). (E ~L) Tissue sections from acute appendicitis specimens. (E~H) Severe inflammation of the appendiceal wall up to the serosa spanning the whole organ. (I~L) Mild inflammation of the mucosa in the apical part of the appendix, without involving the submucosa. In the inflammatory tissue, epithelial cells, lymphocytic infiltrates, endothelia, and smooth muscle vascular walls are positive for DcR3. Germinal centers of the lymphatic follicles (J, K, compared with B) remain largely negative. In L, DcR3 positive lymphocytes can be recognized in vascular lumina. e = Epithelial cells; li = lymphocytic infiltrate; lf = lymphatic follicle; v = vessel; arrows = endothelial cells; * = erythrocytes, negative for DcR3, as expected. Stains: A, E, G, I, H, and E; all other, DcR3 IHC. Original magnifications: A, B, E, I, J, 40; F, K, 100; C, D, G, H, L, 400.
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Discussion In spite of their basal levels of DcR3, all three colonic epithelial cell lines significantly increased DcR3 secretion in response to LPS that is widely known to activate NF-nB, a central regulator of immune and inflammation responses. The activation of NF-nB has been detected in epithelial cells of inflamed intestinal mucosa as well as in intestinal mucosa during endotoxemia [30,31]. Therefore, our data seemed to indicate that the induction of DcR3 would most likely occur in most parts of the human intestinal epithelium in response to LPS challenge. The pathogenesis of acute appendicitis is believed to involve bacterial infection due to the blockage of appendix lumen. Recently, it has been suggested that bacterial LPS may play a central role in the development of appendicitis and in the consequently developing generalized systemic anti-inflammatory response, which is observed in the majority of patients with clinically characterized disease [32]. The extensive DcR3 staining in many different cell types in inflamed appendix appeared to be in line with our findings that LPS treatment results in increases of DcR3 in a variety of human primary cells including monocytes and DC1 [15], and dermal microvascular endothelial cells (HMVEC) and keratinocytes (unpublished data). It is also likely that the cytokines that are released in the course of acute inflammatory reaction are a contributing factor to the extensive DcR3 expression in acute appendicitis. Although not to the extent of LPS stimulation, we found that proinflammatory cytokines such as TNF and IL-1 can also induce soluble DcR3 release in HMVEC (unpublished data). In sharp contrast to intensive DcR3 staining in surrounding epithelial cells, infiltrating inflammatory and endothelial cells, lymphoid follicles appeared to be devoid of DcR3 immunoreactivity. Similarly, we had reported that human primary B and T cells in culture did not express any significant level of DcR3 even in the presence of activators: LPS and S. aureus Cowan I (SAC) for B cells and phytohemagglutinin (PHA) and anti-CD3/CD28 for T cells [15]. The intestinal mucosa is regarded as a first line of defense against invading pathogens in the intestine. The overexpresion of DcR3 in acutely inflamed appendix indicates that DcR3 might play a potentially important role in the innate immune response in the intestine. Similarly to the biological effects that were observed with recombinant soluble DcR3 in culture systems, the endogenously secreted DcR3 might also exert its anti-apoptotic activity via binding to its pro-apoptotic ligands expressed in the course of acute inflammatory responses in the intestine. On the other hand, overly elevated DcR3 in the intestine under certain pathological conditions might also contribute to uncontrolled cell growth resulting in colorectal neoplasia. This might be one of the reasons that DcR3 overexpression was detected in certain colorectal carcinomas.
