Plasmin Triggers Chemotaxis of Monocyte-Derived Dendritic Cells Through an Akt2-Dependent Pathway and Promotes a T-Helper Type-1 Response
Objective— Dendritic cells (DC) accumulate in atherosclerotic arteries where they can modulate atherogenesis. We investigated whether plasmin might alter the function of human DC.
Methods and Results— Stimulation of monocyte-derived DC with plasmin elicited a time-dependent actin polymerization and chemotaxis comparable to that triggered by the standard chemoattractant formyl-methionyl-leucyl-phenylalanine. Plasmin triggered rapid activation of Akt and mitogen-activated protein kinases, followed by phosphorylation of the regulatory myosin light chain and chemotaxis. For the chemotactic DC migration, the activation of Akt and p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases were indispensable, as shown by pharmacological inhibitors. DC express Akt1 and Akt2, but not Akt3. However, in DC, plasmin activates exclusively Akt2 via a p38 mitogen-activated protein kinase-dependent pathway. Accordingly, knockdown of Akt2 with short-hairpin RNA, but not of Akt1, blocked the plasmin-induced extracellular signal-regulated kinase 1/2 activation and the chemotactic response. Moreover, plasmin-stimulated DC induced polarization of CD4+ T cells toward the interferon-γ–producing, proinflammatory Th1 phenotype. Consistent with a role for DC and adaptive immune response in atherogenesis, we demonstrate DC in human atherosclerotic vessels and show that plasmin is abundant in human atherosclerotic lesions, where it colocalizes with DC.
Conclusion— Plasmin generation in the atherosclerotic vessel wall might contribute to accumulation of DC, activation of the adaptive immune response, and aggravation of atherosclerosis.
Atherosclerosis is a chronic inflammatory disease accompanied by activation of the innate and adaptive immune systems.1 Dendritic cells (DC) are the most potent antigen-presenting cells that are able to define immune responses by directing the T-cell differentiation into different T-cell subsets. Small numbers of DC are localized in the intima of apparently normal, nondiseased arteries.2 In advanced human atherosclerotic plaques, the number of DC and their precursors are increased compared with early lesions and they are localized in the exposed plaque shoulder and rupture-prone areas colocalized with T cells, signifying that this interaction could contribute to plaque destabilization.2 There is solid evidence from mouse models of atherosclerosis demonstrating involvement of T-cell subsets, mainly of interferon (IFN)-γ–producing Th1, in the development of atherosclerosis.1,3 Thus, transfer of CD4+ T lymphocytes aggravates atherosclerosis in apolipoprotein E−/− mice, whereas apolipoprotein E−/− or low-density lipoprotein receptor−/− mice deficient in IFN-γ exhibit substantially fewer atherosclerotic lesions.3
The serine protease plasmin is generated from its zymogen plasminogen by either tissue plasminogen activator or urokinase plasminogen activator, which activate plasminogen during fibrinolysis and inflammation, respectively.4 The expression of fibrinolytic genes is increased in the atherosclerotic aorta and the upregulated fibrinolytic activity correlates with the severity of coronary lesions.4 Even though the presence of proteases in atherosclerosis is mainly regarded in terms of plaque destabilization,5 plasmin may also induce intracellular signaling and modulate immune and inflammatory responses.4,6–8 We have previously demonstrated that binding of plasmin to monocytes9 induces expression of proinflammatory cytokines via activation of NF-κB and Janus kinase/signal transducer and activator of transcription pathways.10,11
Any direct effects of plasmin on DC are currently unknown. Yet, it has been shown that DC derived from CD34+CD14+ precursor cells utilize the urokinase plasminogen activator/urokinase plasminogen activator receptor system for transendothelial migration and the invasion of the subendothelial extracellular matrix.12 Although the role of plasmin in this process was not addressed, it was demonstrated that plasmin generation takes place at the surface of DC. Plasmin, when bound to cell membranes, remains protected from its fluid phase inhibitors, such as α2-antiplasmin, and remains proteolytically active.4,13 Therefore, we examined the effects of plasmin on human DC.
