-Mediated Autocrine Activation of Human Dermal FibroblastsFibroblasts constitute a large pool of resident cells in skin, which are an important source for cytokines early in inflammation, before leukocytes have been attracted to the site. Interleukin-1
(IL-1) and IL-1
are induced in fibroblasts upon exposure to pro-inflammatory cytokines, such as tumor necrosis factor (TNF-), or IL-1 itself. IL-1 is a very potent cytokine. Typically, IL-1 concentrations in the pM range induce a robust response in many cell types, triggering the production of multiple inflammatory mediators, including chemokines. These in turn attract leukocytes, which further amplify the inflammatory reaction (Dinarello, 1996;Karmiol and Phan, 1997;Cullinan et al, 1998). IL-1 mRNA and protein expression is tightly regulated at the level of transcription, mRNA stability, and translation. In addition, IL-1 and IL-1 are post-translationally modified by the processing enzymes calpain and caspase-1, respectively (Dinarello, 1996).
IL-1 and IL-1 both act via the same receptor complex, consisting of IL-1 receptor type I, the ligand binding subunit, and IL-1 receptor accessory protein (IL-1RAcP), the signaling subunit. In fibroblasts, IL-1 induces several intracellular signaling pathways that lead to the expression of proteases, prostaglandins, cytokines, and chemokines. Activated fibroblasts express IL-1, which can bind to and activate IL-1 receptors on the same cells in an autocrine fashion. Through this mechanism, fibroblasts could trigger and amplify a local inflammatory response.
Although IL-1 primarily acts as an extracellular cytokine, both IL-1 and IL-1 lack a signal peptide and thus are not secreted along the classical pathway through the endoplasmic reticulum and Golgi apparatus (Rubartelli et al, 1990). An alternative secretory pathway has been proposed that may involve transporters of the ATP binding cassette (ABC) transporter family. In eukaryotes as well as prokaryotes, ABC transporters translocate a wide range of different substrates across cell membranes by an energy-dependent mechanism (Broccardo et al, 1999;Klein et al, 1999). As many as fifty different ABC transporter genes are estimated to exist in humans. Glybenclamide is a known potent inhibitor of ABC1, a member of the ABCA subclass. Using expression of ABC1 in Xenopus laevis oocytes, this compound almost completely blocked ABC1 transporter activity (Becq et al, 1997). It has been suggested that ABC1 is involved in IL-1 secretion, as glybenclamide also blocked the secretion of IL-1 in human monocytes (Hamon et al, 1997;Andrei et al, 1999).
IL-1 is thought to play a role in several inflammatory conditions of the skin, such as contact and atopic dermatitis (Junghans et al, 1998;Murphy et al, 2000;Ulfgren et al, 2000). IL-1-deficient mice display impaired contact hypersensitivity (Shornick et al, 1996), whereas mice that overexpress IL-1 in the epidermis develop spontaneous focal cutaneous inflammation (Groves et al, 1995). Specific inhibition of IL-1 leads to an attenuation of inflammatory processes. Recombinant IL-1 receptor antagonist (IL-1RA) is an efficacious treatment of arthritis, or endotoxin shock, but must be given at large doses due to its low affinity towards IL-1 receptors (Burger et al, 1995;Ohlsson et al, 1990;Dinarello, 1996;Joosten et al, 1999). Transgenic mice that overexpress IL-1 receptor type II, a nonsignaling "decoy receptor", in the epidermis display a weaker inflammatory response in the skin (Rauschmayr et al, 1997). Thus, antagonizing IL-1 activity is a promising way to downregulate inflammation in the skin.
We hypothesized that ABC transporter inhibitors, as suppressors of IL-1 secretion, could interfere with IL-1-mediated autocrine activation in fibroblasts. Using IL-1-transfected fibroblasts, we describe autocrine induction of IL-8. In this system, glybenclamide inhibited IL-8 production. We corroborated the inhibitory effect of glybenclamide in fibroblasts that were stimulated with TNF- to express endogenous IL-1. Finally, we used transfected African green monkey kidney cells (CV1 origin SV40) (COS-1) cells overexpressing either IL-1 or IL-1 to directly demonstrate the inhibition of IL-1 secretion by glybenclamide.
