Annexin II mediates plasminogen-dependent matrix invasion by human monocytes: enhanced expression by macrophages

Monocytes and macrophages participate in a wide variety of host defense mechanisms. Annexin II, a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator (t-PA) independently at the cell surface, thereby enhancing the catalytic efficiency of plasmin production. We demonstrated previously that annexin II on the surface of both cultured monocytoid cells and monocyte-derived macrophages promotes their ability to remodel extracellular matrix. Here, we demonstrate that human peripheral blood monocytes represent the major circulating annexin II-expressing cell. Annexin II supported t-PA-dependent generation of cell surface plasmin and the matrix-penetrating activity of human monocytes. Compared to polymorphonuclear leukocytes, monocytes supported a 12.9-fold greater rate of plasmin generation in the presence of exogenous t-PA, and this activity was largely attributable to annexin II. Likewise, anti-annexin II IgG directed against the t-PA-binding tail domain inhibited plasminogen-dependent, cytokine-directed monocyte migration through extracellular matrix. On differentiation of monocytes to macrophages, there was a 2.4-fold increase in annexin II-specific mRNA, and a 7.9-fold increase in surface annexin II. Thioglycolate-elicited peritoneal macrophages, furthermore, displayed an additional 3.8-fold increase in annexin II surface expression compared with resident cells. Thus, annexin II-mediated assembly of plasminogen and t-PA on monocyte/macrophages contributes to plasmin generation, matrix remodeling, and directed migration.     

Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells

Angiostatin(AS), an internal fragment of plasminogen, is one of the most potent specific inhibitors of angiogenesis. Angiostatin treatment has resulted in the complete regression of human tumors implanted subcutaneously into nude mice and has great therapeutic value (O'Reilly et al., Nat. Med. 2, 689-692, 1996). Despite promising therapeutic value in the treatment of cancer, the mechanism of its action is still unknown. We found that angiostatin binds to a 35-kDa protein in bovine aortic endothelial (BAE) cells (Sharma et al., Proc. Am. Assoc. Cancer Res. 42, 568, A3050, 2002). In an attempt to begin to understand angiostatin's mechanism of action, we have purified and characterized this 35-kDa protein from BAE cells. Internal peptide sequence analysis of purified protein demonstrated (SLYYIQQDTK, SYSPYDMLESIK, and ALLYLXGGDD) 100% sequence identity with tyrosine kinase substrate annexin II. Solid phase binding analysis suggests that angiostatin specifically bound to purified annexin II immobilized on 96-well plastic plates. Hundred-fold molar excess of unlabeled AS and anti-annexin II antibody inhibited bindings 85 and 55%, respectively, suggesting specific interaction. Annexin II is a predominant receptor for angiostatin, since neutralizing the angiostatin by soluble receptor (annexin II) effectively blocks angiostatin's anti-EC activity. Similarly, saturating the annexin II receptor by plasminogen in endothelial cells also blocks angiostatin's activity. Both angiostatin and plasminogen bind to purified annexin II in BAE cells saturably with apparent K(d) values of 101 and 164 nM, respectively, for purified annexin II and K(d) values of 83 and 125 nM, respectively, for BAE cells. Anti-annexin II monoclonal antibody inhibited angiostatin and plasminogen binding to endothelial cells by 68 and 62%, respectively, supporting our in vitro studies that annexin II is a receptor for angiostatin. Angiostatin-binding protein/annexin II specifically expressed in endothelial cells but not in fibroblasts suggests its EC-specific function. Epsilon-aminocaproic acid, a lys analogue, effectively blocks angiostatin and annexin II interaction, indicating that the lysine-binding domain of AS is required for binding to annexin II. These results suggest that the antiangiogenic action of angiostatin may be mediated via interaction with annexin II. Identification of annexin II as a receptor for angiostatin provides further evidence that clotting and fibrinolytic pathways are directly involved in the angiogenic process.     

