To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis.
To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis.
Adenoviral vectors encoding either sIL-1RAcP or IL-1Ra were administered systemically before onset of collagen-induced arthritis in DBA/1 mice. Anti–bovine type II collagen IgG and IL-6 were quantified in serum. Proliferative response of splenic T cells was determined in the presence of sIL-1RAcP or IL-1Ra. The effect on IL-1 inhibition of recombinant sIL-1RAcP and IL-1Ra was further examined in vitro, using NF-κB luciferase reporter cell lines. Quantitative polymerase chain reaction was used to determine the relative messenger RNA expression of the IL-1 receptors.
Adenoviral overexpression of both sIL-1RAcP and IL-1Ra resulted in amelioration of the collagen-induced arthritis. Both IL-1 antagonists reduced the circulating levels of antigen-specific IgG2a antibodies, but only IL-1Ra was able to inhibit lymphocyte proliferation. By using purified lymphocyte populations derived from NF-κB reporter mice, we showed that sIL-1RAcP inhibits IL-1–induced NF-κB activity in B cells but not T cells, whereas IL-1Ra inhibited IL-1 on both cell types. A study in a panel of NF-κB luciferase reporter cells showed that the sIL-1RAcP inhibits IL-1 signaling on cells expressing either low levels of membrane IL-1RAcP or high levels of IL-1RII.
We show that the sIL-1RAcP ameliorated experimental arthritis without affecting T cell immunity, in contrast to IL-1Ra. Our results provide data in support of receptor competition by sIL-1RAcP as an explanation for the different mode of IL-1 antagonism in comparison with IL-1Ra.
The proinflammatory cytokine interleukin-1 (IL-1) is an important mediator controlling local and systemic effects on a wide variety of target cells, thereby regulating immunity and inflammation (1). IL-1 binds to IL-1 receptor type I (IL-1RI) (80 kd) (2, 3), which results in the recruitment of the IL-1 receptor accessory protein (IL-1RAcP) (4–8). IL-1RAcP does not recognize the ligand but stabilizes IL-1 binding to the IL-1RI (4, 7). Furthermore, IL-1RAcP is a crucial coreceptor in this complex by enabling recruitment and binding of intracellular adaptor proteins such as MyD88 and kinases such as IL-1R–associated kinases, ultimately leading to NF-κB activation (9–14). Another member of the IL-1 receptor family is IL-1RII (68 kd) (15), which upon binding of IL-1 also associates with IL-1RAcP. However, this does not lead to signal transduction because this receptor lacks the intracellular Toll–IL-1 receptor domain that is crucial for MyD88 binding (16–18). Therefore, the type II receptor is a decoy receptor as has been described for its transmembrane and soluble forms (19).
Another inhibitor of IL-1 signaling is the IL-1 receptor antagonist (IL-1Ra), which competes with IL-1 for occupation of the IL-1RI. It is generally accepted that occupation of only 1% of the IL-1RI by IL-1 instigates full-blown cell activation, and as a consequence, relatively high amounts of IL-1Ra (100–1,000-fold molar excess) are required to fully block IL-1. Fortunately, IL-1Ra binds poorly to IL-1RII (19), and both IL-1Ra and soluble IL-1RII (sIL-1RII) cooperate in IL-1 inhibition. We have demonstrated that administration of IL-1Ra protein or the IL-1Ra gene ameliorates disease in several models of experimental arthritis, with a profound protective effect against cartilage and bone destruction (20, 21).
Recently, an alternative splice transcript of the membrane IL-1RAcP, encoding a smaller and soluble protein comprising the 3 extracellular Ig domains and a unique C-terminal domain, has been described (22, 23). This sIL-1RAcP is mainly produced by the liver and circulates systemically. We showed that systemic overexpression of sIL-1RAcP by adenoviral gene transfer in mice markedly ameliorates collagen-induced arthritis (CIA) (24, 25). A possible explanation is that sIL-1RAcP can interact with sIL-1RII, thus forming a high-affinity IL-1 scavenger (26). Although the mechanism and efficacy of IL-1 inhibition might be different, it is expected that under optimal conditions both IL-1Ra and sIL-1RAcP may exert similar biologic effects.
