Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages

【摘要】  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- B 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- and IL-6. Inhibitors of JAK, p38, and NF- B revealed that these signaling pathways are indispensable for the plasmin-mediated tumor necrosis factor- 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.

Plasmin, but not catalytically blocked plasmin, induces cytokines such as TNF- and IL-6 in human monocyte-derived macrophages. The plasmin-induced signaling utilizes the annexin A2 heterotetramer as receptor that triggers downstream signaling via JAK/STAT, Akt-dependent NF- B activation, as well as ERK1/2 and the p38 MAPK, leading to proinflammatory gene induction.

【关键词】  IL macrophage plasmin signaling tumor necrosis factor

Introduction

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)- upregulate the expression of adhesion molecules such as selectins and vascular adhesion molecule-1, which are important for monocyte recruitment, but TNF- also regulates apoptosis as well as procoagulant and fibrinolytic activity of endothelial cells. Elevated levels of TNF- 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 cytometry 11; 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 x 10 6 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- B Family Transcription Factor Assays

Activations of STAT1, 3, 5A, 5B, and p65, c-Rel, RelB were determined with DNA-binding TransAM enzyme-linked immunosorbent assays for STAT and NF- B family transcription factors in nuclear extracts from stimulated macrophages. 9,12

Statistical Analysis

Means±SEM are shown. Probabilities calculated with the Newman-Keuls test were considered significant for P <0.05.

Results

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 1 A). We additionally confirmed the expression of the respective subunits on the macrophage surface by flow cytometry ( Figure 1 B). 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 1 C). This cleavage is dependent on the proteolytic activity of plasmin because exposure of macrophages to catalytically inactivated plasmin failed to produce the fragment ( Figure 1 C). These data suggest that macrophages share with monocytes the annexin A2 heterotetramer as signaling plasmin receptor.

Figure 1. Human macrophages express S100A10 and annexin A2. A, Western immunoblot of annexin A2 and S100A10 in whole cell lysates. Lysates from monocytes, macrophages, and neutrophils were separated and blotted onto polyvinylidenefluoride membrane. Actin-loading control. B, Surface expression of S100A10 and annexin A2. The cells were incubated with antibodies against CD14 (monocytes), CD68 (macrophages), CD47 (neutrophils), and S100A10, as well as annexin A2. For the analysis of CD68, the cells were fixed and permeabilized with 0.1% Triton X100. After incubation with the corresponding phycoerythrin-labeled F(ab) 2, the cells were analyzed by flow cytometry. Empty peaks are isotype controls. C, Proteolytic activity of plasmin is required for the cleavage of annexin A2. Macrophages were stimulated for 30 minutes either with 0.43 CTA U/mL plasmin or the equivalent amount of catalytically inactivated plasmin. After treatment, cells were lysed, and proteins were separated and visualized by immunoblotting with antibodies against annexin A2 and S100A10. All results shown are representative of at least 3 independent experiments.

Plasmin Triggers Signaling in Human Macrophages

Plasmin may activate multiple signaling pathways, in particular the JAK1/STAT, the p38 MAPK, 9 and the NF- B 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 2 A). 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 Tyr 705 was observed within 5 minutes ( Figure 2 A). Transcriptionally active STAT3 requires both Tyr and Ser phosphorylation. 9 Plasmin also triggered STAT3 phosphorylation on Ser 727 ( Figure 2 A).