Materials and Methods
A complete Materials and Methods section is available in the online Data Supplement at http://atvb.ahajournals.org.
Cell Isolation and Differentiation
Human monocytes were isolated from buffy coats and differentiated into DC with granulocyte–macrophage colony-stimulating factor and IL-4 for 6 days.14
Protein expression was analyzed by flow cytometry or Western immunoblotting;14 for phosphorylated PDK1, Akt, extracellular signal-regulated kinase (ERK) 1/2, p38, and myosin light chain (MLC) 2, DC were kept in fetal calf serum-free medium for 12 hours before plasmin stimulation. For inhibitor experiments, the cells were pretreated for 15 minutes with either SB203580,15 U0126, or Akt inhibitor VIII.16
DC were stimulated with the chemoattractants plasmin or the positive control formyl-methionyl-leucyl-phenylalanine (FMLP). Permeabilized cells were stained with Alexa 488-labeled phalloidin and analyzed by flow cytometry or fluorescence microscopy.
Migration of DC suspended in RPMI 1640 containing 0.1% bovine serum albumin was evaluated in transwell plates after 4 hours.7 For inhibitor experiments, DC were preincubated for 15 minutes with the compounds.
Downregulation of Annexin A2 and S100A10 by Antisense Oligodeoxynucleotides
For in vitro knockdown of annexin A2 and S100A10, phosphorothioate-modified oligodeoxynucleotides were applied to the DC cultures.9
Analysis of mRNA Expression
Downregulation of Akt Isoforms by RNA Interference
For in vitro knockdown of Akt1 or Akt2, plasmids encoding short-hairpin RNA were transfected by nucleofection; cells were allowed to recover for 48 hours.
Immunoprecipitation of Phosphotyrosine-Containing Proteins
Phosphotyrosine-containing proteins were immunoprecipitated from DC lysates with antiphosphotyrosine antibody. Akt1, Akt2, and P-Akt (S473) were analyzed by Western immunoblotting.
In Vitro Polarization of T Cells
CD3+ T cells isolated from buffy coats were cocultured with washed DC (responder/DC ratio, 10:1). The release of IFN-γ by cocultured cells was determined with the Proteome Profiler array. Alternatively, Brefeldin A-pretreated DC were analyzed for IFN-γ or IL-4 by multicolor flow cytometry.
DC were visualized with anti-S10019 in human atherosclerotic vessel specimens from 9 surgical patients. Sections were double-stained with antiplasmin antibodies, which recognize plasmin and partially plasminogen. The microscopic images were digitally recorded. For fluorescent immunostaining, FITC-coupled and Cy5-coupled F(ab′)2 were used and visualized with an Axioskop 2 plus fluorescence microscope.14
Statistical significance was calculated with the Newman-Keuls test. Differences were considered significant for P<0.05.
Plasmin Elicits Chemotaxis in DC
Plasmin triggered a significant and concentration-dependent migration of DC across polycarbonate membranes (Figure 1A), with a peak response at 0.143 Committee on Thrombolytic Agents (CTA) U/mL. At this maximum effective plasmin concentration, the DC migration was not significantly different from that observed with the standard chemoattractant FMLP at the chemotactic optimal concentration of 10 nmol/L. In contrast, an equivalent amount of catalytically inactivated plasmin (VPLCK-plasmin) failed to induce any significant chemotactic response, indicating a necessity for proteolytic activation of the DC. At higher plasmin concentrations, the DC migration decreased exhibiting the bell-shaped concentration–response curve (Figure 1A), a feature common for chemoattractants. Both FMLP and MCP-3 in DC and plasmin in monocytes induce bell-shaped chemotactic responses.7,20 Autodigestion of plasmin and generation of fragments, acting as plasmin antagonists, might account for the decrease in the chemotactic activity of plasmin at higher concentrations.
Checkerboard analysis revealed that the plasmin-induced immature DC migration required a positive concentration gradient between the lower and the upper compartments of the chemotaxis chamber. The plasmin-induced DC migration therefore mainly was attributable to chemotactic activity, with only minor components attributable to chemokinetic effects (Supplemental Table I, available online at http://atvb.ahajournals.org). Thus, depending on its proteolytic activity, plasmin induces a true chemotaxis in DC.
