Introduction

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Kinins play an important role in the pathophysiological processes that accompany inflammation and tissue damage and repair (Proud and Kaplan, 1988; Dray and Perkins, 1993). The first kinins, bradykinin (BK) and Lys-BK or kallidin (KD), formed from kininogen precursors, are rapidly metabolized by proteolytic enzymes to form fragments, including the biologically active carboxypeptidase products desArg⁹BK and desArg¹⁰KD (Bhoola et al., 1992). Kinins have a remarkably broad repertoire of biological actions, including vasodilatation, smooth muscle spasm, edema, hyperalgesia, pain, modulation of hormone and cytokine release, increased epithelial transport, and cell proliferation. These actions are mediated through two receptor subtypes, B1 and B2 (Regoli and Barabe, 1980). The B2 receptor subtype mediates the action of BK and KD, whereas the B1 receptor subtype mediates the action of desArg⁹BK and desArg¹⁰KD. Both receptor subtypes are members of the superfamily of seven transmembrane-domain, G protein-coupled receptors (GPCR) (Hess et al., 1992; Menke et al., 1994).

The B2 receptor is constitutively expressed in a diverse range of tissues, whereas the B1 receptor is generally not expressed under nonpathological conditions. However, the B1 receptor is rapidly and dramatically induced in vivo under stress conditions (Marceau et al., 1998) and in vitro in some cell types, including lung fibroblasts and alveolar macrophages, after exposure to noxious stimuli, including the cytokines interleukin-1 (IL-1) and tumor necrosis factor- (Phagoo et al., 1999, 2000; Tsukagoshi et al., 1999). Evidence from animal models has suggested that B2 receptor activation is responsible for most of the actions of kinins under normal conditions (Dray, 1997). However, after inflammatory insult, the response to B1 receptor agonists can develop within hours and last for over 3 days (Davis and Perkins, 1994). Therefore, this receptor may become the dominant subtype in mediating chronic inflammation (Dray and Perkins, 1993).

The mechanisms underlying the rapid induction of B1 receptors in chronic inflammatory conditions are relatively unknown. Recently, it was shown that kinin receptor agonists are capable of stimulating the activation of several transcriptional regulatory factors, including nuclear factor-B (NF-B) in fibroblasts and epithelial cells (Pan et al., 1998; Schanstra et al., 1998; Naraba et al., 1999). Furthermore, promoter analysis of the B1 receptor gene has revealed the presence of several putative transcription factor binding motifs (Bachvarov et al., 1996; Yang and Polgar, 1996; Ni et al., 1998). The signaling pathways leading to B1 receptor induction have not been investigated to any significant extent although the mitogen-activated protein kinase (MAPK) cascade has recently been implicated (Larrivee et al., 1998; Campos et al., 1999).

Both B1 and B2 receptors have been located on lung cells (Mak and Barnes, 1991; Nadar et al., 1996), and several lines of evidence suggest that kinins play an important role in chronic inflammatory pulmonary disease (Polosa, 1992; Proud, 1998). Furthermore, attention is being focused on the potential role for B1 receptors in the migration of immune cells to the site of inflammation (McLean et al., 2000).

Recently, we proposed a cellular mechanism in which kinins themselves regulate the expression and activity of their receptors in human lung fibroblasts, and which is compatible with the sequence of activation of these receptors during the inflammatory process (Phagoo et al., 1999). In this mechanism, B2 receptor agonists act on B2 receptors to produce secondary mediators, including IL-1, that increase B1 receptor expression. Subsequent carboxypeptidase action produces the second set of kinins, which activate B1 receptors and feed-forward to optimize the expression of B1 receptors. This is compatible with the events in some chronic pulmonary diseases. The airways of asthmatic subjects contain elevated levels of both kallikrein activity and kinins (Christiansen et al., 1992), and bronchoalveolar lavage fluids in diseased lung from asthmatic subjects contain elevated levels of IL-1. In this study, we have investigated several of the intracellular pathways whereby IL-1 and the B1 receptor agonist desArg¹⁰KD increase B1 receptor expression in human lung fibroblasts and human lung epithelial cells.