Annexin A2: biology and relevance to the antiphospholipid syndrome

Antiphospholipid antibodies (aPL), the majority of which are directed against beta(2)-glycoprotein I (beta(2)GPI), are associated with an increased incidence of venous and arterial thrombosis. The pathogenesis of antiphospholipid/anti-beta(2)GPI-associated thrombosis has not been defined, and is likely multifactorial. However, accumulating evidence suggests an important role for endothelial cell activation with the acquisition of a procoagulant phenotype by the activated endothelial cell. Previous work demonstrated that endothelial activation by antiphospholipid/anti-beta(2)GPI antibodies is beta(2)GPI-dependent. We extended these observations by defining annexin A2 as an endothelial beta(2)GPI binding site. We also observed that annexin A2 plays a critical role in endothelial cell activation induced by anti-beta(2)GPI antibodies, and others have described direct endothelial activation by anti-annexin A2 antibodies in patients with aPL . Similar findings have been reported using human monocytes, which also express annexin A2. Because annexin A2 is not a transmembrane protein, how binding of beta(2)GPI/anti-beta(2)GPI antibodies, or anti-annexin A2 antibodies, to endothelial annexin A2 causes cellular activation is unknown. Recent studies, however, suggest an important role for the Toll-like receptor family, particularly TLR4. In this article, we review the role of these interactions in the activation of endothelial cells by aPL . The influence of these antibodies on the ability of annexin A2 to enhance t-PA-mediated plasminogen activation is also discussed.     

Role of plasminogen activator in degradation of extracellular matrix protein by live human alveolar macrophages

Recent evidence indicates that human alveolar macrophages can degrade purified elastin in vitro by a cell contact-dependent process involving acidic proteinases of the cysteine proteinase class. It is unclear to what extent these cells can degrade elastin within a natural extracellular matrix. To address this question, we cultured live human alveolar macrophages on elastin-rich, 3H-lysine-labeled, extracellular matrices deposited by rat smooth muscle cells in vitro. Under various culture conditions, we then measured release of total radioactivity from the matrices during co-culture with cells as well as net loss of desmosine/isodesmosine as a specific marker of elastin degradation. Live macrophages adhered to and progressively solubilized matrix protein at a slow rate (approximately 5 micrograms/10(6) cells/24 h) but the rate of solubilization increased more than 15-fold in the presence of plasminogen. The elastin component of the complicated matrix was not measurably degraded in the absence of plasminogen, but in medium containing plasminogen, 3.5 X 10(6) macrophages degraded 25 +/- 8 micrograms of elastin in 72 h. After pretreatment of matrices with trypsin to remove glycoprotein elements, live cells degraded 16 +/- 4 micrograms of elastin under plasminogen-free conditions. The addition of serum to the medium (1 to 5%) inhibited degradation of elastin within whole matrices (approximately 50% compared to serum-free medium containing plasminogen) but had no effect on degradation of elastin in trypsin-pretreated matrices. An active site inhibitor of cysteine proteinases, Z-phenylalanine-phenylalanine-diazomethylketone, blocked approximately 50% of the elastin degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Annexin II: a mediator of the plasmin/plasminogen activator system

The annexins constitute a family of calcium-dependent membrane binding proteins. Recently, annexin II has been shown to accelerate the activation of the clot-dissolving protease plasmin by complexing with the plasmin precursor plasminogen and with tissue plasminogen activator. Binding of plasminogen to annexin II is inhibited by the atherogenic lipoprotein, lipoprotein(a), while binding of tissue plasminogen activator to annexin II is blocked by the thiol amino acid homocysteine. Formation of the plasminogen/tissue plasminogen activator/annexin II complex may represent a key regulatory mechanism in fibrinolytic surveillance.     