To demonstrate that the plasmin-induced chemotaxis is not attributable to activation of n-formyl peptide receptors, we used the n-formyl peptide receptor antagonist Boc2.21 Cells preincubated with 10 μmol/L Boc2 failed to migrate in response to FMLP stimulation but did migrate to plasmin (Figure 1B). Plasmin-induced cell migration was, however, totally inhibited by preincubation with pertussis toxin (P<0.01 vs plasmin control; Figure 1A), indicating involvement of Gαi protein-mediated responses.
The ability of immature DC to migrate toward plasmin was greatly diminished on maturation with lipopolysaccharide (100 ng/mL; CI, 1.68±0.2 for mature DC compared to 4.1±0.8 for immature DC). Plasmin treatment for 48 hours did not increase expression of maturation markers on DC (Supplemental Figure I, available online at http://atvb.ahajournals.org) and had no effect on the cell viability (Supplemental Figure II).
Cellular movement requires distinct patterns of actin reorganization of the cells to establish a leading edge of the polarized cells and to generate contractile force to migrate forward. Incubation of DC without plasmin did not lead to any significant changes in F-actin contents during a 5-minute incubation period (Figure 1C). However, stimulation with plasmin led to a time-dependent increase in F-actin formation. As expected, FMLP also induced time-dependent changes in F-actin formation. In contrast, catalytically inactivated VPLCK-plasmin used in an equivalent concentration was unable to elicit any significant F-actin formation (Figure 1C). The plasmin-induced actin polymerization became clearly visible in permeabilized DC stained with fluorescent-labeled phalloidin (Figure 1D). A similar increase in actin polymerization was seen with FMLP (data not shown). Consistent with the previous data, catalytically blocked VPLCK-plasmin remained inactive (Figure 1D).
Plasmin Requires the Annexin A2 Heterotetramer for Chemotaxis Induction
Because we have previously identified the annexin A2 heterotetramer as a receptor involved in the plasmin-mediated signaling in monocytes and macrophages,6,9 we investigated its expression in human DC. DC exhibit expression of subunits of the annexin A2 heterotetramer, annexin A2, and S100A10, with a shift in favor of annexin A2 when compared to monocytes (Figure 2A). The respective subunits were also expressed on the DC surface (Figure 2A). Stimulation of DC with plasmin led to cleavage of ≈20% of annexin A2, yielding a proteolytic fragment of ≈33 kDa, whereas S100A10 remained unaffected. Exposure of DC to catalytically inactivated plasmin failed to produce this fragment (Figure 2B).
To clarify the putative role of the annexin A2 heterotetramer in the plasmin-induced DC chemotaxis, we downregulated the expression of annexin A2 and S100A10 with antisense oligodeoxynucleotides (Figure 2C). Consistent with the notion that annexin A2 stabilizes S100A10,22 downregulation of annexin A2 concomitantly decreased the expression of S100A10. Pretreatment of the DC with annexin A2 and S100A10 antisense oligodeoxynucleotides profoundly inhibited the chemotactic response (Figure 2D). Treatment of DC with oligodeoxynucleotides without plasmin stimulation had no effect on the DC migration (data not shown). These data suggest that DC share with monocytes and macrophages6,9 the annexin A2 heterotetramer as signaling plasmin receptor.