This study was conducted to compare the inhibitory effects of sIL-1RAcP and IL-1Ra in CIA, and to discern their modes of action. Systemic overexpression of either sIL-1RAcP or IL-1Ra using adenoviral vectors before onset of CIA resulted in amelioration of CIA, with both demonstrating their potential as IL-1 inhibitors in vivo. Treatment with both inhibitors resulted in a clear reduction of circulating levels of antigen-specific IgG2a and IL-6. However, sIL-1RAcP treatment did not affect T cell function, whereas IL-1Ra inhibited both antigen- and mitogen-induced lymphocyte proliferation. Next, we studied the effect of both inhibitors on enriched T and B cells obtained from mice expressing an NF-κB reporter gene. Recombinant sIL-1RAcP protein was unable to block IL-1 signaling on T cells, while IL-1Ra inhibited IL-1–induced NF-κB activation in both T and B cells. A study of an array of reporter cell types that differ in their expression of the IL-1 receptors (IL-1RI, IL-1RII, and the coreceptor IL-1RAcP) provided a possible explanation for the cell type–specific inhibitory effect of sIL-1RAcP, i.e., receptor competition with the membrane IL-1RAcP.
Male 10–12-week-old DBA-1/bom mice were obtained from Bomholtgård (Ry, Denmark). C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany); all mice were housed in low-pressure isolator cages during the experiments. The animals were fed a standard diet with water and food ad libitum. All in vivo studies complied with national legislation and were approved by local authorities for the Care and Use of Animals with related codes of practice.
The sIL-1RAcP or IL-1Ra gene was obtained from murine liver complementary DNA (cDNA) and cloned into the first-generation E1/E3-deleted serotype-5 adenoviral vectors and produced according to the method described by Chartier et al (27). All constructed viruses contained the RGD amino acid motif, which was incorporated into the H1 loop of the adenoviral fiber knob (28, 29). Purified recombinant adenoviral vector DNA was linearized through digestion with Pac I and transfected into 293 viral packaging cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The Ad sIL-1RAcP, Ad IL-1Ra, and Ad luciferase were purified using 2 × CsCl gradient purification and stored in small aliquots at −80°C. Viral titer of the purified viral vectors was determined on 911 indicator cells by immunohistochemical detection of viral capsid protein 20 hours after transfection (30).
C-terminal FLAG-tagged sIL-1RAcP recombinant protein was obtained from a stable transduced NIH3T3 cell line and purified as described previously (24). Briefly, sIL-1RAcP was purified at 4°C from conditioned supernatants through affinity chromatography using anti-FLAG M2 affinity gel beads (Sigma, St. Louis, MO), followed by competitive elution with a solution containing 100 μg/ml 3 × FLAG peptide (Sigma) in 50 mM Tris HCl, 150 mM NaCl (pH 7.4). The protein was concentrated using molecular weight cutoff filters of 10 kd (Amicon; Millipore, Bedford, MA) and stored in silicon-coated reaction tubes at 4°C for later usage. Purified protein was quantified using Coomassie Protein Assay Reagent according to the manufacturer's instructions (Pierce, Rockford, IL), and the average production of sIL-1RAcP by stably transfected NIH3T3 fibroblasts was ∼500 ng/24 hours/1 × 106 cells.
The IL-1–responsive murine fibroblast NIH3T3 cell line, the EL.4 NOB-1 thymocytic cell line (American Type Culture Collection, Rockville, MD), and the H4 chondrocyte cell line (31) were all stably transfected with an NF-κB luciferase reporter construct. Reporter cells were seeded in Krystal 2000 96-well plates (Thermo Labsystems, Brussels, Belgium) at a concentration of 2–4 × 104 cells/well, and cultured for 1 day at 37°C in 5% CO2/95% air. Cells were incubated for 24 hours with either sIL-1RAcP or IL-1Ra (Amgen Boulder, Boulder, CO) followed by addition of different concentrations of murine recombinant IL-1α (1 ng/ml or 10 ng/ml). Six hours after IL-1α addition, the intracellular luciferase activity was quantified using a Bright-Glo luciferase assay system (Promega, Madison, WI) followed by luminometric detection according to the manufacturer's protocol (Polarstar Galaxy, Offenburg, Germany).