Figure 2. Plasmin triggers activation of JAK/STAT3, MAPK, and NF- B signaling pathways. Macrophages (1 x 10 6 /assay) were stimulated with plasmin (0.43 CTA U/mL) in serum-free RPMI 1640 for the indicated time. Whole cell lysates were separated and analyzed by Western immunoblots. A, Time-dependent activation of JAK/STAT3. Western immunoblots of phospho-JAK1 (P-JAK1), phospho-TYK2 (P-TYK2), phospho-STAT3-(Tyr 705 ), and phospho-STAT3-(Ser 727 ). Expression levels of JAK1, TYK2, and actin-loading controls. B, Time-dependent activation of MAPK. Western immunoblots of phospho-p38 and phospho-ERK1/2. Immunostaining of p38 or ERK2-loading control. C, Time-dependent activation of the NF- B pathway. Immunoblots of phospho-Akt-(Ser 473 ) and phospho-I B from whole cell lysates from macrophages stimulated with plasmin. D, Effects of the JAK inhibitor, AG490, on the plasmin-induced MAPK and NF- B signaling pathways. Cells pretreated with 50 µmol/L AG490 for 30 minutes were stimulated with 0.43 CTA U/mL plasmin for 30 minutes. Immunoblots of phospho-ERK1/2, phospho-Akt-(Ser 473 ), phospho-I B, and phospho-p38. Staining of nonphosphorylated proteins and actin-loading controls. All results shown are representative of at least 3 independent experiments.

In addition to STAT3 activation, plasmin elicits phosphorylation of p38 MAPK after 5 minutes of stimulation with a marked increase after 15 minutes ( Figure 2 B). 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- B signaling, 8 we analyzed the phosphorylation state of the NF- B inhibitor I B by immunoblotting. Plasmin induced phosphorylation of I B, targeting the inhibitor to proteosomal degradation, consequently allowing nuclear translocation of NF- B. Because the Akt kinase is an upstream effector of NF- B signaling, 13 we checked its activation status by immunoblotting; plasmin induced a time-dependent phosphorylation of Ser 473 that is required for the full activation of Akt1 14 ( Figure 2 C). These data show that in macrophages plasmin triggers several signaling pathways, particularly JAK1/STAT3, p38, and ERK1/2 MAPK, and NF- B.

To address the role of JAK1 and TYK2 activation in the regulation of downstream targets such as the p38 MAPK, ERK1/2, Akt1, and I B, we used the JAK inhibitor AG490 ( Figure 2 D). At 50 µmol/L AG490 inhibits JAK1 15 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 I B, but not that of p38 MAPK ( Figure 2 D), 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 2 D, see supplemental Figure I (available at http://atvb.ahajournals.org).

Plasmin Induces Nuclear Translocation of STAT and NF- B Transcription Factors

To define the profile of the plasmin-induced nuclear translocation of STAT and NF- B 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 3 A). Similarly, plasmin-stimulated macrophages exhibit binding of p65, but not of RelB or c-Rel ( Figure 3 B). Thus, stimulation of macrophages with plasmin yields nuclear translocation of transcriptionally active STAT3 and p65 transcription factors.

Figure 3. Plasmin induces nuclear transclocation of STAT3 and NF- B transcription factors. A, STAT-1, 3, 5A, and 5B activation were assayed with the STAT family TransAM. Nuclear extracts were prepared from macrophages stimulated with plasmin for 15 minutes. B, p65, c-Rel, and RelB activation were assayed with the NF- B family TransAM. Nuclear extracts were from macrophages stimulated with plasmin for 1 hour. The results represent the mean±SEM fold activation of translocated transcription factors compared with control cells of 3 to 4 independent experiments. ** P <0.01 vs control.

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- and IL-6 mRNA ( Figure 4 A). Lipopolysaccharide (LPS) (1 µg/mL) stimulation induced the expression of both TNF- and IL-6 mRNA quicker with significant induction of TNF- and IL-6 mRNA already after 1 hour. The induction of TNF- and IL-6 mRNA in macrophages stimulated with plasmin for 3 hours was also concentration-dependent ( Figure 4 B). Although 0.043 CTA U/mL plasmin increased TNF- 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.