Plasmin Elicits Chemotactic Signaling Via Mitogen-Activated Protein Kinases and Akt Activation
Mitogen-activated protein kinases (MAPK) ERK1/2, c-Jun N-terminal kinase, and p38 have important roles in cell proliferation, differentiation, and stress responses. Recent studies have shown that these kinases might also play a role in cell migration by phosphorylating MLC kinase, calpain, or the focal adhesion kinase FAK.23 Plasmin triggered a rapid, time-dependent, and concentration-dependent phosphorylation of p38 and ERK1/2, but not of c-Jun N-terminal kinase (Figure 3A). The PI3K/Akt pathway is also required for efficient cell polarization and chemotaxis in response to several chemoattractants, including FMLP.24,25 Akt colocalizes with F-actin and the activation state of Akt correlates with the chemotactic activity of the cell.26 Stimulation of DC with plasmin led to a rapid, prolonged phosphorylation of Akt and the regulatory MLC2 (Figure 3A). MLC2 regulates the interaction of myosin with actin, essential for cell movement.23 MLC2 phosphorylation, in turn, is regulated by the Ca2+/calmodulin-regulated MLC kinase. Activated ERK is able to phosphorylate and activate the MLC kinase directly,23 explaining the observed phosphorylation of MLC2 on activation of ERK (Figure 3A).
To address the role of Akt and MAPK in the plasmin-induced migration, we used pharmacological inhibitors of Akt, p38, and the MEK/ERK pathway. The concentrations of the inhibitors were validated in preliminary experiments with negative controls for the inhibitors. Inhibition of p38 with SB203580 at a concentration that specifically inhibits p38 resulted in reduced activation of Akt and ERK1/2, indicating that p38 is upstream of Akt and ERK1/2 (Figure 3B). Consistently, the phosphorylation of Akt on S473 was largely p38-dependent. Inhibition of Akt by a pleckstrin domain-dependent, dual Akt1/Akt2 inhibitor (VIII) inhibited the Akt activation and concomitantly reduced the activation of ERK1/2, whereas the upstream kinases p38 and PDK1 remained unaffected. Therefore, within the plasmin-induced signaling cascade, ERK1/2 acts downstream of Akt. Interestingly, inhibition of the ERK1/2 activity by the MEK inhibitor resulted in an increased phosphorylation of Akt, pointing to a negative feedback between the ERK pathway and Akt (Figure 3B). Consistent with crucial functions of Akt and MAPK in the plasmin-induced signaling, the Akt, p38, and MEK kinase inhibitors impaired the plasmin-induced phosphorylation of MLC2 (Figure 3B) and the chemotaxis (Figure 3C).
A Distinct Role for Akt2 in the Plasmin-Induced Chemotactic Signaling
Of the 3 Akt isoforms known, DC express Akt1 and less Akt2, but no Akt3 (Figure 4A). The tyrosine phosphorylation of Akt is important for its activation by PDK1.27,28 Immunoprecipitation of Tyr-phosphorylated proteins demonstrated that in DC, plasmin exclusively activates Akt2 (Figure 4B).
Silencing of Akt1 with a plasmid encoding siRNA downregulated the Akt1 protein expression and resulted in an increased ERK1/2 phosphorylation in response to plasmin stimulation. When Akt2 was selectively downregulated, the basal ERK1/2 phosphorylation was increased, whereas the plasmin-induced ERK1/2 phosphorylation was abolished (Figure 4C). In contrast to the nondirectional migration of DC that remained unaffected by either Akt1 or Akt2 silencing, the plasmin-induced chemotaxis of DC was totally inhibited in cells with silenced Akt2, but not in those with silenced Akt1 (Figure 4D).
These data indicate that the plasmin-induced activation of ERK1/2 in DC and the chemotactic response are Akt2-dependent, whereas Akt1 is a negative regulator of the plasmin-induced ERK activation.
Plasmin Induces Th1 Polarization of CD4+ Cells
Finally, we compared the ability of plasmin-stimulated DC to trigger production of IFN-γ by CD4+ T cells. More IFN-γ–producing cells were detected when T cells were cocultured with plasmin-stimulated DC as compared with T cells cocultured with unstimulated DC (Figure 5A). Analysis of the intracellular IFN-γ by flow cytometry revealed that the amount of IFN-γ+ CD4+ T-cells in coculture with plasmin-activated DC was increased 3-fold. Different to chemotaxis, this increase was independent of Akt (Figure 5B, C). This process was accompanied by expression of IL-12 by DC (Figure 5D). There was no increase in IL-4+ CD4+ Th2 cells (Figure 5C). Accordingly, there were no Th2 cytokines, such as IL-4, IL-5, and IL-10, as well as IL-2, secreted by the T cells cocultured with the plasmin-stimulated DC (Supplemental Figure III), indicating augmentation of T-cell differentiation to the Th1 subset by plasmin-activated DC.