	Materials and Methods

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Culture of IMR-90 Human Fetal Lung Fibroblasts and A549 Human Lung Epithelial Cells. IMR-90 and A549 cells were obtained from the American Type Culture Collection (Rockville, MD). IMR-90 fibroblasts were cultured in complete growth media comprised of Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Sigma, St. Louis, MO), 50 IU/ml penicillin, 50 µg/ml streptomycin, 4 mM L-glutamine, and 1% nonessential amino acids (Life Technologies). The cells were maintained in a humidified atmosphere in 5% CO₂ at 37°C and were subcultured by incubating with 0.05% trypsin-0.5 mM ethylenediaminetetraacetate (Life Technologies) at a ratio of 1:3, weekly. For all IMR-90 experiments, cells were plated at a density of 150,000 cells/well in six-well plates and used at confluency (4-5 days) between passage 15 and 20. Prior to experimentation, the IMR-90 cells were washed once with growth medium excluding fetal bovine serum (hence referred to as DMEM) before being incubated in the absence and presence of the B1 receptor agonist desArg¹⁰KD (Bachem California, Torrance, CA) and/or IL-1 (R & D Systems, Minneapolis, MN) as described in the figure legends. A549 cells were cultured in RPMI media (Cellgro; Mediatech, Herndon, VA) with 10% heat inactivated fetal bovine serum (Sigma) and plated at a density of 200,000 to 300,000 cells/well in six-well plates for 4 to 5 days before use.

Radioligand Binding. To remove surface receptor-bound agonist by low pH washing, to determine B1 receptor-specific binding on cells that had been exposed to receptor agonist, a previously described acid-stripping technique was used that effectively removes bound ligand from cells by washing with low pH buffer (Munoz et al., 1993). This procedure was performed at 4°C. In short, after exposure of the cells to receptor agonist, bound ligand was removed by first rinsing with PBS, followed by two incubations in 0.05 M glycine-HCl, pH 3.0 for 6 and 0.5 min, and then two brief rinses in PBS. As determined by cell viability staining with trypan blue, this acid washing procedure was not detrimental to the IMR-90 cells and did not significantly alter [³H]desArg¹⁰KD binding (Phagoo et al., 1999).

RT-PCR. Total RNA was extracted from cells using TRIzol reagent as described by the manufacturer (Life Technologies). Single-stranded cDNA was generated using Superscript II reverse transcriptase (100 U; Life Technologies) in a 20-µl reaction mixture containing reaction buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol), 0.5 mM dNTP, 0.5 µg oligo(dT)_12-18 (Life Technologies), 10 U rRNasin (Promega, Madison, WI), and 2 µg of total RNA. The reaction was carried out for 1 h at 42°C. Amplification of cDNA by PCR was performed using oligonucleotide primer pairs (Life Technologies synthesis) for the human B1 receptor (Ni et al., 1998) and -actin as described by Jung et al. (1995). The reactions were carried out using a RoboCycler (Stratagene, La Jolla, CA) in a 50-µl reaction mixture containing reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl₂), 0.2 mM dNTP, 2.5 U Taq polymerase (Life Technologies), and 1 µl of cDNA. Each primer was added at a final concentration of 0.2 µM. PCR was for 30 to 35 cycles, each cycle consisting of 1-min denaturation at 94°C, annealing at 55°C for 50 s, and extension at 72°C for 45 s. PCR reaction products were separated on 1% agarose gels containing 50 µg/ml ethidium bromide and visualized under UV light. The human B1 receptor PCR product was 429 base pairs (Ni et al., 1998).

Data Analysis. Specific binding was processed using ORIGIN (MicroCal Software, North Hampton, MA). Data are reported as the mean ± S.E. and were compared using Student's t test. p values <0.05 were considered to be significant.

	Results

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Bradykinin B1 Receptor Agonist and the Proinflammatory Cytokine IL-1 Synergistically Up-Regulate B1 Receptor Expression

