Published online before print June 18, 2008
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Department of Rheumatology and Inflammation Research, University of Göteborg, Sweden
1 Correspondence: Department of Rheumatology and Inflammation Research, Guldhedsgatan 10A, S-413 46 Göteborg, Sweden. E-mail: maria.bokarewa{at}rheuma.gu.se
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was assessed. Intracellular tranduction mechanisms activated by oligonucleotides were evaluated using specific inhibitors of signaling pathways and genetically modified mice. polyIC efficiently and dose-dependently induced the expression of Plg, IL-6, and IFN-, whereas TF was not induced by polyIC. polyI was unable to trigger IFN- production, and it was efficiently inducing Plg and TF. IFN-R and dsRNA-dependent protein kinase signaling were not required for the polyI-induced production of Plg or TF. Neither polyU nor polyC induced the expression of Plg or TF. Importantly, the presence of U- and C-nucleotide strands in the dsRNA significantly reduced expression of Plg and TF compared with polyI alone. Exposure of splenocytes to polyI activated the NF-
B pathway followed by the expression of TF and IL-6. In contrast, Plg production did not require NF-B, was only partly down-regulated by p38 MAPK inhibitor, and was efficiently inhibited by insulin, indicating a different mechanism for its induction. ssRNA exerts its TF-generating properties through NF-B activation in an IFN--independent manner. The expression of fibrinolytic versus coagulation proteins is regulated through distinctly different transduction pathways. As fibrinolytic and coagulation cascades are important components of inflammatory homeostatis, these findings might have importance for developement of new, targeted therapies.
Key Words: RNA • inflammation • plasminogen • tissue factor • coagulation • fibrinolysis
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B, IFN regulatory factor 3 (IRF-3), c-Jun, and activating transcription factor 2, and they coordinately induce transcription of other genes. Activation of IRF-3 with consequent induction of IFN type I activity has been considered as the main mediator of RNA effects [11
]. It has been recognized recently that structures other than TLRs, e.g., cytosolic RNA helicases, retinoic acid-induced protein I (RIG-1) and melanoma differentiation-associated gene 5 (Mda5), are also required for an antiviral response [12
, 13
]. Viral activation of these structures represents an alternative way of IFN induction. In our previous study, we showed that synthetic dsRNA had potent proinflammatory properties inducing the expression of cytokines in vitro and joint inflammation in vivo [14
] in an IFN--dependent manner [15
].
In the present study, we demonstrate that synthetic polyinosinic acid (polyI) and polyI:polycytiylic acid (polyIC) are potent inducers of the fibrinolytic agent Plg. In contrast, although polyI efficiently activates TF production, polyIC or polyI:polyuridylic acid (polyIU) down-regulates this phenomenon. We show that the expression of TF is regulated by NF-B-dependent mechanisms, and the expression of Plg is NF-B-independent.
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Reagents
The plasmin-specific synthetic substrate H-D-Val-Leu-Lys-Paranitroanilide (S-2251) was purchased from Chromogenix (Mölndal, Sweden). Spectrozyme FVIIa, human factor VII, and human TF/TF pathway inhibitor (TFPI)-depleted plasma were from American Diagnostica (Greenwich, CT, USA). Human Glu-Plg was obtained from Biopool (Umeå, Sweden). Human tissue-Plg activator, acetylase, was obtained from Boehringer Ingelheim (International GmbH, Ingelheim, Germany). Human fibrinogen was purchased from Enzyme Research Laboratories (South Bend, IN, USA). Insulin (Actrapid) was purchased from NovoNordisk (Bagsvaerd, Denmark).
Synthetic dsRNA consisted of ds copolymer, polyIC, or polyIU and ssRNA polymer consisting of polyI, polyU, and polycytidylic acid (polyC). All nucleic acids were purchased from Sigma-Aldrich (Stockholm, Sweden). dsRNA and ssRNA molecules were dissolved in 1 ml sterile water and diluted further in PBS to obtain a stock concentration of 1 mg/ml, which was kept at –20°C until use. sspolyI was annealed with polyU or polyC at 50°C for 30–40 min. Thereafter, the annealed dsRNAs (polyIC, polyIU) were kept at room temperature for 10–20 min before use.