Bovine type II collagen (BII) was dissolved in 0.05M acetic acid to a concentration of 2 mg/ml and was emulsified in equal volumes of Freund's complete adjuvant (Mycobacterium tuberculosis strain H37Ra; Difco, Detroit, MI). The mice were immunized at the base of the tail with an intradermal injection of 100 μl of emulsion (100 μg BII). On day 21, mice without clinical manifestations of arthritis received an intraperitoneal booster injection of 100 μg of BII dissolved in phosphate buffered saline (PBS). To evaluate systemic effects of sIL-1RAcP and IL-1Ra on development of CIA, we applied adenoviral delivery of the transgenes to immunized mice. At the onset of arthritis, we injected 3 × 108 plaque-forming units of adenoviral vector intravenously in immunized mice without clinical signs of CIA (22–24). Arthritis development in the hind paw and ankle joint was macroscopically monitored through day 38, at which point the experiment was terminated and the mice were killed. Arthritis was scored by 2 independent observers in a blinded manner on a scale ranging from 0 to 2 for each limb, based on redness, swelling, and, at later stages, ankylosis, as follows: 0 = no changes; +0.25 = 1–2 toes red or swollen; +0.5 = 3–5 toes red or swollen; +0.5 = swollen ankle; +0.5 = swollen footpad; +0.5 = severe swelling and ankylosis. A cumulative score was derived for all 4 paws, yielding a maximal possible score of 8 per animal. Serum was obtained and stored for further analysis.
IgG2a anti-BII titers were determined by enzyme-linked immunosorbent assay. Briefly, 96-well plates were coated with 10 μg of BII, followed by blocking of nonspecific binding sites. Serial dilutions of the mouse sera were added, followed by incubation with isotype-specific goat anti-mouse peroxidase (Southern Biotechnology Associates, Birmingham, AL) and substrate (5-aminosalicylic acid; Sigma). Absorbance was determined at 492 nm.
Splenocytes were obtained from mice systemically treated with sIL-1RAcP, IL-1Ra, or the control adenoviral vector AdCMV Luciferase. Mice previously immunized against BII were injected with the adenoviral vectors. Spleens from mice which had already developed CIA were used for lymphocyte proliferation assays in the presence or absence of recombinant mouse IL-1Ra (10 μg/ml). Three days after receiving the viral load, mice were killed and their spleens were dissected. Spleens were disintegrated and erythrocytes were removed by osmotic shock. The remaining cell fraction was washed twice and incubated in RPMI 1640 at 37°C in 5% CO2/95% air for 1 hour in order to remove the splenic adherent cell (SAC) fraction. Nonadherent lymphocytic cells were used for the lymphocyte stimulation assay. Lymphocyte stimulation was determined against the T lymphocyte–stimulating concanavalin A and the immunization antigen BII (heated for 10 minutes at 80°C). Thymocytes (1 × 105) were added to the stimuli and incubated for 3 days (37°C in 5% CO2/95% air). Six hours before harvesting of the cells, 0.25 μCi of 3H-thymidine was added. The amount of incorporated 3H-thymidine was quantified using a Micro Beta-plate reader (Perkin-Elmer, Brussels, Belgium).