Previous studies have shown that treatment of human lung fibroblasts with IL-1 results in an up-regulation of B1 receptors in the cells (Menke et al., 1994; Zhou et al., 1998; Phagoo et al., 1999, 2000). These responses are preceded by an increase in the steady-state level of B1 receptor mRNA, indicating that the regulation occurs at the level of gene expression. In the present study the number of B1 receptors, as detected with the B1-selective agonist [³H]desArg¹⁰KD, increased by up to 4-fold within 6 h after stimulation of IMR-90 cells with 500 pg/ml IL-1 (Fig. 1A). Saturation binding isotherms revealed that this increase occurred without any statistically significant change in the equilibrium dissociation constant (K_D) [untreated (DMEM): maximum receptor binding density (B_max) = 18 ± 2 fmol/mg of protein, K_D = 0.44 ± 0.13 nM; IL-1 treated: B_max = 64 ± 1 fmol/mg of protein; K_D = 0.27 ± 0.01 nM; n  3; Fig. 1B]. As demonstrated in Fig. 1C, treatment of the cells for 6 h with IL-1 resulted in a significant increase in the PCR product encoding for B1 receptor mRNA. The IL-1-mediated response was almost completely abrogated in the presence of IL-1 receptor antagonist (data not shown). Exposure of the cells to 100 nM desArg¹⁰KD yielded a 2- to 3-fold induction in the number of B1 receptors available for [³H]desArg¹⁰KD binding (B_max = 33 ± 4 fmol/mg of protein; K_D = 0.18 ± 0.04 nM; n  3; Fig. 1, A and B). This induction by desArg¹⁰KD was significantly inhibited by a B1 receptor antagonist desArg⁹[Leu⁸]BK (100 µM) but not by a B2 receptor antagonist (HOE140, 10 µM; data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Synergistic effect of desArg10KD and IL-1 on B1 receptor expression. IMR-90 cells were treated for 6 h at 37°C with DMEM (Ctrl), 100 nM desArg10KD, and/or 500 pg/ml IL-1, washed with low pH buffer, and then assayed for specific [3H]desArg10KD binding at 4°C or B1 mRNA as described under Materials and Methods. A, cells were treated as indicated and then assayed for binding. The data shown are from five experiments. The results are presented as percentage of control where 100% refers to the response to DMEM treatment alone. Comparison to DMEM treatment: ***p < 0.001. B, cells were treated as indicated for 6 h and then assayed for binding by saturation binding analysis. The result is representative of at least three experiments. C, IMR-90 cells were treated for 6 h as indicated and then analyzed for B1 receptor mRNA using RT-PCR. -Actin mRNA was used as a loading control. The result is a representative of two experiments.

Remarkable synergism in the induction of B1 receptors was observed after coincubation of 100 nM desArg¹⁰KD and 500 pg/ml IL-1 for 6 h. This combination produced up to a 20-fold increase in available B1 receptor binding sites without any statistically significant effect on the K_D value of the binding (B_max = 332 ± 10 fmol/mg of protein, K_D = 0.23 ± 0.03 nM; n  3; Fig. 1, A and B). This synergistic response on B1 expression was always at least 2-fold greater than the additive effect of desArg¹⁰KD and IL-1. To test whether the synergistic increase in B1 receptors was due to the stimulation of B1 receptor gene transcription, IMR-90 cells were treated for 6 h and then analyzed for B1 receptor mRNA. Figure 1C shows that the synergistic up-regulation of B1 receptors by IL-1 and desArg¹⁰KD was paralleled by an increase in B1 receptor mRNA levels. The increased density of the PCR product encoding for B1 mRNA in response to IL-1 and desArg¹⁰KD was 3-fold greater than the additive effect of the two factors alone.

Bradykinin B1 Receptor Agonist and IL-1 Act Synergistically to Alter the Kinetics of B1 Receptor Induction

We next examined the kinetics and concentration-response relationship of the synergistic B1 receptor up-regulation. As shown in Fig. 2, exposure of the cells to IL-1 time dependently increased the expression of the B1 receptor with a maximal response at 4 h, which was sustained for up to 8 h. The binding then declined to 2-fold of basal levels by 24 h after IL-1 stimulation. DesArg¹⁰KD stimulation followed similar kinetics, reaching a maximal response at approximately 4 h and returning to near basal level by 24 h. Interestingly, stimulation of the cells for 0.5 and 2 h with IL-1 and desArg¹⁰KD in combination did not produce an additive effect, whereas at 4 h of exposure a dramatic synergism was observed. This synergistic response was maximal at 6 h and declined slightly after 8 h but was still significantly elevated above the 4-h time point (4 h, 11-fold; 8 h, 18-fold). At 24 h, a small synergistic response remained (4-fold), which was significantly higher than that observed in response to IL-1 (2-fold) at this time point. These results suggest that at time points up to 2 h both desArg¹⁰KD and IL-1 may act via shared pathways to increase B1 receptor gene expression, whereas independent and/or synergistic pathways may be activated at longer time points. Furthermore, B1 receptors remained up-regulated longer in the presence of desArg¹⁰KD and IL-1 than IL-1 alone.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Time course of the synergistic effect of desArg10KD and IL-1 on B1 receptor expression. IMR-90 cells were treated for various times at 37°C with 100 nM desArg10KD and/or 500 pg/ml IL-1 as indicated, washed with low pH buffer, and then assayed for specific [3H]desArg10KD binding at 4°C as described under Materials and Methods. The data shown are from at least three experiments. The results are presented as percentage of control where 100% refers to the response to DMEM treatment alone. Comparison with DMEM treatment at each time point: *p < 0.05; **p < 0.01; ***p < 0.001.