Previously, IEC lines including HT29 were shown to be unresponsive to Gram-positive bacterial antigens primarily due to the lack of TLR-2 protein [33]. Our result also seemed to indicate that IECs selectively induce DcR3 in response to LPS but not to LTA in part due to their preferential expression of TLR-4 transcripts. Unlike monocytes and DC1 that responded to both LPS and LTA to induce DcR3, we had reported that DC2 which was known to express neither TLR-2 nor TLR-4 did not induce DcR3 after LPS or LTA stimulation [15]. DC2, although it was known to express TLR-7 and TLR-9, did not induce DcR3 release in response to single-stranded RNA virus infection or bacterial DNA sequence CpG-oligodeoxynucleotides while it released IFN-a against both agents in the same culture supernatant [15]. In addition, we found that human primary keratinocytes could induce DcR3 in response to both LPS and LTA while human primary HMVEC only responded to LPS (unpublished data). It has been reported that human keratinocytes express both functional TLR-4 and TLR-2 [34] while HMVEC expresses functional TLR-4 but not TLR-2 [35]. Therefore, the regulation of DcR3 in response to LPS and LTA appeared to be critically dependent on the presence of functional TLR-4 and TLR-2 in different human cell types. As it was seen with DC1, the increase in DcR3 release in IECs following LPS was significantly inhibited by the inhibitors of ERK1/2 (p44/p42) MAP kinase. However, inhibitors of p38 MAPK again showed no effects on DcR3 release in IECs with or without LPS stimulation. We had already shown that the p38 inhibitor SB203580 did not inhibit DcR3 release in human primary DC1 after LPS challenge [15]. The MAPK p38 is well known to play a critical role in induction of the proinflammatory cytokines TNF and IL-1 in monocytes, and we had also demonstrated that TNF release in DC1 after LPS stimulation could be significantly inhibited by the p38 MAPK inhibitor [15]. Hence, the lack of p38 involvement might be a characteristic feature in DcR3 regulation, compared to proinflammatory cytokines such as TNF and IL-1. If DcR3 turns out to play a protective role in acute inflammatory conditions induced by LPS, the inhibition of p38 might prove to be more effective in controlling inflammation than the inhibition of upstream MAPK pathway kinases or the downstream NF-nB that indiscriminately control expression of both survival and death molecules that are activated in the course of inflammatory responses. An inhibitor of p38 MAPK, FR167653, which is being actively evaluated for its clinical use, was shown to be highly effective to ameliorate numerous inflammatory symptoms including experimentally induced acute colitis by suppressing the production of TNF and IL-1 [36]. Also, SB203580 was found to reduce mortality in a murine model of LPSinduced septic shock, through its inhibitory effects on LPSinduced TNF increases in mice [37]. LPS is widely known to trigger sepsis, a spectrum of clinical conditions characterized by systemic inflammation
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and coagulation that result from the host immune response to infection. Increasing evidence indicates that apoptosis may be responsible for sepsis-related mortality. Increased apoptosis in columnar epithelial cells in colon and ileum were detected in patients who died from sepsis [38]. It has been reported that patients with severe sepsis and septic shock have elevated plasma levels of nucleosomes, specific markers released by cells during the later stages of apoptosis [39], and elevated serum levels of epithelial cell apoptosis specific markers were also detected in critically ill septic patients [40]. Furthermore, increased serum levels of soluble Fas and FasL were demonstrated in patients with sepsis [41,42] and increased lymphoid tissue apoptosis in experimental baboons with Gram-negative bacteremic shock resulted from multiple cell death pathways including FasL-mediated signaling [43]. Moreover, inhibition of Fas/ FasL signaling was shown to improve septic survival in experimental mice [44]. Therefore, soluble DcR3, having been demonstrated for its therapeutic effects on many FasLmediated pathological conditions, may represent a potential therapeutic candidate in sepsis. The increases in soluble DcR3 release in many different cell types after endotoxin challenge and its overexpression in the acutely inflamed organ suggests that DcR3 may affect the outcome of cellular behavior in the course of acute inflammatory responses presumably via interaction with its ligands, FasL, LIGHT, and TL1A. Therefore, our findings might also aid the understanding of certain pathophysiological conditions mediated by these ligands of DcR3.
Acknowledgment
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The authors thank Dr. Prodromos Hytiroglou for his expert advice in the histological studies in the paper.
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