Spleen lymphocytes were obtained from 3 × NF-κB reporter mice (32). Spleens were disintegrated and erythrocytes were removed by osmotic shock. Remaining cell fraction was washed twice and incubated in RPMI 1640 at 37°C in 5% CO2/95% air for 1 hour in order to remove the SAC fraction. Nonadherent lymphocytic cells were used for purification of T and B cell fractions. T lymphocytes were obtained through negative selection using MACS mouse pan–T cell sorting and purification kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Unlabeled T cell fraction was collected as effluent by placing separation columns in a strong magnetic field. Purity of isolated T cells was >96%. Columns were repeatedly washed using PBS containing 0.5% bovine serum albumin (BSA)/ 2 mM EDTA. In order to obtain the fraction containing the splenic B cell, columns were removed from the magnetic field and labeled cell fraction was flushed out using PBS/0.5% BSA/ 2 mM EDTA. Both the unlabeled T cell fraction and the labeled enriched B cell fraction were washed in PBS/0.5% BSA/2mM EDTA, centrifuged at 1,100 revolutions per minute for 5 minutes, and taken up in RPMI 1640 containing 5% fetal calf serum and gentamicin. For the NF-κB activity assay 1 × 105 T or B cells were seeded in Krystal 2000 96-well plates in the presence of IL-1Ra (1 μg/ml) or sIL-1RAcP (1 μg/ml) for 24 hours, followed by 6 hours of incubation using 10 ng/ml of IL-1α. Activation of NF-κB was quantified through determination of intracellular luciferase production, as described above.
Concentrations of IL-6 in serum were determined using the B9 bioassay as previously described (33). Briefly, serial dilutions of serum samples were made in culture medium in order to quantify IL-6 levels. B9 cells (5 × 103) were added to diluted serum samples, followed by incubation for 3 days (37°C in 5% CO2/95% air). The cells were incubated an additional 4 hours before harvesting in the presence of 0.25 μCi of 3H-thymidine.
Tissue samples from different organs of naive DBA/1 mice were snap-frozen in liquid nitrogen. Total RNA was extracted from these samples using TRIzol reagent, as described by Chomczynski and Sacchi (34). Prior to washing with saline and total RNA extraction with TRIzol, NIH3T3 NF-κB reporter fibroblasts, H4 NF-κB reporter chondrocytes, or EL.4NOB-1NF-κB reporter thymocytes were cultured for 24 hours. Quantitative real time PCR was performed using the ABI/Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). The PCR protocol consisted of 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. All PCRs were performed using SYBR Green master mix (Applied Biosystems), 10 ng of cDNA, and a primer concentration of 300 nmoles/liter in a total reaction volume of 25 μl. Quantification of the PCR signals was achieved by comparing the cycle threshold (Ct) value of the gene of interest of each sample with the Ct value of the reference gene GAPDH. Primer sequences for gene expression analysis were as follows: mouse GAPDH forward 5′-GGCAAATTCAACGGCACA-3′, reverse 5′-GTTAGTGGGGTCGTCCTG-3′; mouse IL-1RI forward 5′-CGAGGTCCAGTGGTATAAGACCTGTA-3′, reverse 5′-TCAGCCACATTCCTCACCAA-3′; mouse IL-1RII forward 5′-GCAGGCTATTACAGATGTGTTATGACA-3′, reverse 5′-GGATGGGTTCCGTGGTTGT-3′; mouse IL-1RAcP forward 5′-TTGGATACAAGGTGTGCATCTTC-3′, reverse 5′-GCTGAGGGTCTCATCTGTGACA-3′.
Nonlinear regression analysis was performed using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). The dose-response curves were analyzed and fitted according to a sigmoidal dose-response model (with variable slope). Significance of differences was determined using Student's t-test or Mann-Whitney U test (GraphPad Software). P values less than 0.05 were considered significant.
Both adenoviral vectors were validated and gene transfer resulted in high production of sIL-1RAcP and IL-1Ra as described previously (24, 35). Systemic overexpression of sIL-1RAcP resulted in circulating amounts of sIL-1RAcP up to 900 ng/ml 3 days after injection of the adenoviral vector (24). Prophylactic treatment through systemic overexpression of both sIL-1RAcP and IL-1Ra resulted in a marked reduction of disease severity in both front and hind paws, which was significant from day 30 through day 38 (Figure 1A). Although sIL-1RAcP treatment reduced the severity of arthritis, progression was still evident. This was in contrast to systemic IL-1Ra gene transfer, which arrested development of CIA.