The IL-1 -induced up-regulation of B1 receptor expression in IMR-90 cells was concentration-dependent (concentration required for half-maximal response, EC₅₀ = 12.5 ± 1.2 pg/ml) with maximal receptor up-regulation plateauing at 100 to 500 pg/ml (Fig. 3A; Table 1). The response to desArg¹⁰KD reached a maximum at 100 to 1000 nM with an EC₅₀ of 8 ± 2 nM (Fig. 3B; Table 1). As shown in Fig. 3, C and D, the level of the synergistic response in IMR-90 fibroblasts was desArg¹⁰KD- and IL-1-concentration dependent, requiring an IL-1 threshold concentration much lower than its EC₅₀ value (1 pg/ml) to produce a clearly greater induction of B1 receptors than the additive effect in cells treated separately with 1 pg/ml IL-1 or 100 nM desArg¹⁰KD. This was not the case for desArg¹⁰KD as a concentration 10 nM was required to produce a synergistic response in the presence of a maximal concentration of IL-1. As shown in Table 1, the presence of 100 nM desArg¹⁰KD caused a >2-fold shift to the left in the IL-1 dose-response curve, thus increasing the potency by which IL-1 up-regulates B1 receptor expression.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Comparison of the individual effects of desArg10KD and IL-1 versus their effect in combination on B1 receptor expression. IMR-90 cells were treated at 37°C for 6 h as indicated, washed with low pH buffer, and then assayed for specific [3H]desArg10KD binding at 4°C as described under Materials and Methods. Cells were treated with various concentrations of IL-1 (A) or desArg10KD (B) and then assayed for binding. C, cells were treated as indicated with 100 nM desArg10KD in the presence or absence of various concentrations of IL-1 and then assayed for binding. D, cells were treated as indicated with 500 pg/ml IL-1 in the presence or absence of various concentrations of desArg10KD and then assayed for binding. The results are presented as percentage of control (Ctrl) where 100% refers to the response to DMEM treatment alone. Comparison to DMEM treatment: *p < 0.05; **p < 0.01; ***p < 0.001. In each study, the data are from at least three independent experiments.

View this table: [in this window] [in a new window]   TABLE 1 EC50 values for B1 receptor up-regulation by IL-1 and desArg18KD EC50 values (mean ± S.E.) were calculated from at least three sets of independently performed dose-response curves of radioligand binding experiments performed in duplicate and processed using ORIGIN.

De Novo Protein Synthesis Is Required for Synergistic Induction of B1 Receptor Expression

The requirement for protein transcription and translation in the up-regulation of [³H]desArg¹⁰KD binding in IMR-90 cells stimulated with IL-1 and desArg¹⁰KD was investigated using the metabolic inhibitors cycloheximide and actinomycin D. Pretreatment with the protein translation inhibitor cycloheximide (10 µg/ml for 1 h; Fig. 4A) prevented the increase in [³H]desArg¹⁰KD binding sites induced by both B1 receptor agonist and IL-1 for 6 h, individually and in combination, reducing the binding to between 5 and 20% of the response in cells stimulated in the absence of cycloheximide. Figure 4B shows that preincubation of the cells with the transcription inhibitor actinomycin D (5 µg/ml) completely prevented the IL-1- and desArg¹⁰KD-mediated increases in B1 receptor mRNA and [³H]desArg¹⁰KD binding (data not shown). The basal level of [³H]desArg¹⁰KD binding was also significantly reduced after cycloheximide and actinomycin D treatments. These results suggest that the synergistic desArg¹⁰KD- and IL-1-mediated up-regulation of B1 receptor expression involves de novo B1 receptor protein synthesis and occurs at the level of gene transcription.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Effect of cycloheximide on B1 receptor expression and actinomycin D on B1 receptor mRNA. A, IMR-90 cells were pretreated for 1 h with cycloheximide (cyclo, 10 µg/ml) before treatment for 6 h with 100 nM desArg10KD and/or 500 pg/ml IL-1 as indicated and then assayed for binding. The results are presented as percentage of agonist where 100% refers to the response to stimulation in the absence of cycloheximide. The data shown are from at least three experiments. Comparison to stimulation in the absence of cycloheximide: ***p < 0.001. B, cells were treated for 6 h with 100 nM desArg10KD and 500 pg/ml IL-1, or pretreated for 1 h with the protein transcription inhibitor actinomycin D (5 µg/ml). The cells were then analyzed for B1 receptor mRNA using RT-PCR as described under Materials and Methods. -Actin mRNA was used as a loading control. The result is a representative of two experiments.