Figure 4. Plasmin triggers time- and concentration-dependent induction of TNF- and IL-6 genes. Time-dependent (A) and concentration-dependent (B) stimulation of the TNF- and IL-6 mRNA expression. Macrophages were stimulated with plasmin (0.43 CTA U/mL) or LPS (1 µg/mL) for the indicated times and the mRNA levels were analyzed by reverse-transcription polymerase chain reaction. C, The gene induction depends on the proteolytic activity of plasmin. Macrophages were stimulated for 3 hours with plasmin (0.43 CTA U/mL) or with equivalent amounts of catalytically inactivated plasmin; TNF- and IL-6 mRNA was analyzed by reverse-transcription polymerase chain reaction. In each case, one representative out of 3 experiments is shown. GAPDH is the loading control.

Because the proteolytic activity of plasmin seems to be indispensable for the activation of signaling pathways, 7,11 we examined its role in TNF- and IL-6 induction. Catalytically inactivated plasmin failed to stimulate the expression of TNF- and IL-6 mRNA ( Figure 4 C). 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 4 C, see supplemental Figure II.

Similar to LPS (1 µg/mL), plasmin induced a time- and concentration-dependent release of TNF- and IL-6 from human macrophages ( Figure 5A, 5 B). The cytokines became detectable as early as 3 hours after stimulation. The release of TNF- 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- and IL-6 in human monocyte-derived macrophages with a somewhat lower potency than the standard stimulus LPS.

Figure 5. Plasmin induces a time- and concentration-dependent release of TNF- and IL-6, which requires activation of JAK1, p38 MAPK, ERK1/2 MAPK, and NF- B. A, Release of TNF- from plasmin-stimulated macrophages. B, Release of the IL-6 from plasmin-stimulated macrophages. Macrophages were stimulated with plasmin or the positive control LPS (1 µg/mL) for the indicated time. The supernatants were recovered and the concentrations of TNF- and IL-6 were measured by enzyme-linked immunosorbent assay. Data are presented as the mean±SEM of 4 independent experiments. ** P <0.01 vs control. C, Effects of pharmacological inhibitors on the TNF- release. D, Effects of pharmacological inhibitors on the IL-6 release. Macrophages preincubated with the indicated inhibitors for 30 minutes were stimulated with plasmin (0.43 CTA U/mL); TNF- and IL-6 were analyzed by enzyme-linked immunosorbent assay after 3 hours and 6 hours of stimulation, respectively. AG490 50 µmol/L (JAK1 inhibitor), SB203580 1 µmol/L (p38 MAPK inhibitor), U0126 10 µmol/L (MEK inhibitor), and AKβBA 10 µmol/L (NF- B inhibitor). The data represent the mean±SEM compared with control cells treated with solvent of 3 (TNF- ) to 8 (IL-6) independent experiments. ** P <0.01 vs control.

Plasmin-Induced Activation of JAK1, p38, ERK1/2, and NF- B Is Indispensable for the Cytokine Expression

To gain evidence for the role of the initiated signaling pathways in the plasmin-induced release of TNF- 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- B (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- 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- 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- ( Figure 5 C). Similar to the TNF- 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 5 D; P <0.05). Not unexpected, the plasmin-induced activation of NF- B 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- 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 5 D).

These data imply pivotal roles of the JAK1/STAT3, MAPK, and NF- B signaling pathways in the plasmin-mediated induction of TNF- 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 6 A). For quantitative densitometric analysis of the gels, see supplemental Figure III.

Figure 6. The annexin A2 heterotetramer is indispensable for the plasmin-stimulated release of cytokines by the macrophages. A, Specific antisense ODN decrease the expression of annexin A2 and S100A10. Macrophages were treated for 48 hours with 10 µmol/L of the corresponding ODN (sense or antisense). After 12-hour recovery in RPMI 1640 without lysine, the cells were lysed, and the proteins were separated and analyzed by immunoblotting. B, Downregulation of the annexin A2 and S100A10 expression by antisense ODN inhibits the plasmin-induced release of TNF-. C, Downregulation of the annexin A2 and S100A10 expression by antisense ODN inhibits the plasmin-induced release of IL-6. Macrophages (controls or treated with sense or antisense ODN) were stimulated with plasmin (0.43 CTA U/mL) for 3 hours and 6 hours for TNF- and IL-6 release, respectively. Supernatants were recovered and TNF- and IL-6 were measured by enzyme-linked immunosorbent assay. All the data represent the mean percentage ±SEM of 4 independent experiments. ** P <0.01 vs controls.