Parthenolide (NF-B inhibitor), staurosporin (tyrosine kinase inhibitor), and H7 [protein kinase C (PKC) inhibitor] were purchased from Sigma-Aldrich. SB203580 (p38-MAPK inhibitor), LY294002 (PI-3K inhibitor), and PD98059 (p42/44 MAPK inhibitor) were purchased from Biosource (Nivelles, Belgium). SP600125 (JNK inhibitor) was purchased from Calbiochem (Darmstadt, Germany).
Cell preparation and culture conditions
Spleens from all mice were aseptically passed through a nylon mesh. Erythrocytes were lysed using ammonium chloride. The resulting single-cell suspension was resuspended in Iscove’s medium supplemented with 10% FCS, 5 x 105 M β-ME, 2 mM L-glutamine, and 50 µg/ml gentamicin. Subsequently, 1 x 106 cells/ml were incubated with different concentrations of dsRNA and ssRNA. Murine spleens cells were preincubated with the inhibitors of intracellular signaling 90 min prior to stimulation with polyI, polyC, polyU, polyIC, or polyIU. The cultures were maintained in 24-well plates (Nunc, Roskilde, Denmark) at 37°C in 5% CO2 and 95% humidity. Nuclear extracts for the EMSA were prepared following 2 h of stimulation. The supernatants were collected after 12 h or 48 h, centrifuged at 1500 rpm for 5 min, and kept frozen at –20°C until analyzed.
EMSA
Nuclear extracts for the EMSA were prepared following 2 h of stimulation of mouse splenocytes with polyI and polyU as well as from nonstimulated splenocytes as described [14
]. An equal protein amount of the nuclear extracts was incubated with ds oligonucleotides containing a NF-B-binding site (sense 5'-GGCTCAAACAGGGGGCTTTCCCTCCTCAATAT-3', antisense 5'-GGATATTGAGGAGGGAAAGCCCCCTGTTTGAG-3') and labeled with [32P]-deoxynucleotide (Amersham Pharmacia Biotech, Uppsala, Sweden) as described [14
]. For the mobility shift assay, antisera to p65 and p50 subunits of NF-B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were introduced to the reaction mixture.
IL-6
IL-6 levels were measured by a bioassay with cell clone B13.29, subclone B9, which is dependent on IL-6 for growth, as described previously [16
]. The samples were tested in 50-fold dilutions and compared with a standard curve obtained using mouse recombinant (mr)IL-6 (Genzyme, Kent, UK).
IFN-
IFN- levels were measured by a sandwich ELISA as described [15
]. In short, 96-well plates were coated with polyclonal sheep anti-mouse IFN- diluted 1/2000 in PBS overnight at 4°C. Blocking was performed for 1 h at 37°C using a buffer containing 0.1 M NaH2PO4/Na2HPO4, pH 6.8, 0.025% merthiolat, 0.05% Tween 20. Thereafter, plates were washed in PBS, and supernatants (diluted 1:5) or mrIFN- were added in a total volume of 50 µl. Following incubation and washing, detection of rat anti-mouse IFN- (clone 4E-A1, 0.25 µg/ml) antibodies was added. Biotinylated sheep anti-rat IgG (1 µg/ml) was used as a secondary antibody. The amount of IFN- in the supernatants was quantified using serial dilutions of the mouse IFN-. The detection limit of the assay was 10 U/ml.