Signaling Pathways Involved in Synergistic Up-Regulation of B1 Receptors

Involvement of Protein Tyrosine Kinase (PTK) Pathways. It has been suggested that the activation of some PTKs are significant in the induction of B1 receptors (Zhou et al., 1998; Campos et al., 1999). To determine whether tyrosine kinase activity was involved in synergistic desArg¹⁰KD- and IL-1-induced B1 receptor up-regulation, cellular tyrosine kinase activity was blocked using the broad-range inhibitor genistein. Cells were pretreated with genistein (50, 75, and 100 µM) for 1 h and then exposed to desArg¹⁰KD and IL-1 for 6 h. Figure 5A shows that genistein concentration dependently provided significant protection against both desArg¹⁰KD- and IL-1-mediated B1 receptor induction in IMR-90 fibroblasts reducing their responses to 32 ± 6 and 71 ± 1%, respectively, at a genistein concentration of 100 µM. Clearly, the desArg¹⁰KD response was more sensitive than the IL-1 response to genistein. Inhibition of the synergistic response by genistein was also concentration-dependent (Fig. 5B).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Effect of the PTK inhibitor genistein on B1 receptor expression induced by exposure to desArg10KD and IL-1. A, cells were pretreated for 1 h with genistein at the concentrations indicated, stimulated with either 100 nM desArg10KD or 500 pg/ml IL-1 for 6 h, and then assayed for binding. The results are presented as percentage of the agonist response where 100% refers to the response in cells without inhibitor treatment. The data shown are from at least four experiments. B, cells were pretreated with genistein at various concentrations before exposure to desArg10KD and IL-1 for 6 h and then assayed for binding. The results are presented as percentage of the synergistic response to desArg10KD and IL-1 where 100% refers to the response in cells without inhibitor treatment. The data shown are from at least four experiments. Comparison to agonist treatment in the absence of inhibitor: *p < 0.05; **p < 0.01.

Involvement of MAPK Pathways. Prominent among the intracellular kinase cascades activated by GPCR are the MAP kinases, including the extracellular signal-regulated kinases (ERKs), stress-activated kinases, and p38 kinase (Lopez-Ilasaca, 1998). Figure 6A shows the effect of inhibiting ERK and p38 MAP kinase on B1 receptor expression induced by desArg¹⁰KD and IL-1. Pretreatment of IMR-90 cells for 1 h with the specific p38 inhibitor SB 203580 (20 µM) significantly attenuated the response to desArg¹⁰KD and the synergistic response (81 ± 11 and 66 ± 3%, respectively; Fig. 6A). The effect of SB 203580 on the synergistic response was dose-dependent with significant inhibition occurring at 10 µM (Fig. 6B). Blocking ERK MAP kinase signaling using the specific inhibitor PD 98059 (20 µM) had a modest (to 83 ± 11% of agonist response) although insignificant effect on the up-regulation of B1 receptor expression by the combination of desArg¹⁰KD and IL-1 (Fig. 6A).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Effect of MAP kinase (ERK and p38) inhibitors on the expression of B1 receptors induced by exposure to desArg10KD and IL-1. A, IMR-90 cells were treated for 6 h at 37°C with 100 nM desArg10KD and/or 500 pg/ml IL-1 as indicated, or pretreated for 1 h with the p38 MAP kinase inhibitor SB 203580 (20 µM) or the ERK MAP kinase inhibitor PD 98059 (20 µM) in the presence of 100 nM desArg10KD and/or 500 pg/ml IL-1 for 6 h. Cells were washed with low pH buffer and then assayed for specific [3H]desArg10KD binding at 4°C as described under Materials and Methods. The results are from at least four experiments and presented as percentage of the agonist response where 100% refers to the response in cells without inhibitor treatment. Comparison to agonist treatment in the absence of inhibitor: *p < 0.05; ***p < 0.001. B, cells were pretreated for 1 h with SB 203580 at the concentrations indicated, stimulated with 100 nM desArg10KD and 500 pg/ml IL-1 for 6 h, and then assayed for binding. The results are presented as percentage of the synergistic response where 100% refers to the response in cells exposed to 100 nM desArg10KD and 500 pg/ml IL-1 for 6 h alone. The result is representative of at least three experiments. Comparison to treatment in the absence of inhibitor: **p < 0.01; ***p < 0.001.

