Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages
Objective— Fibrinolytic activity is upregulated in atherosclerotic lesions, yet little is known about the role of plasmin in macrophage function. We postulated a direct effect of plasmin on human monocyte-derived macrophages.
Methods and Results— Plasmin activates macrophages via the annexin A2 heterotetramer composed of annexin A2 and S100A10 with subsequent stimulation of Janus kinase JAK1/TYK2 signaling. JAK1/TYK2 leads to STAT3 activation, Akt-dependent NF-κB activation, and phosphorylation of extracellular signal-regulated kinase 1/2 and mitogen-activated kinase p38. These signaling pathways trigger nuclear translocation of STAT3 and p65 transcription factors and the induction of the proinflammatory cytokines tumor necrosis factor-α and IL-6. Inhibitors of JAK, p38, and NF-κB revealed that these signaling pathways are indispensable for the plasmin-mediated tumor necrosis factor-α and IL-6 induction. By contrast, the extracellular signal-regulated kinase 1/2 activation is essential only for the IL-6 expression. The activation clearly depends on the proteolytic activity of plasmin, which cleaves the A2 subunit of the annexin A2 heterotetramer. Downregulation of each of the receptor subunits by antisense oligodeoxynucleotides abolished the plasmin-induced expression of proinflammatory cytokines stressing the crucial role the annexin A2 heterotetramer.
Conclusions— Plasmin generated at sites of inflammation such as atherosclerotic lesions will trigger cytokine expression in human macrophages.
Inflammation is central to all stages of atherosclerosis. Macrophages particularly are the main source of cytokines in atherosclerotic plaques.1,2 Proinflammatory cytokines such as tumor necrosis factor (TNF)-α upregulate the expression of adhesion molecules such as selectins and vascular adhesion molecule-1, which are important for monocyte recruitment, but TNF-α also regulates apoptosis as well as procoagulant and fibrinolytic activity of endothelial cells. Elevated levels of TNF-α are associated with an elevated risk of recurrent myocardial infarction and cardiovascular death after a first myocardial infarction.1,2 The role of IL-6 in atherosclerosis appears more ambivalent; exogenously applied, it enhances fatty lesions in mouse models, whereas in apolipoprotein E/IL-6 double-knockout mice rather a reduced recruitment of inflammatory cells into the atherosclerotic plaque was observed. However, IL-6 is crucial for the acute phase response and IL-6 plasma levels appear to be predictive of future coronary artery disease.2
Although excessive inflammatory and immune responses driven by proinflammatory cytokines are held responsible for cardiovascular events associated with atherosclerosis, the initiation of the cytokine induction is still poorly understood and the initial triggers for the cytokine production remain obscure. Several recent intervention trials with antibiotics suggest that contrary to earlier belief, pathogens such as Chlamydia pneumoniae infection are not an etiologic agent for atherosclerosis.1,2 Oxidized low-density lipoprotein, while instrumental in triggering the early atherosclerotic events, seems to be less critical in upholding the inflammatory environment leaving the primary trigger of cytokine induction in atherosclerosis largely unexplored.2
Proteases, specifically serine proteases, play also a major role in atherosclerosis. Contact activation takes place in any type of chronic inflammation including atherosclerosis and is invariably associated with the generation of plasmin.3,4 Consistently in human atherosclerotic lesions, fibrinolytic activity is upregulated and positively correlates with the severity of coronary lesions.4,5 In addition, studies in plasminogen knockout mice indicated that plasmin/plasminogen alters the vascular response to injury. During remodeling plasmin/plasminogen is essential for this compensatory response.4,5 At the molecular level, apart from its fibrinolytic function, plasmin is a potent cell activator. In platelets plasmin induces aggregation and in endothelial cells it promotes biosynthesis of platelet-activating factor and chemotaxis.4 Moreover, in human monocytes, plasmin triggers release of proinflammatory lipid mediators, induces chemotaxis, and triggers the expression and release of cytokines, but also of CD40 and the macrophage chemoattractant protein-1.6–9
On this background, we hypothesized that plasmin generated in atherosclerotic lesion could induce macrophage activation.
Materials and Methods
A complete Materials and Methods section is available in the online data supplement at http://atvb.ahajournals.org.