Plasmin and DC Colocalize in Atherosclerotic Lesions
The precise function of DC in the vessel wall and their role in atherogenesis are currently not well-defined. Immunohistochemical analysis of atherosclerotic vessel sections confirmed that intima areas adjacent to the atherosclerotic lesions of abdominal aorta specimens were highly enriched in DC (Figure 6A). In all layers of the human atherosclerotic aorta sections, we also observed high amounts of plasmin (Figure 6B). Double staining of the atherosclerotic tissues revealed that clusters of DC colocalize with plasmin (Figure 6C, D), suggesting plasmin-mediated stimulation of DC in atherosclerotic lesions.
For proper immune function, the migration and accurate positioning of DC are of utmost importance. Therefore, a large number of external signals, many of them chemokines but also nonchemokine chemoattractants, navigate DC in concert with adhesion molecules through the body. Many chemotactic and maturation factors are rapidly produced within inflamed or damaged tissue, thereby playing a pivotal role in stimulating recruitment of DC or targeting them for localization to the lymph nodes or other tissues.29,30 Our data demonstrate that plasmin, which is generated in the context of contact activation taking place in inflammatory and allergic reactions13 and also in atherosclerotic vessel wall lesions,4,31 also classifies as a potent DC chemoattractant.
Consistent with our previous studies in monocytes and macrophages,6,9 we also found that in DC, the annexin A2 heterotetramer serving as a receptor for plasmin is a crucial component of the signaling cascade. Conceptually similar yet biochemically different from protease-activated receptors, the plasmin-induced signaling is critically dependent on the proteolytic activity of the serine protease. Annexin A2 is targeted by plasmin through lysine in position 27,9 which yields a 33-kDa fragment of annexin A2. Because the first 14 amino acids of the N-terminal domain of annexin A2 are required as binding domain for S100A10,32 cleavage in position 27 inevitably leads to dissociation of the annexin A2 heterotetramer, a process apparently required for further downstream signaling.6,9
Plasmin generated on the cell surface is bound to annexin A2 heterotetramer, which has been shown to facilitate the plasmin autoproteolysis.33 However, the kinetic of this process is slower than that of plasmin-induced cell activation. Thus, plasmin-induced actin polymerization was observed within seconds, whereas no significant reduction of plasmin activity occurred within the first minutes of incubation.33,34 Later, plasmin fragments generated via autoproteolysis and containing the lysine-binding sites but lacking the protease domain might prevent binding of plasminogen to the cell surface and therefore might downregulate the plasmin-induced cell activation.
Regarding the mechanistic aspect of DC chemotaxis, several key intracellular pathways and signaling molecules usually downstream of chemokine receptors have begun to be recognized. Generally, chemotactic signaling in DC is thought to be regulated primarily through Gαi proteins downstream of receptors for chemoattractants, such as chemokines. Also, the plasmin-induced chemotaxis of DC obviously proceeds via a pertussis toxin-sensitive Gαi protein. On receptor engagement, the βγ subunit is released and subsequently activates downstream effectors such as PI3K, which regulates the Akt pathway that plays a pivotal role in regulating chemotaxis in leukocytes, including DC.29,30 How Akt may regulate the cell motility is not well understood. Akt may directly phosphorylate actin24 or the actin-binding protein girdin, thus facilitating its binding to F-actin and the formation of stress fibers in migrating cell.35 Accordingly, cells with constitutively active Akt exhibit enhanced migration.26
Consistent with the signaling cascade identified for chemokine receptors, the plasmin-mediated signaling in DC proceeds via Akt. The signaling mechanisms utilizing Akt in DC have not been thoroughly investigated yet. Of the 3 Akt isoforms known, only Akt1 and Akt2 are expressed by human DC. However, unexpectedly, it is solely the Akt2 isoform that is selectively activated by the plasmin stimulation. We show that the plasmin-induced activation of ERK1/2 in DC and the chemotactic response are Akt2-dependent, whereas Akt1 is a negative regulator of the plasmin-induced ERK activation, which might be attributable to phosphorylation of c-Raf at S259 promoting its association with 14-3-3 and Raf/ERK inactivation.36
In contrast to plasmin, lipopolysaccharide or CD40 stimulation of murine bone marrow-derived DC activates almost exclusively Akt1, which plays a major role for DC survival and maturation.37 However, the selective Akt2 stimulation associated with the plasmin-induced chemotactic response is in line with a recent study38 showing that Akt2 is essential for the colony-stimulating factor-1–induced and the MCP-1–induced chemotaxis in mouse peritoneal macrophages.