Involvement of Transcription Factor NF-B. Recently, it was suggested that the induction of B1 receptors by IL-1 is regulated via a transcriptional mechanism involving the activation of NF-B (Schanstra et al., 1998). Furthermore NF-B binding site-like sequences have been identified on the B1 receptor gene, and activity in response to noxious stimuli appears to be sensitive to NF-B-like binding sites in the promoter (Ni et al., 1998; Schanstra et al., 1998). To determine the role of NF-B in the synergistic B1 receptor induction response, cells were pretreated for 1 h with an antioxidant inhibitor of NF-B, pyrrolidine dithiocarbamate (PDTC). As demonstrated in Fig. 7A, PDTC (500 µM) provided minimal, if any protection against the up-regulation of B1 receptor expression induced by exposure to desArg¹⁰KD (95 ± 10% of agonist response) or IL-1 (82 ± 25% of response). In contrast, PDTC at the same concentration almost completely inhibited the synergistic response mediated by the combination of IL-1 and desArg¹⁰KD at both the level of B1 receptor mRNA (Fig. 7B) and B1 receptor protein (24 ± 5% of response; Fig. 7A). This effect of PDTC was concentration-dependent with a significant inhibitory effect occurring at 100 µM (Fig. 7C).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Effect of NF-B inhibition on B1 receptor up-regulation induced by exposure to desArg10KD and IL-1. A, IMR-90 cells were treated with 100 nM desArg10KD and/or 500 pg/ml IL-1, or pretreated for 1 h with the NF-B inhibitor PDTC (500 µM) in the presence or absence of desArg10KD and/or IL-1 for 6 h as indicated. Cells were washed with low pH buffer and then assayed for specific [3H]desArg10KD binding at 4°C as described under Materials and Methods. The results are from eight experiments and are presented as percentage of the agonist response where 100% refers to the response in cells without inhibitor treatment. Comparison to treatment in the absence of inhibitor: ***p < 0.001. B, cells were treated for 6 h with 100 nM desArg10KD and 500 pg/ml IL-1, or pretreated for 1 h with 500 µM PDTC. The cells were then analyzed for B1 receptor mRNA using RT-PCR as described under Materials and Methods. -Actin mRNA was used as a loading control. The result is a representative of two experiments. C, cells were pretreated for 1 h with PDTC at the concentrations indicated, stimulated with 100 nM desArg10KD and 500 pg/ml IL-1 for 6 h, and then assayed for binding. The results are presented as percentage of the synergistic response where 100% refers to the response in cells exposed to 100 nM desArg10KD and 500 pg/ml IL-1 for 6 h alone. The data are representative of at least four experiments. Comparison to treatment in the absence of inhibitor: *p < 0.05, **p < 0.01, ***p < 0.001.

Induction of B1 Receptor Expression in Human Lung Epithelial Cells by desArg¹⁰KD and IL-1. It has been reported that kinins are stimulatory for NF-B activation in lung epithelial cells (Pan et al., 1998). Considering that desArg¹⁰KD and IL-1 synergize to activate B1 receptor expression through an NF-B-mediated mechanism, we were curious as to whether B1 agonist and IL-1 are able to induce B1 receptors in A549 human lung epithelial cells. Treatment of the cells with IL-1 (500 pg/ml) produced a 2-fold induction of B1 receptors (Fig. 8A) and a significant increase in B1 receptor mRNA (Fig. 8B) from an almost undetectable basal level. A549 cells exposed to desArg¹⁰KD (100 nM) stimulated a smaller, although significant increase in B1 binding sites. In contrast to IMR-90 fibroblasts, the combination of B1 receptor agonist and IL-1 did not produce either an additive or synergistic response (Fig. 8A). Furthermore, B1 receptor mRNA was not significantly greater than that obtained with IL-1 alone (Fig. 8B).

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Up-regulated expression of B1 receptors and B1 receptor mRNA in A549 human lung epithelial cells induced by exposure to desArg10KD and IL-1. A, human A549 cells were treated with RPMI medium (Ctrl), 100 nM desArg10KD, and/or 500 pg/ml IL-1 as indicated. Cells were washed with low pH buffer and then assayed for specific [3H]desArg10KD binding at 4°C as described under Materials and Methods. The data shown are from at least four experiments. The results are presented as percentage of control where 100% refers to the response to RPMI treatment alone. Comparison with RPMI treatment: *p < 0.05; **p < 0.01; ***p < 0.001. B, A549 cells were treated for 6 h with 500 pg/ml IL-1 in the presence or absence of 100 nM desArg10KD before analysis for human B1 receptor mRNA using RT-PCR as described under Materials and Methods. -Actin mRNA was used as a loading control. The result is representative of two experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The bradykinin B1 receptor is a unique GPCR that is highly induced by inflammatory stimuli such as lipopolysaccharide and IL-1 (Marceau, 1995). Even more intriguing is the fact that B1 receptors are autoinduced by agonists (Schanstra et al., 1998; Phagoo et al., 1999). In other words, kinin agonists promote the B1 receptor up-regulation rather than receptor desensitization and internalization, which occurs with the bradykinin B2 receptor subtype (Mathis et al., 1996; Austin et al., 1997). Little if anything is known about the interaction of kinins and cytokines and the intracellular signaling pathways involved in B1 receptor induction in response to stress and inflammation. Here, we report the novel finding that B1 agonist autoinduction and the IL-1-mediated induction of B1 receptors in human lung fibroblasts occurs through apparently distinct but synergistic mechanisms involving MAP kinase and NF-B. These mechanisms may also mediate the generation of additional proinflammatory mediators, thus, producing a feed-forward system of inflammation mediated through B1 receptor expression and activation. This further strengthens the idea that the induction of B1 receptors is involved in the chronic phase of the inflammatory response.