Role of annexin II in estrogen-induced macrophage matrix metalloproteinase-9 activity: the modulating effect of statins

Annexin II (ANXII) is a receptor for tissue plasminogen activator and plasminogen for the conversion to plasmin, which, in turn, induces metalloproteinase-9 (MMP-9). 17beta-Estradiol (E(2)) is reported to decrease plasminogen activity inhibitor-1 and increase plasmin and matrix metalloproteinase activity. However, the combined effects of estrogen and statins on macrophage MMP-9 activity and ANXII expression remain unclear. Treatment of J774A.1 macrophages with 1.0-100 nM of E(2) for 24h increased both MMP-9 activity and ANXII expression in a dose-dependent manner (p<0.05). Preincubation with EGTA (10mM) released ANXII from the cell membrane and inhibited the E(2)-mediated MMP-9 activity as did incubation of macrophages with anti-annexin IgG. In the presence or absence of E(2) (5 nM), simvastatin treatment in the range of 0.1-5.0 microM significantly reduced macrophage MMP-9 enzymatic activity (p<0.005) in a dose-dependent manner. In the presence or absence of E(2), simvastatin also decreased ANXII expression (p<0.05). These findings indicate that ANXII plays a central role in modulating the enzymatic activity of MMP-9 in response to E(2) and that E(2)-mediated ANXII expression and MMP-9 activity can be prevented by simvastatin. Prevention of E(2)-mediated activation of MMP-9 by simvastatin suggests that concurrent statin use may account for early event risk of myocardial infarction seen with hormone therapy in recent clinical trials.     

LDL oxidized with iron in the presence of homocysteine/cystine at acidic pH has low cytotoxicity despite high lipid peroxidation

Fe(III) can have a strong oxidizing effect in the presence of reductants at acidic pH, which may occur under anaerobic conditions or in regions of inflammation. Low density lipoprotein (LDL) oxidation with Fe(III) and homocysteine/cystine at acidic pH provoked mainly formation of lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS) in the absence of significant protein modification. Even when oxidized to a high TBARS content, LDL oxidized at acidic pH was not cytotoxic when added to THP-1 monocytes in a concentration causing cell death when LDL was oxidized to a similar TBARS content at plasma pH with Fe(III) or Cu(II) in the presence or absence of homocysteine/cystine. Inducible nitric oxide production by RAW264.7 mouse macrophages was only weakly inhibited by LDL oxidized at acidic pH, even if acetylated before oxidation to increase uptake, as compared to LDL oxidized with Cu(II) at plasma pH to a similar TBARS content or anodic electrophoretic mobility. LDL oxidized at acidic pH may mainly induce protective mechanisms against oxidative stress while causing little acute damage of cells.     

Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

BACKGROUND: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. AIMS: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. METHODS: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. RESULTS: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. CONCLUSION: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.     

Histone H2B as a functionally important plasminogen receptor on macrophages

Plasminogen (Plg) facilitates inflammatory cell recruitment, a function that depends upon its binding to Plg receptors (Plg-Rs). However, the Plg-Rs that are critical for cell migration are not well defined. Three previously characterized Plg-Rs (alpha-enolase, annexin 2, and p11) and a recently identified Plg-R (histone H2B [H2B]) were assessed for their contribution to Plg binding and function on macrophages. Two murine macrophage cell lines (RAW 264.7 and J774A.1) and mouse peritoneal macrophages induced by thioglycollate were analyzed. All 4 Plg-Rs were present on the surface of these cells and showed enhanced expression on the thioglycollate-induced macrophages compared with peripheral blood monocytes. Using blocking Fab fragments to each Plg-R, H2B supported approximately 50% of the Plg binding capacity, whereas the other Plg-Rs contributed less than 25%. Anti-H2B Fab also demonstrated a major role of this Plg-R in plasmin generation and matrix invasion. When mice were treated intravenously with anti-H2B Fab, peritoneal macrophage recruitment in response to thioglycollate was reduced by approximately 45% at 24, 48, and 72 hours, with no effect on blood monocyte levels. Taken together, these data suggest that multiple Plg-Rs do contribute to Plg binding to macrophages, and among these, H2B plays a very prominent and functionally important role.     