Cell Preparation and Culture
Monocyte-derived human macrophages, monocytes, and neutrophils were isolated and cultured as described.10,11
Analysis of Protein Expression
Proteins were analyzed by Western immunoblot and flow cytometry11; cytokine levels were quantified with enzyme-linked immunosorbent assays.
Reverse Transcription and Polymerase Chain Reaction Analysis
Cytokine mRNA was quantified by semi-quantitative reverse-transcription polymerase chain reaction from total RNA of 1×106 macrophages.11,12
Inhibition of Annexin A2 and S100A10 Expression by Antisense Oligodeoxynucleotides
For in vitro knockdown phosphorothioate-modified oligodeoxynucleotides (ODN; Thermo Hybaid, Ulm, Germany) were applied to macrophage cultures.11
STAT and NF-κB Family Transcription Factor Assays
Activations of STAT1α, 3, 5A, 5B, and p65, c-Rel, RelB were determined with DNA-binding TransAM enzyme-linked immunosorbent assays for STAT and NF-κB family transcription factors in nuclear extracts from stimulated macrophages.9,12
Means±SEM are shown. Probabilities calculated with the Newman-Keuls test were considered significant for P<0.05.
Human Macrophages Express the Annexin A2 Heterotetramer
The macrophage phenotype was confirmed by flow cytometry showing reduction of the monocyte-specific surface marker CD14, increased expression of CD68, and exposure of CD71 (data not shown), which is characteristic for macrophages.10
Because we have previously identified the annexin A2 heterotetramer as receptor transducing the plasmin-mediated signaling in monocytes,11 we investigated its expression in human macrophages. Immunoblots of whole cell lysates revealed that macrophages in comparison to monocytes exhibit an increased expression of both subunits of the annexin A2 heterotetramer, annexin A2 and S100A10 (143±8% and 230±6%, respectively). By contrast, polymorphonuclear neutrophils, which do not respond to plasmin,7 likewise do not express annexin A2 (Figure 1A). We additionally confirmed the expression of the respective subunits on the macrophage surface by flow cytometry (Figure 1B). Stimulation of macrophages with plasmin led to cleavage of ≈6% of annexin A2 as detected in whole cell lysates yielding a proteolytic fragment of ≈33 kDa, whereas S100A10 remained unaffected (Figure 1C). This cleavage is dependent on the proteolytic activity of plasmin because exposure of macrophages to catalytically inactivated plasmin failed to produce the fragment (Figure 1C). These data suggest that macrophages share with monocytes the annexin A2 heterotetramer as signaling plasmin receptor.
Plasmin Triggers Signaling in Human Macrophages
Plasmin may activate multiple signaling pathways, in particular the JAK1/STAT, the p38 MAPK,9 and the NF-κB pathway.8 To explore the activation of JAK kinases we used antibodies against phosphorylated JAK1, JAK2, JAK3, and TYK2 for Western immunoblotting of macrophage whole cell lysates (Figure 2A). Stimulation of macrophages with 0.43 CTA U/mL plasmin triggered tyrosine phosphorylation of JAK1 and TYK2 within 5 minutes, reaching a maximum after 15 minutes. By contrast, plasmin did not induce phosphorylation of JAK2 or JAK3 (data not shown). As a consequence of the JAK1/TYK2 activation, phosphorylation of STAT3 on Tyr705 was observed within 5 minutes (Figure 2A). Transcriptionally active STAT3 requires both Tyr and Ser phosphorylation.9 Plasmin also triggered STAT3 phosphorylation on Ser727 (Figure 2A).
In addition to STAT3 activation, plasmin elicits phosphorylation of p38 MAPK after 5 minutes of stimulation with a marked increase after 15 minutes (Figure 2B). Plasmin also activates ERK1/2 within 5 minutes with the maximum reached after 15 minutes. During the whole observation time, we did not detect any activation of SAP/JNK, the third family member of the MAP kinases (data not shown).
Because plasmin may activate NF-κB signaling,8 we analyzed the phosphorylation state of the NF-κB inhibitor IκBα by immunoblotting. Plasmin induced phosphorylation of IκBα, targeting the inhibitor to proteosomal degradation, consequently allowing nuclear translocation of NF-κB. Because the Akt kinase is an upstream effector of NF-κB signaling,13 we checked its activation status by immunoblotting; plasmin induced a time-dependent phosphorylation of Ser473 that is required for the full activation of Akt114 (Figure 2C). These data show that in macrophages plasmin triggers several signaling pathways, particularly JAK1/STAT3, p38, and ERK1/2 MAPK, and NF-κB.