Our data demonstrate that the plasmin-induced ERK1/2 activation is mediated by Akt. There are conflicting data on the Akt-mediated activation of the Ras–ERK pathway. Akt negatively regulates ERK1/2 activity in transfected HEK 293.39 However, spontaneous Ras/ERK activation has been observed in PTEN-null cells and the Ras activation level was decreased by treatment with the PI3K inhibitor LY294002. A positive PI3K/F-actin feedback might be responsible for the increased activation of the Ras/ERK axis.26
It is difficult to dissect the importance of each MAPK toward a specific biological end point because MAPK play important roles in many DC functions, including maturation, migration, cell proliferation, cytokine production, and survival. Phosphorylation of p38 MAPK seems to be required for DC maturation and the synthesis of cytokines; however, consistent with our findings in plasmin-stimulated DC, DC are at least partially competent to chemotax when p38 is inhibited.30 It is possible that when p38 MAPK is blocked, another MAPK may take over. An integrated signaling module was recently proposed to regulate chemotaxis in CCR7-stimulated DC. This pathway is induced by Gαi-mediated activation of either p38 or ERK1/2, and this activation was thought to be upstream of c-June N-terminal kinase. Interestingly, inhibition of all these kinases does not completely blunt CCR7-dependent chemotaxis, suggesting that additional unidentified molecules or signaling pathways may also regulate chemotaxis of DC.30,40
The precise function of DC in the vessel wall and their involvement in the atherosclerotic process still remains unclear. The abundance of DC in the intima directly correlates with the susceptibility to atherosclerosis.41,42
In all layers of human atherosclerotic aorta sections, we observed large amounts of plasmin and DC. Intima areas adjacent to the atherosclerotic lesions of abdominal aorta specimens were highly enriched in DC clusters colocalized with plasmin. Such DC clusters are thought to be a source of the proinflammatory cytokines,41 which initiate and amplify the inflammatory response crucial for the atherogenesis. Consistent with our immunohistochemical findings, others found plasmin and plasminogen in the core and shoulder of culprit plaques, ie, atherosclerotic lesions associated with clinical complications. Using microdissection and inhibitor-controlled chromogenic assays for the determination of plasmin activity, these authors also demonstrated that this is the site where plasminogen and enzymatically active plasmin is predominantly located in the atherosclerotic lesion.31,43 Therefore, it is reasonable to expect occurrence of plasmin-mediated activation of DC in atherosclerotic lesions, where both plasminogen and plasmin colocalize with DC.
Vascular DC exhibit a predominantly inflammatory phenotype.1,44 This view is further supported by the observation that DC stimulate autologous CD4+ T cells to produce IFN-γ and to infiltrate the arterial wall, thereby breaking T-cell tolerance to self-antigens and initiating vascular inflammation.1 Plasmin would further contribute to the inflammatory response by inducing DC capable of the direction of T-cell polarization toward the IFN-γ–producing Th1 subset. IFN-γ primarily produced by activated T cells would further activate macrophages and endothelial cells1 to enhance the inflammatory response and aggravate atherosclerosis.
To summarize, our data identify plasmin as a novel and potent activator of DC leading to Akt2-mediated migration and Th1 polarization of T cells. These findings shed new light on DC activation under conditions of enhanced local plasmin generation as found in atherosclerotic lesions.