Plg level
Plg level was determined using an amidolytic assay as described [17
]. Plates were filled with Tris/HCl buffer (50 mM, pH 7.4), and then supernatants were added and activated using tissue-type Plg activator (tPA; acetylase; 2 µg/ml) for 10 min at 37°C. The activation of Plg present in the supernatants into plasmin was followed by plasmin-dependent hydrolysis of 0.5 µM S-2251 substrate (Chromogenix). The abosorbance was registered at 405 nm (A405), and color development was compared with standard dilutions of human Glu-Plg ranging from 100 to 1.6 µg/ml.
TF activity
TF activity was measured using reagents from American Diagnostica. Plates were filled with 50 mM Tris/HCl buffer (pH 8.6), and supernatants were added and incubated for 10 min at 37°C with equal volumes of TF/TFPI-depleted plasma and human factor VII. The activation of factor VII was monitored by the cleavage of Spectrozyme FVIIa (10 µM). The sample was analyzed at A405 and compared with the values obtained from the standard curve obtained using lapidated, recombinant human (rh)TF (ranged from 100 pM to 1.6 pM).
Turbidimetric fibrinolytic assay
Turbidimetric fibrinolytic assay was carried out as described previously [18
]. Briefly, fibrin gel was prepared in a microplate well at 37°C by incubation of the mixture containing 2.5 g/l human fibrinogen (Enzyme Research Laboratories, South Bend, IN, USA), 1 NIH U/ml thrombin, and Plg (1–10 µM) in 10 mM imidazole buffer containing 150 mM NaCl (pH 7.4). The test supernatant was mixed with 3 µg tPA and applied on the surface of the clot. The microplates were incubated in a humid chamber at 37°C and vigorously shaken. Lysis of fibrin clots was monitored by light A340. The lysis time (t1/2), defined as the time needed to reduce the turbidity of the clot to half-maximal value, was used as a quantitative parameter of fibrinolytic activity.
Statistical analysis
Statistical comparison between the groups was made by using the Mann-Whitney. Results of the paired samples were compared using the t-test. All values are reported as the mean ± SEM. Values of P < 0.05 are considered significant.
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Figure 1. Expression of Plg and TF in murine leukocytes following stimulation with ss- and dsRNA. (A) Levels of Plg were measured in spleen cell cultures (n=19, 1x106/ml) following 48 h stimulation with polyI, polyIC, and polyIU (0, 50, 150 µg/ml). polyI as well as polyIC and polyIU induced expression of Plg dose-dependently. (B) Levels of TF produced in vitro by spleen cell cultures (n=6, 1x106/ml) after 12 h stimulation with polyI, polyIC, and polyIU (0, 50, 150 µg/ml). polyI induced production of TF dose-dependently. In contrast, stimulation of cells with polyIC did not significantly change the level of TF in the cell medium, and stimulation with polyIU had an inhibitory effect on TF production.
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Figure 2. Effect of ssRNA consisting of different oligonucleotides for the expression of TF by murine leukocytes. (A) Levels of Plg were measured in spleen cell cultures (n=19, 1x106/ml) following 48 h stimulation with polyI, polyC, and polyU (0, 50, 150 µg/ml). polyI, but not polyC and polyU, induced expression of Plg dose-dependently. (B) Levels of TF produced in vitro by spleen cell cultures (n=6, 1x106/ml) after 12 h stimulation with polyI, polyC, and polyU (0, 50, 150 µg/ml). polyI induced production of TF dose-dependently. In contrast, stimulation of cells with polyC or polyU did not significantly change the level of TF in the cell medium.
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Figure 3. Newly expressed Plg is functionally active in lysing fibrin clot, and fibrin clots containing 2.5 g/l human fibrinogen have been prepared as described in Materials and Methods. The lysing of clots was monitored by addition of 100 µl culture medium from splenocyte stimulation (polyI, 150 µg/ml; polyU, 150 µg/ml; and unstimulated) mixed with 3 µg tPA applied on the surface of the clot. The microplates were incubated in a humid chamber at 37°C and vigorously shaken. The change in clot turbidity was measured by A340. The t1/2, defined as the time needed to reduce the turbidity of the clot to half-maximal value, was used as a quantitative parameter of fibrinolytic activity. *, Significant (P<0.05) difference between the supernatants from polyI- and polyU-stimulated cells at different time-points.