Materials and methods
Cell culture, expression plasmids, transfection
Primary human dermal fibroblasts (HDF), established from a surgical specimen of a 6-y-old boy, were kindly provided by Dr. Ulrich Wiesmann, University of Bern, Switzerland. COS-1 cells were a kind gift of Dr. Erwin Sterchi, Institute of Biochemistry and Molecular Biology, University of Bern. HDF and COS-1 cells were cultured in HEPES-buffered (25 mM) minimum essential medium (Gibco BRL Life Technologies, Paisley, U.K.), supplemented with 10% fetal bovine serum (Seromed/Biochrom, Berlin, Germany), 2 mM glutamine, and antibiotics (100 IE per ml penicillin, 100 
g per ml streptomycin). Non-essential amino acids (100 M each) were additionally supplied to the medium for HDF cultures. HDF were used at passages below 20.
Plasmids containing full-length cDNAs for human IL-1 and IL-1 (GenBank accession numbers M15329 and M15330) (Nishida et al, 1987) were kindly provided by Dr. Christoph Müller, Institute of Pathology, University of Bern. Coding sequences were amplified by polymerase chain reaction (PCR) using primers that included BamHI restriction sites and were cloned into the eukaryotic expression vector pSG5 (Stratagene, La Jolla, CA). Primers were GAC ATG GAT CCA AAG AAG TCA AGA GGC/GTT AAG GAT CCT ACG CCT GGT TTT CCA G for IL-1, and GAC ATG GAT CCG CAG CCA TGG CAG AAG TAC C/GTT AAG GAT CCT TAG GAA GAC ACA AAT TGC for IL-1. IL-1 coding regions in vector constructs were verified by sequencing. HDF were transfected using Effectene (Qiagen, Basel, Switzerland) according to the manufacturer's instructions. COS-1 cells were transfected using diethylaminoethyl dextran as described previously (Dumermuth et al, 1993). Transfection efficiency was monitored using green fluorescent protein (Stauber et al, 1998) (plasmid GFPsg25 was a gift from Dr. Volker Heussler, Institute of Animal Pathology, University of Bern).
Treatment of cell cultures with cytokines, antibodies, and glybenclamide
One day after transfection, fibroblasts were incubated overnight with either of the following agents: neutralizing anti-IL-1 monoclonal antibody (MAB601), IL-1RA (both from R&D Systems, Abingdon, U.K.), glybenclamide [Sigma, St. Louis, MI, prepared as a 100 mM stock solution in dimethylsulfoxide (DMSO)]. In a separate set of experiments, glybenclamide was added to confluent HDF cultures that were stimulated for 24 h with 10 ng per ml TNF- (Peprotech, London, U.K.) or 1 ng per ml IL-1 (R&D Systems). TNF- treatment was initiated overnight, and cells were subsequently incubated with glybenclamide in the continuous presence of the cytokine. In IL-1-treated cells, glybenclamide was present over the whole 24 h incubation period. Where glybenclamide was used, DMSO was adjusted to the same concentration in the culture medium, never exceeding 0.8%. Supernatants were collected and supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 200 g per ml pepstatin, 200 g per ml aprotinin, 1 mg per ml leupeptin, 3.5 mg per ml benzamidine, from Sigma). Cells were harvested in cell culture lysis reagent (Promega, Madison, WI) containing protease inhibitors. To exclude a cytotoxic effect of glybenclamide and/or DMSO, lactate dehydrogenase (LDH) activities were measured in cell supernatants.
Enzyme-linked immunosorbent assay (ELISA)
ELISA for IL-8 and IL-1 were performed using a matched pair of antibodies (R&D Systems): anti-IL-8 MAB208 and anti-IL-1 MAB601 primary antibodies were used in combination with biotinylated secondary antibodies. Bound antibody was detected using streptavidin peroxidase (Southern Biotechnology Associates, Birmingham, AL) and ABTS (Sigma) as chromogenic substrate. Samples were measured in double from multiple dilutions and quantified against standard curves obtained with recombinant cytokines (standard 10 ng per ml to 14 pg per ml).
Western blot
For IL-1 and IL-1, equal amounts of cell lysates (100 ng protein) and supernatants (10 l) from IL-1- and IL-1-transfected COS-1 cells were separated by 14% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and blotted onto PVDF membrane. For caspase-1, protein extracts (100 g) from HDF treated with TNF- and/or glybenclamide and from control HDF were separated by 12% SDS-PAGE. Membranes were blocked with 5% skim milk in isotonic Tris-buffered saline, pH 7.4, and incubated with either antihuman IL-1 polyclonal antibody (Peprotech), antihuman IL-1 monoclonal antibody (MAB601, R&D Systems), or antihuman caspase-1 polyclonal antibody (Santa Cruz), and corresponding peroxidase-coupled anti-IgG polyclonal antibody (AmershamPharmaciaBiotech). Immunoreactive protein bands were visualized onto radiographic films using enhanced chemiluminescence (SuperSignal West Femto, Pierce, Rockford, IL). Densitometric measurements were performed on digitized radiographs using the image analysis software Gel-Base/GelBlot-Pro (Synoptics, Cambridge, U.K.).