To address the role of JAK1 and TYK2 activation in the regulation of downstream targets such as the p38 MAPK, ERK1/2, Akt1, and IκBα, we used the JAK inhibitor AG490 (Figure 2D). At 50 μmol/L AG490 inhibits JAK115 but does not inhibit TYK2 (data not shown). Immunoblots of whole cell lysates from macrophages pretreated for 30 minutes with AG490 and stimulated with plasmin for 30 minutes revealed that the inhibitor impairs the phosphorylation of ERK1/2, Akt1, and the subsequent phosphorylation of IκBα, but not that of p38 MAPK (Figure 2D), suggesting that both ERK1/2 and Akt, but not the p38 MAPK, are directly dependent on JAK1 activation.
For quantitative densitometric analysis of the various Western immunoblots shown in Figure 2A to 2D, see supplemental Figure I (available at http://atvb.ahajournals.org).
Plasmin Induces Nuclear Translocation of STAT and NF-κB Transcription Factors
To define the profile of the plasmin-induced nuclear translocation of STAT and NF-κB proteins, we analyzed nuclear extracts from plasmin-stimulated or control macrophages with enzyme-linked immunosorbent assays for the respective transcription factors.12 Plasmin triggered activation and nuclear translocation of STAT3, but not of STAT1, STAT5A, or STAT5B (Figure 3A). Similarly, plasmin-stimulated macrophages exhibit binding of p65, but not of RelB or c-Rel (Figure 3B). Thus, stimulation of macrophages with plasmin yields nuclear translocation of transcriptionally active STAT3 and p65 transcription factors.
Plasmin Elicits Induction of Proinflammatory Cytokines
Because activated STAT3 and p65 can induce gene expression, we analyzed the ability of plasmin to elicit expression of proinflammatory cytokines in macrophages. Plasmin (0.43 CTA U/mL) induced a time-dependent increase of TNF-α and IL-6 mRNA (Figure 4A). Lipopolysaccharide (LPS) (1 μg/mL) stimulation induced the expression of both TNF-α and IL-6 mRNA quicker with significant induction of TNF-α and IL-6 mRNA already after 1 hour. The induction of TNF-α and IL-6 mRNA in macrophages stimulated with plasmin for 3 hours was also concentration-dependent (Figure 4B). Although 0.043 CTA U/mL plasmin increased TNF-α and IL-6 mRNA, the optimum response was reached at 0.43 CTA U/mL; at 1.43 CTA U/mL plasmin, there was almost no further increase.
Because the proteolytic activity of plasmin seems to be indispensable for the activation of signaling pathways,7,11 we examined its role in TNF-α and IL-6 induction. Catalytically inactivated plasmin failed to stimulate the expression of TNF-α and IL-6 mRNA (Figure 4C). Thus, activation of the proinflammatory cytokines by plasmin in macrophages requires proteolytically active plasmin. For quantitative densitometric analysis of gels used in Figure 4A to 4C, see supplemental Figure II.
Similar to LPS (1 μg/mL), plasmin induced a time- and concentration-dependent release of TNF-α and IL-6 from human macrophages (Figure 5A, 5B). The cytokines became detectable as early as 3 hours after stimulation. The release of TNF-α reached a maximum within 3 hours after plasmin and within 6 hours after LPS stimulation. The time course of IL-6 release appeared somewhat delayed, starting after 3 hours, but continuing up to 6 to 12 hours. Generally, LPS triggered a more rapid and extensive release of cytokines, indicating differences between the LPS- and plasmin-mediated activation of macrophages. These data show that plasmin triggers expression and release of proinflammatory cytokines such as TNF-α and IL-6 in human monocyte-derived macrophages with a somewhat lower potency than the standard stimulus LPS.