Systemic overexpression of sIL-1RAcP in mice after immunization resulted in a significant reduction of circulating levels of IgG2a antibodies directed against BII after 7 days (Figure 1B). Therapeutic intervention using IL-1Ra also resulted in a significant reduction of circulating anti-BII IgG2a (Figure 1B). Reduced levels of the B cell–activating cytokine IL-6 preceded the observed reduction of circulating anti-BII IgG levels, which was evident 3 days after either sIL-1RAcP or IL-1Ra adenoviral injection. Mean ± SEM levels of IL-6, determined in serum from at least 3 treated animals per group, were as follows: control 527 ± 47 pg/ml, sIL-1RAcP 188 ± 31 pg/ml (P < 0.05 versus control), IL-1Ra 217 ± 23 pg/ml (P < 0.05 versus control).
Splenocytes were isolated from BII-immunized mice 3 days after systemic injection with Ad sIL-1RAcP, Ad IL-1Ra, or the control vector adenovirus luciferase. Lymphocytes obtained from IL-1Ra–treated animals showed a significantly reduced overall proliferative response toward the immunization antigen BII and the mitogen concanavalin A. Although both IL-1Ra and sIL-1RAcP ameliorated CIA, sIL-1RAcP treatment did not reduce lymphocyte proliferative activity, in contrast to IL-1Ra treatment (Figure 1C). To confirm that sIL-1RAcP was unable to inhibit lymphocyte proliferation, we performed an in vitro experiment using spleen-derived T lymphocytes from untreated DBA/1 mice with CIA. These lymphocytes were cultured in the presence or absence of either recombinant IL-1Ra or sIL-1RAcP protein and stimulated with the pan–T cell activator concanavalin A. Recombinant IL-1Ra (10 μg/ml) significantly inhibited the concanavalin A–induced lymphocyte proliferation in vitro, with 47% and 53% concentrations of concanavalin A at 1.25 μg/ml and 0.6 μg/ml, respectively. This revealed the importance of IL-1 in lymphocyte function, and confirmed our in vivo findings of IL-1Ra as a functional antagonist of T cell proliferative activity. Interestingly, and in contrast to IL-1Ra, sIL-1RAcP at a high dose (10 μg/ml) was not able to inhibit lymphocyte activity.
The different effect of sIL-1RAcP treatment on immunity, in which sIL-1RAcP mainly inhibited humoral immunity without affecting T cell function, might reflect a discrepancy of sIL-1RAcP inhibition of IL-1 signaling on T and B cells. To demonstrate that in vivo effects of sIL-1RAcP were the result of a direct effect of sIL-1RAcP–induced modulation of IL-1 signaling on B cells, we tested the IL-1 responsiveness of T and B cells in the presence or absence of sIL-1RAcP, using IL-1Ra as a control. Purified T and B cell populations derived from transgenic mice expressing a 3 × NF-κB reporter gene were shown to be sensitive to IL-1Ra treatment, which resulted in reduced NF-κB activity upon IL-1 stimulation (Figure 2). Interestingly, incubation with a similar dose of sIL-1RAcP in the presence of IL-1 did not affect NF-κB activation in purified T cell populations, whereas the enriched B cell fraction was shown to be sensitive to sIL-1RAcP treatment, although these differences did not reach statistical significance (Figure 2).
T and B cell populations expressed all 3 IL-1 receptor subtypes. Interestingly, B cells strongly expressed IL-1RII, revealing 21 times more IL-1RII messenger RNA (mRNA) compared with IL-1RI, while T cells expressed 5 times more IL-1RII compared with IL-1RI (Figure 2A). The levels of expression of the different types of IL-1 receptors may contribute to the antagonistic activity of sIL-1RAcP. Therefore, we wanted to further substantiate the mechanism of action in vitro using NF-κB reporter cell lines. For these experiments, the reporter cell lines were characterized by their expression of IL-1RI, IL-1RII, and IL-1RAcP. Detection of the IL-1 receptor components on cell lines using immunohistochemistry or fluorescence-activated cell sorting analysis was not possible due to low surface expression of all 3 IL-1 receptor components, which hampered their detection. Therefore, we used the more sensitive quantitative PCR for analysis of mRNA expression. On H4 chondrocytes, which, compared with B cells, also highly express IL-1RII, sIL-1RAcP efficiently inhibited IL-1 signaling (Figure 3A). A concentration of 1,000 ng/ml sIL-1RAcP inhibited the IL-1 (10 ng/ml) response by 47%; the maximal observed inhibition of 88% was observed at a concentration of 10,000 ng/ml sIL-1RAcP (Figure 3C).