Caspase-1 assay
Caspase-1 activity in HDF was measured using a kit from R&D Systems. This assay is based on the specific peptide substrate WEDH-p-nitroanilide.
Results
IL-1
-transfected fibroblasts release IL-8
As treatment of fibroblasts with exogenous IL-1 induces IL-8, these cells may enhance IL-8 expression through the autocrine activity of endogenously expressed IL-1. To demonstrate such a mechanism, we transfected HDF with the IL-1 precursor and monitored the consecutive daily IL-8 production. IL-1 protein expression in IL-1-transfected cells peaked after 3-4 d. In marked contrast, IL-1 was hardly expressed in vector-transfected control cells Figure 1a. Protein concentration in cell extracts did not differ between IL-1- and vector-transfected cell populations (not shown), indicating that cell densities were similar. IL-1-transfected HDF released nine to 20 times more IL-8 than vector-transfected control cells (Figure 1b, reaching a maximum after 3-4 d, coincident with maximum IL-1 expression. Incubation with IL-1RA or neutralizing antibodies against IL-1 led to a 75% reduction of IL-8 (Figure 1b, inset). Thus, IL-1 is present extracellularly as a soluble or cell-surface-associated molecule to allow induction of IL-8 in an autocrine fashion. In the medium, IL-1 was only detectable 3 d after transfection at concentrations close to the detection limit of the ELISA used (15 pg per ml, not shown).
Figure 1.
Interleukin-8 is induced in IL-1-transfected human primary fibroblasts. HDF were transfected with the expression vector pSG5 containing the coding sequence of the IL-1 precursor under the control of an SV40 promoter (closed symbols) or with vector alone (open symbols). Using ELISA, IL-1 and IL-8 were measured in cells and supernatants over 2-6 d after transfection. (A) IL-1 expression in cells. (B) Daily release of IL-8. Inset: Inhibition of IL-8 production in the presence of IL-1RA (25 ng per ml) and anti-IL-1 monoclonal antibody (10 g per ml) relative to control cells. Results shown are representative for experiments repeated three times.
Glybenclamide reduces IL-8 production in IL-1-transfected and TNF-
-treated fibroblasts
The ABC1 transporter inhibitor glybenclamide, as a suppressor of IL-1 secretion (Becq et al, 1997), dose-dependently reduced IL-8 production in IL-1-transfected fibroblasts Figure 2b. After an overnight incubation with 100 M glybenclamide, IL-8 in the supernatant was 40% compared to IL-1-transfected control cells. In contrast, glybenclamide did not affect IL-8 levels in supernatants of cells that were transfected with vector alone (13% and 12% in the presence of 100 M glybenclamide and DMSO, respectively). IL-8 was not detectable in cellular extracts, indicating that IL-8 did not accumulate intracellularly and was secreted efficiently in the presence of glybenclamide (not shown), confirming previous findings that glybenclamide does not affect secretion of proteins along the classical pathway (Hamon et al, 1997). Thus, reduction of IL-8 in the supernatant by glybenclamide is attributable to decreased production. Slightly decreased cellular IL-1 levels were observed in the presence of the highest glybenclamide dose Figure 2.
Figure 2.
Glybenclamide reduces IL-8 production in IL-1-transfected fibroblasts. HDF were transfected with IL-1 (pSG5-IL-1), with vector alone (pSG5), or were processed for transfection without addition of DNA (none). The following day, media were changed, and cells were incubated overnight with glybenclamide at indicated concentrations, or with DMSO alone. IL-1 in cells (A) or IL-8 levels in supernatants (B) are given as relative values compared to IL-1-transfected cells incubated without glybenclamide. n.d. = not detectable. IL-1 was not detectable in cells transfected with vector alone. Experiments were repeated three times, and a representative example is shown.
HDF were treated overnight with TNF-, which strongly induces IL-1 concomitantly with IL-8 (Sporri et al, 1998). Subsequent incubation with glybenclamide dose-dependently reduced IL-8 production, revealing a similar inhibitory pattern as in IL-1-transfected cells Figure 3a. In the presence of 100 M glybenclamide, IL-8 in the supernatant was 58% compared to control cells. The inhibitory effect of glybenclamide was not restricted to IL-8, as IL-6 was reduced to a similar extent (not shown). Glybenclamide did not cause unspecific hyporesponsiveness in HDF, however. In a control experiment, glybenclamide-treated cells were stimulated with exogenous IL-1 (1 ng per ml), which is expected to override autocrine stimulation from endogenous IL-1. These cells produced similar amounts of IL-8 compared to control cells. As a cytotoxic assay, we further determined LDH activities in cell culture supernatants. No significant LDH activity could be detected in either control HDF or HDF treated with glybenclamide and/or TNF-, excluding an unspecific inhibitory effect of glybenclamide on IL-8 production due to cytotoxicity.