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PolyI-induced expression of TF is dependent on NF-B, and expression of Plg is partly dependent on p38 MAPK
dsRNA activates NF-B signaling leading to the expression of proinflammtory cytokines, one of which is IL-6 [14
]. The effect of ssRNA on NF-B signaling was evaluated following stimulation of splenocytes with polyI and polyU polymers using EMSA. A translocation of the p65 unit of NF-B by polyI- but not polyU-stimulated cells into the cell nuclei was demonstrated (Fig. 4
). The NF-B activation was followed by up-regulation of IL-6 (not shown), Plg, and TF (Fig. 1)
.
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Figure 4. Binding of nuclear extracts from mouse spleen cells stimulated with ssRNA to NF-B-specific binding sites, assessed by EMSA. Lane 1, Nonstimulated spleen cells. Lanes 2 and 3, Binding of nuclear extracts from spleen cells stimulated with polyI, 50 and 150 µg/ml, respectively. Lane 4, Nuclear extract as in Lane 3 following preincubation with an excess of competitive-binding, unlabeled NF-B oligonucleotides. Lane 5, Nuclear extract as in Lane 3 following preincubation with anti-p65 antibodies. A decreased mobility of the NF-B complexed with anti-p65 is shown. Lanes 6, Binding of nuclear extracts from spleen cells stimulated with polyU, 150 µg/ml. Lane 7, Nuclear extract as in Lane 6 following preincubation with anti-p65 antibodies.
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B and MAPKs for the production of Plg and TF was further studied by preincubating the cells with specific pathway inhibitors prior to polyI stimulation. We used parthenolide (25 µM) as the NF-B inhibitor, SB203580 (10 and 25 µM) as the p38 MAPK inhibitor, PD98059 (10 and 25 µM) as the p42/44 MAPK inhibitor, and SP600125 (10 µM) as the JNK inhibitor. The concentration of the inhibitors was adjusted to assure cell survival and minimal effect on background production of Plg and TF.
To elucidate intracellular pathways activated by polyI, the levels of IL-6 were measured in splenocyte cultures following introduction of synthetic inhibitors. The polyI-induced synthesis of IL-6 was dependent on NF-B activation and on the activity of p44/42 and p38 MAPK (Fig. 5
). In contrast, inhibition of JNK had no significant effect on synthesis of IL-6.
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Figure 5. Effect of NF-B and MAPK inhibitors on the production of Plg and TF induced by polyI nucleotides in mouse leukocytes. Plg and TF production was evaluated in spleen cell cultures (n=8, 1x106/ml) following stimulation with polyI (150 µg/ml). Cells were treated with synthetic inhibitors of NF-B (parthenolide, 25 µM) or inhibitors of MAPK, SB203580 (25 µM) for p38, PD98059 (25 µM) for p44/42, and SP600125 (10 µM) for JNK for 90 min at 37ºC and stimulated with polyI. Production of Plg and TF in the cell cultures stimulated with polyI (100%) was compared with the cells exposed to a combination of polyI and an inhibitor as indicated. Treatment of spleen cells with the chosen concentrations of inhibitors had no effect on the basal production of Plg and TF. *, Significant difference (P<0.05) between the cells treated with an inhibitor as compared with those exposed directly to polyI stimulation.
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B, p44/42, and JNK MAPKs (Fig. 5)
. Only exposure of cells to the inhibitor of p38 MAPK slightly reduced Plg production. The intracellular ways required for polyIC-induced synthesis of Plg demonstrated a similar pattern being independent on NF-B and MAPK (not shown). The exposure of splenocytes to the NF-B inhibitor resulted in a significant reduction of TF expression in response to polyI (Fig. 5)
, whereas the inhibitors of p38 and JNK MAPK could not significantly change the expression of TF in response to polyI. These results suggest that NF-B is an important step regulating polyI-triggered expression of TF. In contrast, the expression of Plg is regulated independently of this pathway.