热休克预处理对脂多糖所致RAW 264.7细胞迁移的影响

目的 观察热休克预处理对脂多糖所致巨噬细胞迁移的影响.方法 采用ELISA观察脂多糖预处理对RAW264.7巨噬细胞中肿瘤坏死因子α和白细胞介素1β释放的影响;采用趋化实验观察脂多糖处理对RAW264.7巨噬细胞迁移的影响;采用趋化实验观察热休克预处理对RAW264.7巨噬细胞迁移的影响.结果 用500μg/L脂多糖作用RAW264.7巨噬细胞1 h,肿瘤坏死因子α和白细胞介素1β的释放较对照组明显增加(P<0.05);用500μg/L脂多糖处理细胞24 h, RAW264.7巨噬细胞的迁移较对照组明显增加(P<0.05);给予细胞42.5℃热休克预处理1 h后,再予500μg/L脂多糖处理细胞,RAW264.7巨噬细胞迁移较单纯脂多糖处理组明显减少(P<0.05).结论 热休克预处理能抑制脂多糖所致RAW264.7巨噬细胞迁移.     

Annexin A5 polymorphism (-1C-->T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome

BACKGROUND: Annexin A5 is thought to have a role in the pathophysiology of the antiphospholipid syndrome (APS)-a syndrome characterised by recurrent thrombosis and pregnancy morbidity. OBJECTIVE: To investigate whether anti-annexin A5 immunoglobulin (Ig)M or IgG antibodies, or the -1C-->T polymorphism of annexin A5, is a risk factor for thrombosis or miscarriage, and whether the -1C-->T polymorphism is correlated with APS. METHODS: A cohort study was carried out with a population of 198 patients with primary APS, systemic lupus erythematosus or lupus-like disease. For the detection of anti-annexin A5 antibodies and the measurement of annexin A5 plasma levels, ELISA-type methods were used. The annexin A5 -1C-->T mutation was detected by restriction fragment length polymorphism. RESULTS: 71 patients were positive for annexin A5 IgM or IgG antibodies, of whom 53 patients were positive for anti-annexin A5 IgG antibodies and 27 of 198 patients were positive for anti-annexin A5 IgM antibodies. The prevalence of IgM or IgG anti-annexin A5 antibodies was not significantly associated with thrombosis or miscarriage on multivariate analysis. The prevalence of the -1C-->T mutation in the annexin A5 gene (46/198 patients) was significantly associated with miscarriage (odds ratio 2.7, 95% confidence interval 1.1 to 6.7, independent risk factor). CONCLUSION: The detection of anti-annexin A5 antibodies does not seem relevant for estimating the risk for thrombosis or miscarriage in APS. The -1C-->T mutation was an independent risk factor for miscarriage, which is independent of APS.     

Annexin V autoantibodies in rheumatoid arthritis.

OBJECTIVE: To investigate the occurrence of anti-annexin V autoantibodies in sera of patients with rheumatoid arthritis to assess involvement with the disease and any relation to glucocorticoid treatment. METHODS: Anti-annexin V antibodies were measured by an enzyme linked immunosorbent assay (ELISA) which used the purified human recombinant protein as antigen. RESULTS: Concentrations of anti-annexin V autoantibodies, predominantly of the IgG class, were significantly raised in sera from patients with rheumatoid arthritis compared to normal controls. This was not correlated with other indices of disease activity such as erythrocyte sedimentation rate or C reactive protein and was unrelated to glucocorticoid treatment. CONCLUSIONS: Extracellular annexin V provides an antigenic stimulus for autoantibody production and its in vivo expression is independent of glucocorticoid control. Such autoantibodies may have a detrimental role in the arthritic condition by interfering with putative functions of annexin V, including collagen type II binding, inhibition of phospholipase A2 activity, and Fc receptor activity.     

巨噬细胞集落刺激因子对RAW264.7细胞谷胱甘肽过氧化物酶活性及其基因表达的影响

为探讨巨噬细胞集落刺激因子抗单核巨噬细胞过氧化损伤的机理,以富含巨噬细胞集落刺激因子的Ｌ９２９细胞条件培养基作为细胞因子来源,采用酶活性测定、反转灵聚合酶链反应等技术,研究了巨噬细胞集落刺激因子对小鼠巨噬细胞系ＲＡＷ２６４．７细胞谷胱甘肽过氧化物酶活性及ｍＲＮＡ表达的影响。结果发现,巨噬细胞集落刺激因子可以提高ＲＡＷ２６４．７细胞硒谷胱革肽过氧化物酶及非硒谷胱甘肽过氧化物酶活性,并增强ＲＡＷ２６４     