Pretreatment of macrophages with annexin A2 antisense ODN profoundly inhibited TNF- and IL-6 release by 86±3% and 61±11%, respectively ( P <0.01; Figure 6B, 6 C). Similarly, pretreatment with S100A10 antisense ODN resulted in significant inhibition of TNF- and IL-6 release by 73±16% and 64±8%, respectively ( P <0.01; Figure 6B, 6 C). Treatment of macrophages with ODN without plasmin stimulation had no effect neither on the TNF- or the IL-6 release (data not shown). These data imply that plasmin stimulates human macrophages through the annexin A2 heterotetramer.

Discussion

Macrophages possess crucial effector functions in a variety of chronic inflammatory diseases including atherosclerosis 1 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- and IL-6 in human macrophages. Plasmin induces activation of multiple signaling pathways including JAK/STAT, p38 MAPK, ERK1/2, and the NF- B 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 Tyr 705 (<5 minutes) followed by a second activation phase starting 15 minutes after plasmin stimulation involving NF- B signaling initiated by phosphorylation of Akt and I B. 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 Ser 727, 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 I B degradation through phosphorylation of Akt. JAK proteins stimulate the phosphoinositide 3-kinase, 16 subsequently activating Akt and the aforementioned activation of NF- B signaling through the stimulation of the I B kinase complex. 13 Our data showing plasmin-induced Akt activation coinciding with I B phosphorylation and subsequent translocation of NF- B are in agreement with activation of the I B 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- –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- B transcription factor p65 triggering expression of TNF- and IL-6. At the transcriptional level, TNF- and IL-6 genes are regulated by recruitment of NF- B to their promoters. The promoter region of TNF- contains 2 binding regions for NF- B. 20 Apart from the expected effect of the NF- B inhibitor AKβBA, 12 our data with SB203580 indicate that the p38 MAPK is also involved in the plasmin-induced expression of TNF-. In human macrophages the p38 MAPK not only is involved in the translational control of TNF- because of mRNA stabilization by regulation of the polyadenylation status and the translation rate but also contributes to transcriptional regulation through NF- B. 21–23 Besides single binding sites for C/EBPβ, the IL-6 promoter likewise contains functional canonical binding sites for NF- B, which both can synergistically activate transcription. 24 Similar to macrophages, during myoblast differentiation cross-talk occurs between the p38 MAPK and NF- B in terms of IL-6 induction; here, the p38 MAPK participates in the regulation of IL-6 in dual ways, by increasing the I B 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- 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- 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- double-knockout mice show a significant decrease in relative lesion size. 27 Likewise, IL-6 is associated with atherosclerosis; it is expressed in atherosclerotic plaques 2 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-, 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

Acknowledgments

Sources of Funding

This work was supported by the Deutsche Forschungsgemeinschaft SFB 451 to T.S. and Th. S.

Disclosures

None.

【参考文献】
  Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.

Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiol Rev. 2006; 86: 515–581.

Kaplan AP, Silverberg M. The coagulation-kinin pathway of human plasma. Blood. 1987; 70: 1–15.

Syrovets T, Simmet T. Novel aspects and new roles for the serine protease plasmin. Cell Mol Life Sci. 2004; 61: 873–885.

Nicholl SM, Roztocil E, Davies MG. Plasminogen activator system and vascular disease. Curr Vasc Pharmacol. 2006; 4: 101–116.

Weide I, Tippler B, Syrovets T, Simmet T. Plasmin is a specific stimulus of the 5-lipoxygenase pathway of human peripheral monocytes. Thromb Haemost. 1996; 76: 561–568.

Syrovets T, Tippler B, Rieks M, Simmet T. Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway. Blood. 1997; 89: 4574–4583.