Source of Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (to T. Syrovets and T. Simmet).
X. Li and T. Syrovets contributed equally to this work.
Received May 24, 2009; revision accepted December 22, 2009.
Garcia-Touchard A, Henry TD, Sangiorgi G, Spagnoli LG, Mauriello A, Conover C, Schwartz RS. Extracellular proteases in atherosclerosis and restenosis. Arterioscler Thromb Vasc Biol. 2005; 25: 1119–1127.
Li Q, Laumonnier Y, Syrovets T, Simmet T. Plasmin triggers cytokine induction in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol. 2007; 27: 1383–1389.
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.
Weide I, Römisch J, Simmet T. Contact activation triggers stimulation of the monocyte 5-lipoxygenase pathway via plasmin. Blood. 1994; 83: 1941–1951.
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, 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-κB 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.
Ferrero E, Vettoretto K, Bondanza A, Villa A, Resnati M, Poggi A, Zocchi MR. uPA/uPAR system is active in immature dendritic cells derived from CD14+CD34+ precursors and is down-regulated upon maturation. J Immunol. 2000; 164: 712–718.
Li X, Syrovets T, Paskas S, Laumonnier Y, Simmet T. Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction. J Immunol. 2008; 181: 1215–1223.
Lali FV, Hunt AE, Turner SJ, Foxwell BM. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 2000; 275: 7395–7402.
Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel RJ, Roth RA. Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem. 1999; 274: 21528–21532.
Visser J, van Boxel-Dezaire A, Methorst D, Brunt T, de Kloet ER, Nagelkerken L. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood. 1998; 91: 4255–4264.
Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood. 1997; 90: 3245–3287.
Sozzani S, Sallusto F, Luini W, Zhou D, Piemonti L, Allavena P, Van Damme J, Valitutti S, Lanzavecchia A, Mantovani A. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol. 1995; 155: 3292–3295.
MacLeod TJ, Kwon M, Filipenko NR, Waisman DM. Phospholipid-associated annexin A2–S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin. J Biol Chem. 2003; 278: 25577–25584.
Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci. 2004; 117: 4619–4628.
Kölsch V, Charest PG, Firtel RA. The regulation of cell motility and chemotaxis by phospholipid signaling. J Cell Sci. 2008; 121: 551–559.
Chen R, Kim O, Yang J, Sato K, Eisenmann KM, McCarthy J, Chen H, Qiu Y. Regulation of Akt/PKB activation by tyrosine phosphorylation. J Biol Chem. 2001; 276: 31858–31862.
Conus NM, Hannan KM, Cristiano BE, Hemmings BA, Pearson RB. Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase. J Biol Chem. 2002; 277: 38021–38028.
Falcone DJ, Borth W, McCaffrey TA, Mathew J, McAdam K. Regulation of macrophage receptor-bound plasmin by autoproteolysis. J Biol Chem. 1994; 269: 32660–32666.
Zhang B, Ma Y, Guo H, Sun B, Niu R, Ying G, Zhang N. Akt2 is required for macrophage chemotaxis. Eur J Immunol. 2009; 39: 1–8.
Galetic I, Maira SM, Andjelkovic M, Hemmings BA. Negative regulation of ERK and Elk by protein kinase B modulates c-Fos transcription. J Biol Chem. 2003; 278: 4416–4423.
Riol-Blanco L, Sanchez-Sanchez N, Torres A, Tejedor A, Narumiya S, Corbi AL, Sanchez-Mateos P, Rodriguez-Fernandez JL. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immunol. 2005; 174: 4070–4080.
Jongstra-Bilen J, Haidari M, Zhu SN, Chen M, Guha D, Cybulsky MI. Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis. J Exp Med. 2006; 203: 2073–2083.
Martin-Ventura JL, Nicolas V, Houard X, Blanco-Colio LM, Leclercq A, Egido J, Vranckx R, Michel JB, Meilhac O. Biological significance of decreased HSP27 in human atherosclerosis. Arterioscler Thromb Vasc Biol. 2006; 26: 1337–1343.