IFN- signaling is not required for polyI-induced production of Plg and TF
The up-regulation of intracellular signaling pathways having IFN-β production as the end-point [11
] is an important step mediating effects of RNA. Thus, we analyzed the IFN- production following exposure of murine splenocytes to ssRNA. The up-regulation of IFN- was only observed in the splenocyte cultures stimulated with polyIC but not with polyI, -U, or -C (Fig. 6
).
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Figure 6. IFN- production in mouse leukocytes following stimulation with ds- and ssRNA. Levels of IFN- were measured in spleen cell cultures (n=5, 1x106/ml) following 48 h stimulation with polyIC, polyI, polyU, and polyC (0, 50, 150 µg/ml). Stimulation with polyIC induced IFN- production in mouse leukocytes. Exposure of mouse leukocytes to ssRNA polynucleotides did not induce IFN- production. n.s., Not significant.
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R knockout mice, PKR knockout mice, and their congenic littermates (Fig. 7
). In addition, spleen cells from wild-type mice were directly stimulated with rhIFN- (200 and 1000 units/ml). No increase of Plg or TF expression was observed in response to IFN- stimulation, indicating that IFN- has no direct effect on the production of these proteins (not shown).
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Figure 7. IFN signaling is not required for the production of plasminogen or TF following polyI stimulation. Spleen cell cultures (1x106/ml) were prepared from mice deficient for IFN-R (n=5), PKR (n=6), or their congenic littermates (n=6) and stimulated with polyI (0, 50, 150 µg/ml). The production of Plg (A) and TF (B) in the supernatants was measured following 48 and 12 h, respectively. *, Significant difference (P<0.05) between the cells treated with polyI as compared with those untreated.
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Insulin serves as a potent inhibitor of polyI-induced production of Plg
The murine Plg gene promotor contains several C/EBPβ-binding sites [19
]. Transactivation of C/EBPβ could therefore be dependent on the activity of PI-3K [20
, 21
]. To assess the role of the PI-3K axis for the expression of polyI-induced Plg, splenocytes were exposed to the inhibitors of PI-3K, LY294002 (25 mM), and PKC inhibitor H7 (25 mM) and to the C/EBPβ inhibitor, insulin (in concentrations 0.01–1 U/ml). Treatment of splenocytes with LY294002 prior to polyI stimulation resulted in a slight increase of Plg production (Fig. 8
). In contrast, treatment of splenocytes with insulin resulted in total abrogation of Plg production in response to polyI (Fig. 8)
. Interestingly, neither LY294002 nor insulin affected the background production of Plg in splenocyte cultures.
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Figure 8. The role of PI-3K and insulin in the expression of Plg following polyI stimulation. Murine splenocytes (n=6, 1x106/ml) were exposed to PI-3K inhibitor LY294002 (25 µM) or to insulin (1 U/ml), followed by stimulation with polyI (150 µg/ml). The levels of Plg were measured in supernatants after 48 h of stimulation. Exposure of spleen cells to insulin efficiently abrogated polyI-induced production of Plg, and the production of Plg in nonstimulated cells in the presence of insulin remained unchanged. *, Significant difference (P<0.05) between the cells treated with polyI as compared with those untreated.
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Most of the immunological and proinflammatory properties of dsRNA are related to the intracellular signaling through the activation of type I IFN responses or the NF-B-dependent cascade [11
, 14
, 15
]. In agreement with this, we observed that the dsRNA induced expression of IFN- and IL-6. However, IFN expression was not inducible when dsRNA was split into its ss components. In contrast, the expression of IL-6 was unaffected following ssRNA stimulation. These observations indicate independent pathways of cell activation in response to stimulation with ds- and ssRNA structures, respectively. The activation of IL-6 expression had a clear NF-B-dependent pattern, as it was reduced following abrogation of NF-B signaling.