The kinetic studies of B1 up-regulation strongly suggest that at early time points (2 h) B1 receptor agonist and IL-1 may act via similar mechanisms to increase B1 receptor gene expression as the effect of these factors was neither additive nor synergistic. On the other hand, the dramatic synergistic effect at later time points (4 h) suggests that these factors act at least in part through distinct mechanisms. The synergistic response significantly increased the effectiveness of IL-1 to up-regulate B1 receptor gene expression. By this mechanism, the potency of IL-1 in the presence of desArg¹⁰KD was increased 2-fold compared with its effectiveness in the absence of desArg¹⁰KD. This modification was not shared by a similar shift in the potency of desArg¹⁰KD in the presence of IL-1, again suggestive of independent and synergistic signaling mechanisms to optimize the expression of B1 receptors in IMR-90 cells.

The up-regulated [³H]desArg¹⁰KD binding sites were of the B1 receptor subtype as they could be displaced using a specific B1 receptor antagonist (Phagoo et al., 1999). Furthermore, a clear increase in human B1 receptor mRNA was obtained after induction. We investigated whether transcriptional and/or post-transcriptional mechanisms could underlie the increased expression of B1 receptors by IL-1 and desArg¹⁰KD. De novo protein synthesis was involved, as the protein translation inhibitor cycloheximide prevented this increase. Furthermore, the transcription inhibitor actinomycin D inhibited both the increase in B1 receptor mRNA and B1 receptor protein. These results suggest that B1 receptor up-regulation in IMR-90 fibroblasts by both IL-1 and desArg¹⁰KD separately or synergistically occurs directly at the level of gene transcription and not through the synthesis of intermediate proteins (Ni et al., 1998; Schanstra et al., 1998; Zhou et al., 1998).

To examine the signaling mechanisms of IL-1 or B1 agonist-mediated receptor up-regulation, we first investigated the potential involvement of cellular protein kinases, as GPCRs are able to induce a variety of inflammatory responses through the activation of several kinase cascades (Lopez-Ilasaca, 1998). The present study is the first to demonstrate that the B1 agonist-promoted receptor response occurs through a tyrosine-phosphorylating step as the response was concentration dependently inhibited to >65% by the wide-ranging tyrosine kinase inhibitor genistein. Furthermore, this pathway also appeared to be involved in the IL-1-promoted receptor increase as suggested recently in lung fibroblasts (Zhou et al., 1998). Although there were differences in the relative level of genistein inhibition of the responses, suggesting distinct pathways of signaling, in general these results indicate that PTK pathways are common to both the B1 agonist and IL-1 response, and consequently contribute in the synergistic response.

Various cellular stresses are known to activate several MAPK pathways, which act as effectors for inflammatory cellular responses (Kyriakis and Avruch, 1996). Inhibitors of these cascades are effective in preventing the induction of proinflammatory genes. To investigate whether the MAPK group of specific protein kinases was involved in B1 receptor up-regulation in human lung fibroblasts, we examined the ERK MAPK and p38 MAPK pathways using specific inhibitors. Our results show for the first time that the p38 MAPK pathway is involved in the B1 agonist-promoted up-regulation of human B1 receptors as the response was partially inhibited using the specific p38 inhibitor SB 203580. This identifies an independent signaling pathway for the autoinduction of B1 receptors that clearly differs from IL-1, as the IL-1-promoted increase in B1 binding sites was unaffected by SB 203580. A synergistic signaling mechanism involving p38 MAPK was indicated by the further significant inhibition of the response. Our results are consistent with the recent finding that the spontaneous sensitization to the B1 agonist desArg⁹BK in isolated rabbit aorta is partially inhibited by blocking p38 MAPK (Larrivee et al., 1998). Animal models have also implicated a role of ERK MAPK in B1 induction. In the present study, blocking the ERK pathway using the specific inhibitor PD 98059 suppressed the synergistic B1 response, however the decrease was not significant. This result is consistent with the finding that IL-1 signaling does not require ERK MAPK in IMR-90 cells (Zhou et al., 1998).