Figure 3.
Glybenclamide reduces IL-8 production in TNF--stimulated primary fibroblasts, but does not change caspase-1 expression. (A) Confluent HDF cultures were stimulated for 24 h with 10 ng per ml TNF- (black bars), or with 1 ng per ml IL-1 (white bars) in the presence of glybenclamide, or with solvent alone. IL-8 production of TNF-- and IL-1-treated cells in the presence of glybenclamide is expressed relative to respective cytokine-treated cells without glybenclamide. Results shown are representative for experiments repeated three times. n.d., not done. (B) Western blot for caspase-1. Cell extracts of HDF (100 g protein, lanes 1–6) and human macrophages (20 g protein, lanes 7, 8) were immunoblotted for caspase-1. Some cells were cultured overnight with 10 ng per ml TNF- (lanes 4–6) or with 1 g per ml lipopolysaccharide (lane 8). HDF were treated with 10 M (lanes 2, 5), or 100 M glybenclamide, or with DMSO as a control (lanes 1, 4). Arrow indicates the activated form of caspase-1 (p20) in human macrophages treated with lipopolysaccharide (lane 8).
An important regulatory step for IL-1 is thought to be the processing and concomitant activation of the precursor form by caspase-1 (Black et al, 1989;Kostura et al, 1989). We therefore investigated the effect of glybenclamide on the expression and activity of caspase-1 in HDF. Caspase-1 was expressed in fibroblasts exclusively as a 35 kDa precursor form Figure 3b, irrespective of treatments with TNF- and/or glybenclamide. Accordingly, no caspase-1 activity was detected in cell lysates using a specific chromogenic peptide substrate (not shown).
Glybenclamide inhibits secretion of IL-1 and IL-1 in transfected COS-1 cells
As IL-1 in the supernatant was hardly detectable in fibroblasts, we used COS-1 cells transfected with IL-1 or IL-1 precursors to directly demonstrate inhibition of IL-1 secretion by glybenclamide in an overexpressing system Figure 4. On Western blots, the IL-1 precursor (34–35 kDa) was expressed in cell lysates, whereas the processed mature form (18 kDa) appeared in supernatants Figure 4a. As mature IL-1 was not detected in cell lysates, processing of IL-1 probably occurred concomitantly with its secretion. In contrast, IL-1 was not processed and was secreted as the 33 kDa precursor form Figure 4b. Cleavage of IL-1 and IL-1 precursors by calpain and caspase-1 is cell type specific, however, depending on the expression and activity of these processing enzymes.
Figure 4.
Glybenclamide inhibits IL-1 secretion in IL-1- and IL-1-transfected COS-1 cells. COS-1 cells were transfected with IL-1 precursor (A, lanes 2, 3), IL-1 precursor (B, lanes 2, 3), or vector alone (A, B, lane 1). Two days after transfection, media were changed, and cells were incubated overnight with 100 M glybenclamide (lane 3) or with DMSO (lanes 1, 2). Identical amounts of protein from cell lysates and supernatants were analyzed on Western blots.
Independently from processing, glybenclamide (100 M) inhibited the secretion of both mature IL-1 and IL-1 precursor, whereas cellular IL-1 levels remained unchanged (Figure 2a, b, lanes 2, 3). Densitometric analysis revealed that signals for mature IL-1 and IL-1 precursor in supernatants of glybenclamide-treated cells relative to control cells were 51% and 41%, respectively. Measurements of IL-1 and IL-1 in supernatants using ELISA confirmed these results, showing reductions to 34% and 51%, respectively, in glybenclamide-treated cells (100 M) relative to control cells. On the other hand, brefeldin, an inhibitor of the "classical" secretory pathway, did not reduce secretion of IL-1 or IL-1 in transfected COS-1 cells (not shown), which is in accordance with a previous study using monocytes (Rubartelli et al, 1990).
Discussion
In primary human dermal fibroblasts, IL-1 acts as an autocrine factor to enhance expression of IL-8. The expression of several other genes depends on the autocrine activity of IL-1 in fibroblasts, such as hepatocyte growth factor, stromelysin, Mn-superoxide dismutase, and collagenase. Furthermore, TNF--treated fibroblasts express less IL-6 in the presence of neutralizing antibodies against IL-1 receptor. Similarly, IL-1RAcP-deficient fibroblasts, which cannot respond to IL-1, also display reduced IL-6 expression levels upon TNF- treatment, indicating that gene induction by TNF- in fibroblasts in part depends on the autocrine activity of IL-1 (Kumar et al, 1992;West-Mays et al, 1995;Cullinan et al, 1998;Miyazaki et al, 1998).