The EL4.NOB-1 thymoma cell line expressed very low amounts of the IL-1RII (Figure 4A) ; even at the highest concentration of 10,000 ng/ml, sIL-1RAcP was unable to inhibit IL-1–induced NF-κB activity (Figure 4C). In contrast to EL4.NOB-1 thymocytes and H4 chondrocytes, the NIH3T3 cell line showed no expression of mRNA for IL-1RII and low expression of the membrane IL-1RAcP (Figure 5A). However, the NIH3T3 cells were clearly susceptible to dose-dependent IL-1 inhibition by sIL-1RAcP, revealing a 48% inhibition of the IL-1 activation at a concentration of 1,000 ng/ml in the presence of 10 ng/ml IL-1α. A 1,000-fold concentration of IL-1Ra (1,000 ng/ml IL-1Ra in the presence of 1 ng/ml IL-1α) was able to completely inhibit IL-1–induced NF-κB activity with all 3 reporter cell lines.
Membrane IL-1RAcP is a crucial component of the IL-1 signaling complex, stabilizing the IL-1–IL-1RI complex and mediating IL-1 signaling (7, 16, 17, 36). The recently discovered naturally occurring alternative splice transcript of the membrane IL-1RAcP comprises the 3 extracellular Ig-like domains (4, 22, 23). Due to the conservation of the ligand-binding domains of sIL-1RAcP, this molecule may still share the receptor complex stabilizing function, thereby acting as an inhibitor of IL-1 signaling through formation of an IL-1 trap. This action of sIL-1RAcP could therefore be distinct from IL-1Ra–mediated inhibition of IL-1 signaling, since binding of IL-1Ra to the IL-1RI does not lead to recruitment of IL-1RAcP (4, 37, 38). In this study, we compared the effect of both inhibitors on CIA.
Adenovirus-mediated intravenous transfer of sIL-1RAcP or IL-1Ra gene resulted in a clear-cut protective effect on bone and cartilage in murine CIA, as was shown previously (20, 21, 24, 25). Accordingly, we expected similar effects of both inhibitors on the parameters of immunity and inflammation. Indeed, both inhibitors reduced circulating levels of antigen-specific IgG and IL-6, a well-known B cell activating and maturating cytokine (39). This observation supports the contention that IL-1 is an adjuvant for antibody production (40, 41). In contrast, IL-1Ra gene transfer in mice inhibited both mitogen- and antigen-induced lymphocyte proliferation, whereas sIL-1RAcP gene transfer has no effect on this parameter. It is known that IL-1 plays an important role in the activation of T lymphocytes in CIA (42). Furthermore, the proliferative activity of T cells is enhanced in IL-1Ra−/− mice while decreased in IL-1α/β−/− mice, highlighting the importance of IL-1 in T cell activation and proliferation (43, 44). However, this unexpected discrepancy in T cell modulation could be due to differences in the pharmacokinetics of the IL-1 inhibitors. Therefore, we tested both inhibitors in vitro using recombinant protein.
Splenocytes from mice with CIA were obtained and incubated with either sIL-1RAcP or IL-1Ra. Recombinant IL-1Ra reduced mitogen-specific T cell proliferation, in contrast to sIL-1RAcP, which did not affect the T cell proliferative response. Likewise, in experiments using enriched T and B cell populations from NF-κB reporter mice, sIL-1RAcP inhibited IL-1–induced NF-κB activity in B cells but had no effect on IL-1 signaling in T cells. Notably, IL-1Ra inhibited IL-1–induced NF-κB activity in both T and B lymphocytes. These results confirmed the selective mode of action of sIL-1RAcP compared with IL-1Ra as observed in vivo. A possible explanation is that sIL-1RAcP acts as an IL-1 antagonist by competing for the association of IL-1RI with membrane IL-1RAcP. Therefore, differential expression of the IL-1 receptor components between cell types could determine the antagonistic activity of sIL-1RAcP. This was further emphasized by the fact that T and B cell populations are known to differ in their receptor expression (15, 45). B cells derived from BII-immunized mice revealed abundant expression of the IL-1RII, in contrast to purified T cells.