RNA exerts its immunomodulatory effects through activating TLR. Different preparations of RNA chose different receptors as gates for their signaling. Indeed, dsRNA acts predominantly through TLR3, requiring Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) as an adaptor molecule [11 ], and ssRNA acts through TLR7 and -8 using adaptor molecule MyD88 [6 ]. In our study, the expression of Plg and TF occurs in a MyD88-independent manner, suggesting that ssRNA-induced production of TF and Plg was not dependent on interaction with TLR7/TLR8. However, other adaptor molecules, such as TRIF, TRIF-related adaptor molecule, and MyD88 adaptor-like protein, may not be excluded, as their participation in TF and Plg expression is not covered by present examinations. Additionally, the role of TLR-independent ways of RNA recognition, e.g., by interaction with cytosolic RNA helicases RIG-1 and Mda5, is not evaluated.
In our set of experiments, neither the expression of TF nor that of Plg was dependent on the activation of the IFN- pathway. Indeed, stimulation of cells with ssRNA did not lead to expression of IFN-. In contrast, when dsRNA (polyIC) was split into polyI and polyC nucleotide strands, a difference in their activity was observed. polyI but not polyC was active with respect to activation of Plg and TF. Also, the expression of TF and Plg was preserved in the IFNR knockout mice and in the absence of PKR signaling. Finally, stimulation of cells with rIFN- was unable to induce TF and Plg expression by mouse splenocytes. We proved that TF expression in response to ssRNA was dependent on the NF-B-mediated transcription. This may by considered as a direct effect of RNA signaling through TLR or a secondary effect as a result of TNF- or thrombin expression. In both cases, TF synthesis has been shown to be mediated through the NF-B signaling pathway [23
, 24
].
We also demonstrated that Plg and TF activation triggered by ssRNA is mediated through distinctly different intracellular pathways. Experiments with specific inhibitors of signaling pathways indicated that RNA-induced Plg expression was in contrast to TF, not dependent on NF-B activation. The promoter of the Plg gene contains several C/EBPβ-binding sites [19
], which have been shown important for Plg induction. Activation of C/EBPβ could be achieved by IL-6 in a MAPK-dependent manner and suppressed by insulin activation of PI-3K [20
, 21
]. We achieved a partial reduction of ssRNA-induced Plg expression by disrupting p38 MAPK signaling. The latter observation is supported by previous reports of MAPK-dependent Plg expression [21
]. An indirect stimulation of Plg production following IL-6 up-regulation is a less-probable mechanism. The up-regulation of IL-6 was prevented by the NF-B inhibitor, and Plg production was unaffected. This indicates that ssRNA induces a direct activation of Plg expression independently of IL-6. We have also shown that insulin could totally abrogate polyI-induced expression of Plg, suggesting the role of PI-3K signaling.
To conclude, the present study shows that besides proinflammatory cytokines, RNA induces expression of coagulation initiator TF and fibrinolytic protease precursor Plg. The ssRNA sequence consisting of the polyI motif is essential for this activation. ssRNA polyI induced Plg and TF, and dsRNA induced only Plg. Plg and TF production was mediated independently of IFN signaling. TF expression was dependent on the NF-B pathway, and expression of Plg is suggested to be dependent on PI-3K activity. The intriguing finding showing that polyC is a potent inhibitor of a polyI-triggered coagulation/fibrinolysis cascade might constitute a basis of novel therapy in virus-induced disseminated intravascular coagulation syndrome.
Received May 23, 2006; revised April 14, 2008; accepted April 23, 2008.
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and 
interferon, but not hemorrhage or pathogen burden, dictate levels of protective fibrin deposition during infection Infect. Immun. 74,1181-1188-defensins neutralize fibrinolytic activity exerted by staphylokinase Thromb. Haemost. 91,991-999[Medline]B and nitric oxide in responders and nonresponders to IFN therapy J. Viral Hepat. 10,360-366[CrossRef][Medline]
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