Plasmin-Induced Activation of JAK1, p38, ERK1/2, and NF-κB Is Indispensable for the Cytokine Expression
To gain evidence for the role of the initiated signaling pathways in the plasmin-induced release of TNF-α and IL-6, we pretreated macrophages with inhibitors for JAK1 (AG490), p38 MAPK (SB203580), the MAPK/ERK kinase pathway (U0126), and an inhibitor of NF-κB (acetyl-11-keto-β-boswellic acid [AKβBA])12 before stimulation with plasmin. We collected the supernatants 3 and 6 hours after plasmin (0.43 CTA U/mL) stimulation for TNF-α and IL-6, respectively, and determined the cytokine release. Pretreatment of macrophages with 50 μmol/L AG490, 1 μmol/L SB203580, and 10 μmol/L AKβBA for 30 minutes inhibited the plasmin-induced TNF-α release by 63±6%, 75±6%, and 70±14% (P<0.01), respectively. In contrast, inhibition of ERK1/2 by the MEK inhibitor U0126 10 μmol/L did not affect the plasmin-mediated expression of TNF-α (Figure 5C). Similar to the TNF-α production, pretreatment of macrophages with 50 μmol/L AG490 and 1 μmol/L SB203580 significantly decreased the plasmin-induced release of IL-6 by 76±11% and 79±11%, respectively (Figure 5D; P<0.05). Not unexpected, the plasmin-induced activation of NF-κB was also essential for the expression of IL-6, because it was inhibited by AKβBA by 66±11% (P<0.05). At variance to the TNF-α induction, activation of ERK1/2 was indispensable to the plasmin-induced release of IL-6, because the MEK inhibitor U0126 reduced its release by 86±5% (P<0.01; Figure 5D).
These data imply pivotal roles of the JAK1/STAT3, MAPK, and NF-κB signaling pathways in the plasmin-mediated induction of TNF-α and IL-6 in human macrophages.
The Annexin A2 Heterotetramer Is a Plasmin Receptor in Macrophages
We addressed the role of the annexin A2 heterotetramer in the plasmin-induced expression of cytokines in macrophages by antisense ODN experiments. Treatment of the cells for 48 hours with antisense ODN directed against the annexin A2 or the S100A10 proteins induced a marked decrease of annexin A2 or S100A10 protein expression, which was not observed in untreated macrophages or cells treated with the control sense ODN (Figure 6A). For quantitative densitometric analysis of the gels, see supplemental Figure III.
Pretreatment of macrophages with annexin A2 antisense ODN profoundly inhibited TNF-α and IL-6 release by 86±3% and 61±11%, respectively (P<0.01; Figure 6B, 6C). Similarly, pretreatment with S100A10 antisense ODN resulted in significant inhibition of TNF-α and IL-6 release by 73±16% and 64±8%, respectively (P<0.01; Figure 6B, 6C). Treatment of macrophages with ODN without plasmin stimulation had no effect neither on the TNF-α or the IL-6 release (data not shown). These data imply that plasmin stimulates human macrophages through the annexin A2 heterotetramer.
Macrophages possess crucial effector functions in a variety of chronic inflammatory diseases including atherosclerosis1 and rheumatoid arthritis, where, because of contact activation, generation of plasmin occurs.3,4 Here we show for the first time that the serine protease plasmin upregulates proinflammatory cytokines such as TNF-α and IL-6 in human macrophages. Plasmin induces activation of multiple signaling pathways including JAK/STAT, p38 MAPK, ERK1/2, and the NF-κB signaling pathway. At the molecular level we demonstrate that similar to monocytes,11 the annexin A2 heterotetramer mediates the plasmin signaling in macrophages, and that its cleavage is a critical event for subsequent cell activation.
Plasmin induces gene expression in macrophages via JAK1 activation, as shown by the use of the JAK inhibitor AG490. The activation kinetics show an early activation of JAK1, p38, and STAT3 on Tyr705 (<5 minutes) followed by a second activation phase starting 15 minutes after plasmin stimulation involving NF-κB signaling initiated by phosphorylation of Akt and IκBα. The JAK family members, encompassing JAK1 to JAK3 and TYK2, are generally considered as early activated kinases in the type I and II interferon receptor signaling.16 Such an early role of JAK signaling also occurs in macrophages and may drive the crucial phosphorylation events. The classical downstream targets of JAK1 are the STAT proteins, which become phosphorylated at their tyrosine residues. In addition, we observed phosphorylation of STAT3 on Ser727, which is induced by ERK1/2 and p38 MAPK, and which is pivotal for full transcriptional activity of STAT3.9,17 In addition, JAK1 can also target IκBα degradation through phosphorylation of Akt. JAK proteins stimulate the phosphoinositide 3-kinase,16 subsequently activating Akt and the aforementioned activation of NF-κB signaling through the stimulation of the IκB kinase complex.13 Our data showing plasmin-induced Akt activation coinciding with IκB phosphorylation and subsequent translocation of NF-κB are in agreement with activation of the IκB kinase complex via Akt.