Syrovets T, Jendrach M, Rohwedder A, Schüle A, Simmet T. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKβ-mediated NF- B activation. Blood. 2001; 97: 3941–3950.

Burysek L, Syrovets T, Simmet T. The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways. J Biol Chem. 2002; 277: 33509–33517.

Colognato R, Slupsky JR, Jendrach M, Burysek L, Syrovets T, Simmet T. Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells. Blood. 2003; 102: 2645–2652.

Laumonnier Y, Syrovets T, Burysek L, Simmet T. Identification of the annexin A2 heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes. Blood. 2006; 107: 3342–3349.

Syrovets T, Büchele B, Krauss C, Laumonnier Y, Simmet T. Acetyl-boswellic acids inhibit lipopolysaccharide-mediated TNF- induction in monocytes by direct interaction with I B kinases. J Immunol. 2005; 174: 498–506.

Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. NF- B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature. 1999; 401: 82–85.

Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. Embo J. 1996; 15: 6541–6551.

Zhang Y, Turkson J, Carter-Su C, Smithgall T, Levitzki A, Kraker A, Krolewski JJ, Medveczky P, Jove R. Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity. J Biol Chem. 2000; 275: 24935–24944.

Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 2005; 5: 375–386.

Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, Messing RO, Bolli R. Role of the protein kinase C- -Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation. 2005; 112: 1971–1978.

Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J. 2003; 374: 1–20.

Uddin S, Lekmine F, Sharma N, Majchrzak B, Mayer I, Young PR, Bokoch GM, Fish EN, Platanias LC. The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon -dependent transcriptional activation but not serine phosphorylation of Stat proteins. J Biol Chem. 2000; 275: 27634–27640.

Kuprash DV, Udalova IA, Turetskaya RL, Kwiatkowski D, Rice NR, Nedospasov SA. Similarities and differences between human and murine TNF promoters in their response to lipopolysaccharide. J Immunol. 1999; 162: 4045–4052.

Campbell J, Ciesielski CJ, Hunt AE, Horwood NJ, Beech JT, Hayes LA, Denys A, Feldmann M, Brennan FM, Foxwell BM. A novel mechanism for TNF- regulation by p38 MAPK: involvement of NF- B with implications for therapy in rheumatoid arthritis. J Immunol. 2004; 173: 6928–6937.

Dean JL, Sarsfield SJ, Tsounakou E, Saklatvala J. p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation. J Biol Chem. 2003; 278: 39470–39476.

Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity. 1999; 10: 387–398.

Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6 and NF- B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A. 1993; 90: 10193–10197.

Baeza-Raja B, Munoz-Canoves P. p38 MAPK-induced nuclear factor- B activity is required for skeletal muscle differentiation: role of interleukin-6. Mol Biol Cell. 2004; 15: 2013–2026.

Li H, Freeman MW, Libby P. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines. J Clin Invest. 1995; 95: 122–133.

Brånén L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor- reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2004; 24: 2137–2142.

Tzoulaki I, Murray GD, Lee AJ, Rumley A, Lowe GD, Fowkes FG. C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study. Circulation. 2005; 112: 976–983.

Keidar S, Heinrich R, Kaplan M, Hayek T, Aviram M. Angiotensin II administration to atherosclerotic mice increases macrophage uptake of oxidized LDL: a possible role for interleukin-6. Arterioscler Thromb Vasc Biol. 2001; 21: 1464–1469.

Alonso J, Sanchez de Miguel L, Monton M, Casado S, Lopez-Farre A. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor. Mol Cell Biol. 1997; 17: 5719–5726.

Saura M, Zaragoza C, Bao C, Herranz B, Rodriguez-Puyol M, Lowenstein CJ. Stat3 mediates interleukin-6 inhibition of human endothelial nitric-oxide synthase expression. J Biol Chem. 2006; 281: 30057–30062.

作者单位：Institute of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Germany.