MAPK pathways conceivably may serve as mediators between direct cell injury and the activation of multiple transcription factors. Indeed, a further downstream event recently postulated to control the regulation of B1 receptors in vivo and in vitro is through the factor NF-B (Marceau et al., 1998; Ni et al., 1998; Schanstra et al., 1998; Campos et al., 1999). In the current study, the synergistic response was strongly inhibited by the antioxidant inhibitor of NF-B PDTC. In contrast, the desArg¹⁰KD- and IL-1-mediated increase in B1 receptor protein was little affected by PDTC. These results strongly suggest that the synergistic up-regulation of B1 receptors may depend critically on NF-B activation and that this signaling pathway is relatively less important in the independent up-regulation by either desArg¹⁰KD or IL-1. Many genes that are implicated in the initiation of inflammatory lung responses are regulated at the level of transcription by NF-B (Rahman and MacNee, 1998). Indeed the enhancement of B1 mRNA by treatment with cycloheximide previously observed in lung fibroblasts (Phagoo et al., 2000) may be related to blocking the synthesis of the inhibitor protein I-B, thereby superinducing NF-B activity (Newton et al., 1996). As such, the involvement of this factor strengthens the idea that the induction of B1 receptors is allied with tissue injury and inflammation (Dray and Perkins, 1993; Marceau et al., 1998). Furthermore, promoter studies of the B1 receptor gene have suggested that it contains all of the elements necessary for its induction by a variety of inflammatory stimuli. As such, promoter analysis has revealed the presence and functional involvement of NF-B response elements (Ni et al., 1998; Schanstra et al., 1998). Several other elements, including activator protein-1 and cAMP response element sites, have been proposed based on sequence analysis, however their roles in B1 receptor inducibility are postulated to be minor (Ni et al., 1998).

In addition to lung fibroblasts, responses to B1 receptor agonists have been demonstrated on human lung epithelial cells. Recently, it was reported that BK-induced NF-B activation occurs in an immortalized lung epithelial cell line, A549, which has the properties of type II alveolar epithelial cells (Pan et al., 1998). Treatment of A549 cells with desArg¹⁰KD or IL-1 for 6 h induced a small, although significant increase in B1 receptor expression. However, in contrast to IMR-90 lung fibroblasts, a synergistic or even additive effect was not observed. Lung epithelial cells may contribute significantly to the up-regulation of B1 receptors in neighboring cells such as fibroblasts indirectly through the B2 receptor-mediated endogenous release of IL-1 (Pan et al., 1998). This mechanism also appears to occur in lung fibroblasts (Pan et al., 1996; Phagoo et al., 1999). Our results in A549 cells suggest that synergistic responses may be cell specific and depend on the location and the role of particular cell lineages in the lung inflammatory response. A clearly synergistic response to desArg¹⁰KD and IL-1 was shared by several other human embryonic fibroblast lung cell lineages (such as WI-38), indicating that the phenomenon was not specific to IMR-90 cells alone.

There is substantial evidence that the kininogen-kallikrein-kinin system is important in manifestations of airway inflammation (Polosa, 1992; Proud, 1998). The precise role of an inducible B1 receptor subtype in airway inflammation is however unclear at present. Several lines of evidence suggest that kinins may be proinflammatory in the lung. An inflammatory effect mediated through B1 receptors was demonstrated by using B1 receptor antagonists to inhibit airway hyperresponsiveness in vivo (Huang et al., 1999). Recently, B1 receptors have been proposed to be involved in leukocyte chemotaxis (Perron et al., 1999; McLean et al., 2000) and fibrotic tissue formation (Nadar et al., 1996). Elevated levels of both kinins (Christiansen et al., 1992) and proinflammatory cytokines, including IL-1, are found in some forms of airway inflammation, suggesting that the potential exists for the synergistic up-regulation of B1 receptors in chronic lung pathologies. Kinins have the ability to induce the secretion of this cytokine in lung-derived cells (Pan et al., 1996, 1998; Phagoo et al., 1999) and these responses may feed forward to up-regulate B1 receptors. Furthermore, MAP kinase and NF-B activation through direct agonist stimulation of up-regulated B1 receptors may have an important role in the coordination of events in lung inflammation, including the expression of genes that encode proteins involved in proinflammatory mediator synthesis.

In conclusion, the present study clearly demonstrates the involvement of protein kinases such as MAPK and the transcription factor NF-B as having important roles in controlling the synergistic up-regulation of B1 receptors. Unlike B2 receptors, up-regulated B1 receptors do not desensitize or internalize after agonist binding, but instead are constitutively active and enhance second messenger systems, which may be dependent on the level of functional receptor expression (Phagoo et al., 1999; Leeb-Lundberg et al., 2001). These may then respond continuously to sustain and perpetuate the inflammation through the kinin-induced release of secondary mediators.