JAK1 stimulates the MEK/ERK1/2 pathway through the recruitment of SHP-2/Grb2/SOS and activation of the Ras/Raf/MEK pathway.18 The stimulation of macrophage with plasmin triggers ERK1/2 activation and our data with AG490 show that this activation, but not that of p38 MAPK, depends on JAK1. Besides, our data are consistent with a phosphorylation of the p38 MAPK through TYK2 similar to interferon-α–activated and interferon-β–activated pathways,16 where activation of TYK2 stimulates the p38 MAPK through activation of Rac1.19
The signaling pathways induced by plasmin in macrophages are converging at the nuclear translocation of the STAT3 and NF-κB transcription factor p65 triggering expression of TNF-α and IL-6. At the transcriptional level, TNF-α and IL-6 genes are regulated by recruitment of NF-κB to their promoters. The promoter region of TNF-α contains 2 binding regions for NF-κB.20 Apart from the expected effect of the NF-κB inhibitor AKβBA,12 our data with SB203580 indicate that the p38 MAPK is also involved in the plasmin-induced expression of TNF-α. In human macrophages the p38 MAPK not only is involved in the translational control of TNF-α because of mRNA stabilization by regulation of the polyadenylation status and the translation rate but also contributes to transcriptional regulation through NF-κB.21–23 Besides single binding sites for C/EBPβ, the IL-6 promoter likewise contains functional canonical binding sites for NF-κB, which both can synergistically activate transcription.24 Similar to macrophages, during myoblast differentiation cross-talk occurs between the p38 MAPK and NF-κB in terms of IL-6 induction; here, the p38 MAPK participates in the regulation of IL-6 in dual ways, by increasing the IκBα degradation and by potentiating the transactivation of p65 through the coactivator CBP/p300 of the basal transcriptional machinery.25
The plasmin-induced activation of macrophages is expected to be of particular importance for cardiovascular disease, where the severity of atherosclerotic lesions positively correlates with the expression of the plasminogen activator urokinase plasminogen activator.4,5 The release of cytokines like TNF-α and IL-6 is a key feature in chronic inflammatory disease. Both cytokines may act as autocrine or paracrine molecules on the cells involved in the atherosclerotic process. TNF-α regulates the expression of receptors involved in lipid uptake such as the oxidized low-density lipoprotein receptor LOX1 in macrophages and the scavenger receptor ScR of smooth muscle cells,2,26 thereby promoting foam cell formation. Consistently, apolipoprotein E/TNF-α double-knockout mice show a significant decrease in relative lesion size.27 Likewise, IL-6 is associated with atherosclerosis; it is expressed in atherosclerotic plaques2 and enhanced plasma levels have been observed in patients with progressive disease.28 In mice, IL-6 participates in the macrophage lipid uptake by increasing the expression of CD36.29 Consistently, IL-6 was shown to also enhance fatty lesion development in mice.2 Further, endothelial dysfunction can be triggered by the cytokine induction, because endothelial nitric oxide synthase expression can be decreased either by TNF-α, which represses the transcription and destabilizes the mRNA,30 or by IL-6, which induces a STAT3-mediated transcriptional repression.31 Finally, by inducing the generation of growth factors, like vascular endothelial growth factor or chemokines such as monocyte chemoattractant protein-1, IL-6, generated by plasmin-activated macrophages, might promote vascular smooth muscle cell migration as well as monocyte recruitment into the atherosclerotic lesions.2
Our data demonstrate that the serine protease plasmin triggers proinflammatory cytokine induction in human macrophages via the annexin A2 heterotetramer and several downstream signaling pathways. These findings significantly expand the relevance of plasmin generated in inflamed tissues and atherosclerotic lesions beyond its roles in proteolysis and matrix metalloproteinase activation.1,5
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft SFB 451 to T.S. and Th. S.
Original received January 9, 2007; final version accepted March 16, 2007.
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