巨噬细胞集落刺激因子对RAW264.7细胞谷胱甘肽过氧化物酶活性及其基因表达的影响

为探讨巨噬细胞集落刺激因子抗单核巨噬细胞过氧化损伤的机理 ,以富含巨噬细胞集落刺激因子的L92 9细胞条件培养基作为细胞因子来源 ,采用酶活性测定、反转录聚合酶链反应等技术 ,研究了巨噬细胞集落刺激因子对小鼠巨噬细胞系RAW2 6 4.7细胞谷胱甘肽过氧化物酶活性及mRNA表达的影响。结果发现 ,巨噬细胞集落刺激因子可以提高RAW2 6 4.7细胞硒谷胱甘肽过氧化物酶及非硒谷胱甘肽过氧化物酶活性 ,并增强RAW2 6 4.7细胞血浆谷胱甘肽过氧化物酶mRNA的表达 ,而对RAW2 6 4.7细胞磷脂氢过氧化物谷胱甘肽过氧化物酶的表达无诱导作用     

抗膜联蛋白A2抗体在抗磷脂综合征中的作用

目的 研究抗膜联蛋白A2抗体在抗磷脂综合征(APS)、系统性红斑狼疮(SLE)的血栓/病态妊娠中的可能作用.方法 先用分子克隆方法表达纯化出重组膜联蛋白A2,然后以重组膜联蛋白A2为抗原,采用酶联免疫吸附试验(ELISA)法分别检测了,101例APS患者,41例SLE合并血栓患者,124例无血栓的SLE患者及120名健康人的血清中IgG型抗膜联蛋白A2抗体水平.结果 APS组和SLE合并血栓组的IgC型抗膜联蛋白A2抗体阳性率分别为21.8%,26.8%,均品著高于单纯SLE组(6.5%)(P值均<0.0.).IgG型抗膜联蛋白A2抗体与血栓/病态妊娠有关联(P<0.01).IgG型抗膜联蛋白A2抗体对血栓/病态妊娠诊断的敏感性、特异性、预测值分别为0.232、0.935、0.805.结论 IgG型抗膜联蛋白A2抗体与APS和SLE患者的血栓/病态妊娠表现相关,将有助于一些潜在的APS患者的诊断.     

Monocyte Fc gamma receptor recognition of cell-bound and aggregated IgG

Monocyte and macrophage Fc gamma receptors are important components in the recognition of IgG-coated cells and IgG-containing immune complexes. Two proteins have been identified on human peripheral blood monocytes that can function as Fc gamma receptors, Fc gamma RI (70 Kd) and Fc gamma RII (40 Kd). We studied the role of Fc gamma RI and Fc gamma RII on human monocytes by examining their binding of IgG-sensitized cells (human IgG anti-D-coated RBCs and rabbit IgG-sensitized sheep RBCs) and their binding of human trimeric IgG. To examine the function of monocyte Fc gamma RII, we used an anti-Fc gamma RII monoclonal antibody (MoAb) that competes for the Fc gamma RII ligand binding site. Preincubation of monocytes with saturating concentrations of anti-Fc gamma RII MoAb did not alter the recognition of IgG (anti-D)-sensitized human RBCs by monocytes. Furthermore, ligand-binding studies demonstrated that anti-Fc gamma RII antibody altered neither the number nor the affinity of monocyte-binding sites for human IgG trimer. Anti-Fc gamma RII inhibited monocyte binding of rabbit IgG-sensitized sheep RBCs, but only at low ionic strength or temperature when increased numbers of monocyte Fc gamma RII were expressed. At low ionic strength and 4 degrees C, anti-Fc gamma RII also partially inhibited monocyte binding of human trimeric IgG. Thus, monocyte Fc gamma RII does not appear to recognize IgG-sensitized RBCs or trimeric IgG at physiologic temperatures and ionic strength. The data suggest that Fc gamma RI is the primary Fc gamma receptor on monocytes involved in the binding of IgG (anti-D)-sensitized erythrocytes and low mol wt complexes of IgG.