Published data suggest that, in contrast to human monocytes, fibroblasts do not secrete IL-1 (Fuhlbrigge et al, 1988;Young et al, 1988). We also did not detect IL-1 in the medium of TNF--treated HDF, and IL-1 was hardly detectable in the medium of transfected HDF. At first glance, these findings are in conflict with IL-1 as an autocrine factor in fibroblasts. IL-1 does not need to be present as a soluble factor in order to activate neighboring cells, however. Using biochemical methods, biologic assays, fluorescence-activated cell sorting, or immunoelectron microscopy, IL-1 and IL-1 have been demonstrated to be localized on the plasma membrane (Kurt-Jones et al, 1985;Brody and Durum, 1989;Zola et al, 1993;Singer et al, 1995;Fukushima et al, 1997). Using binding studies with radiolabeled soluble IL-1 receptor, it has been estimated that 1000 high affinity sites per cell reside on the plasma membrane of TNF--treated fibroblasts (Sporri et al, 1998). Therefore, cell surface-associated IL-1 on fibroblasts could be engaged in cell-contact-dependent cell activation.
In transfected COS-1 cells glybenclamide inhibited the secretion of both IL-1 and IL-1. In contrast to our data, it has been described previously that only secretion of IL-1, but not of IL-1, was impaired in glybenclamide-treated monocytes (Hamon et al, 1997). This suggests that (i) several ABC transporters might be capable of IL-1 and/or IL-1 translocation, (ii) specific cell types could be equipped with different sets of ABC transporters, and (iii) regulation of ABC transporters could differ from one cell type to another.
It remains to be resolved which ABC transporter(s) translocate IL-1 and IL-1. Recently, several mutations in the ABC1 transporter have been identified to cause Tangier disease in humans (Bodzioch et al, 1999;Brooks-Wilson et al, 1999;Lawn et al, 1999;Remaley et al, 1999;McNeish et al, 2000). Interestingly, secretion of IL-1 is not impaired in these patients (G. Schmitz, personal communication). Glybenclamide may also inhibit other ABC transporters. As glybenclamide did not completely block secretion of IL-1 and IL-1 in COS-1 cells Figure 4, alternative IL-1 transport systems may exist.
Glybenclamide did not change the expression or activation of caspase-1 in fibroblasts. In fact, only a 33 kDa form of caspase-1 was detected in fibroblasts, and processing to activated forms was not induced by TNF- or glybenclamide. Furthermore, no activity for caspase-1 was detected using a specific chromogenic peptide substrate. The 33 kDa form thus corresponds to one of the previously described inactive precursor forms, which range from 45 to 30 kDa (Alnemri, 1995). IL-1 in fibroblasts may be processed independently of caspase-1, however. Such an alternative processing pathway was proposed in a report on caspase-1 knockout mice (Li, 1997): these animals are still able to produce residual amounts of mature IL-1.
Glybenclamide is widely used as an antidiabetic drug in patients with type II diabetes. Therapeutic glybenclamide concentrations in the serum of diabetic patients in vivo are well below the concentrations necessary to inhibit ABC1 transporters in vitro. Clearly, induction of insulin secretion via the sulfonylurea receptor and membrane depolarization (Boyd et al, 1991;Aguilar-Bryan et al, 1995) occurs independently from inhibition of ABC1 transporters (Becq et al, 1997). Glybenclamide, as a suppressor of IL-1 secretion, interrupts IL-1-mediated autocrine activation in fibroblasts, reducing the expression of IL-8 and possibly other IL-1 inducible pro-inflammatory genes, including IL-1 itself. From this point of view, glybenclamide might have anti-inflammatory properties. Interestingly, IL-18, another member of the IL-1 family, also lacks a signal peptide, and therefore could also be secreted via ABC transporters. Could more potent and specific ABC transporter inhibitors become a new group of anti-inflammatory drugs?
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Acknowledgments
We thank Catherine Solioz and Nicolas Delaleu for their excellent technical assistance and fruitful discussions during the project. This study was supported in part by the Program for Biomedical Research of the Department of Clinical Research, University of Bern (Z.B.), and by Grant 198 of the Swiss Society of Odontology SSO (M.B. and D.L.).
D.L. and Z.B. contributed equally to this work.