To elucidate the mechanism behind the cell-specific antagonistic activity of sIL-1RAcP, we compared 3 NF-κB luciferase reporter cell lines that differ in their IL-1 receptor composition (IL-1R expression in relation to antagonist activity is summarized in Table 1). We demonstrated that sIL-1RAcP was able to inhibit IL-1–induced NF-κB activation in cell lines that express either low levels of membrane IL-1RAcP or high levels of IL-1RII. It has already been shown that membrane- anchored sIL-1RAcP functions as a coreceptor and competitive IL-1 inhibitor (23). Those same authors were, however, unable to observe IL-1 inhibition using sIL-1RAcP on Hep-G2 cells, which they attributed to a low sIL-1RAcP concentration at the membrane level. Alternatively, the inability of sIL-1RAcP to inhibit IL-1 signaling on Hep-G2 cells could be a consequence of the high expression of membrane IL-1RAcP (46), in comparison with the low expression of NIH3T3 fibroblasts, for which we were able to induce sIL-1RAcP–mediated IL-1 antagonism.
|Relative mRNA expression||% IL-1 inhibition|
|IL-1R type I||IL-1R type II||IL-1RAcP||IL-1Ra||sIL-1RAcP|
|Splenic T cells||+/−||+||+/−||39†||0†|
|Splenic B cells||+/−||+++||+||44†||44†|
|NIH3T3 fibroblasts||+||− ND||+/−||84†||48†|
Taken together, our results show that the underlying mechanism of cell specificity is probably dependent on the availability of membrane IL-1RAcP for IL-1 signaling. As previously reported, sIL-1RAcP can interact with soluble IL-1RII, thus forming an IL-1 scavenging molecule (26). Hence, the formation of this IL-1 trap, together with the observed inhibition of IL-1 at the cell membrane, are both mechanisms of IL-1 antagonism which could operate simultaneously in vivo. We observed IL-1 antagonism by sIL-1RAcP on cells highly expressing IL-1RII (B cells and H4 chondrocytes). Since we cannot exclude the formation of a membrane-bound IL-1 decoy receptor comprised of IL-1RII and sIL-1RAcP, this could still be a possible mechanism of IL-1 antagonism on cells expressing high levels of IL-1RII. However, the IL-1RII can also indirectly determine the availability of the IL-1RAcP. The amount of IL-1RII expression on the membrane determines the sensitivity of the cell toward IL-1, and membrane IL-1RAcP is able to interact with the IL-1RII (18, 47). Therefore, abundant expression of the IL-1RII will not only lead to scavenging of IL-1, but will also reduce the available IL-1RAcP to form a signaling complex with the type I receptor, shifting the balance in favor of the sIL-1RAcP for receptor competition with the IL-1RAcP for formation of receptor complexes with IL-1RI.
This study provides evidence for an additional antagonistic mechanism by which sIL-1RAcP could directly inhibit IL-1 signaling at the cell membrane level. Due to the distinct action of sIL-1RAcP compared with IL-1Ra, both functionally and mechanistically, combining both antagonists offers additive therapeutic effects (25). With the sIL-1RAcP, we can now distinguish the roles of IL-1 in T cell– and B cell–driven processes, which may provide opportunities for treatment of diseases in which B cell derailment plays a prominent role.
We acknowledge the Central Animal facility (Faculty of Medicine, University of Nijmegen, The Netherlands) for the animal care. Furthermore, we would like to acknowledge D. T. Curiel and I. P. Dmitriev (Human Gene Therapy Center, University of Alabama at Birmingham) for constructing the adenoviral vectors, and W. Falk (Department of Internal Medicine, University of Regensburg, Regensburg, Germany) for supplying the plasmid containing the 5 × NF-κB luciferase reporter construct.