Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

The Role of Autophagy as a Trigger of Post-Translational Modifications of Proteins and Extracellular Vesicles in the Pathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent joint inflammation, leading to cartilage and bone destruction. Autoantibody production is directed to post-translational modified (PTM) proteins, i.e., citrullinated or carbamylated. Autophagy may be the common feature in several types of stress (smoking, joint injury, and infections) and may be involved in post-translational modifications (PTMs) in proteins and the generation of citrullinated and carbamylated peptides recognized by the immune system in RA patients, with a consequent breakage of tolerance. Interestingly, autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy combines the autophagy machinery with the secretion of cellular content via extracellular vesicles (EVs). A role for exosomes in RA pathogenesis has been recently demonstrated. Exosomes are involved in intercellular communications, and upregulated proteins and RNAs may contribute to the development of inflammatory arthritis and the progression of RA. In RA, most of the exosomes are produced by leukocytes and synoviocytes, which are loaded with PTM proteins, mainly citrullinated proteins, inflammatory molecules, and enzymes that are implicated in RA pathogenesis. Microvesicles derived from cell plasma membrane may also be loaded with PTM proteins, playing a role in the immunopathogenesis of RA. An analysis of changes in EV profiles, including PTM proteins, could be a useful tool for the prevention of inflammation in RA patients and help in the discovery of personalized medicine.

Role of a Novel Heparanase Inhibitor on the Balance between Apoptosis and Autophagy in U87 Human Glioblastoma Cells.

BACKGROUND: Heparanase (HPSE) is an endo-ß-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. METHODS: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. RESULTS: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. CONCLUSIONS: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.

Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells.

Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Anti-Inflammatory Activity of a CB2 Selective Cannabinoid Receptor Agonist: Signaling and Cytokines Release in Blood Mononuclear Cells.

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1ß, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.

Raft-like lipid microdomains drive autophagy initiation via AMBRA1-ERLIN1 molecular association within MAMs.

Mitochondria-associated membranes (MAMs) are essential communication subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. We previously demonstrated that, upon macroautophagy/autophagy induction, AMBRA1 is recruited to the BECN1 complex and relocalizes to MAMs, where it regulates autophagy by interacting with raft-like components. ERLIN1 is an endoplasmic reticulum lipid raft protein of the prohibitin family. However, little is known about its association with the MAM interface and its involvement in autophagic initiation. In this study, we investigated ERLIN1 association with MAM raft-like microdomains and its interaction with AMBRA1 in the regulation of the autophagic process. We show that ERLIN1 interacts with AMBRA1 at MAM raft-like microdomains, which represents an essential condition for autophagosome formation upon nutrient starvation, as demonstrated by knocking down ERLIN1 gene expression. Moreover, this interaction depends on the "integrity" of key molecules, such as ganglioside GD3 and MFN2. Indeed, knocking down ST8SIA1/GD3-synthase or MFN2 expression impairs AMBRA1-ERLIN1 interaction at the MAM level and hinders autophagy. In conclusion, AMBRA1-ERLIN1 interaction within MAM raft-like microdomains appears to be pivotal in promoting the formation of autophagosomes.Abbreviations: ACSL4/ACS4: acyl-CoA synthetase long chain family member 4; ACTB/ß-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATG14: autophagy related 14; BECN1: beclin 1; CANX: calnexin; Cy5: cyanine 5; ECL: enhanced chemiluminescence; ER: endoplasmic reticulum; ERLIN1/KE04: ER lipid raft associated 1; FB1: fumonisin B1; FE: FRET efficiency; FRET: Förster/fluorescence resonance energy transfer; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GD3: aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)ceramide; HBSS: Hanks' balanced salt solution; HRP: horseradish peroxidase; LMNB1: lamin B1; mAb: monoclonal antibody; MAMs: mitochondria-associated membranes; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MYC/cMyc: proto-oncogene, bHLH transcription factor; P4HB: prolyl 4-hydroxylase subunit beta; pAb: polyclonal antibody; PE: phycoerythrin; SCAP/SREBP: SREBF chaperone; SD: standard deviation; ST8SIA1: ST8 alpha-N-acetyl-neuraminide alpha-2,8 sialyltransferase 1; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUBB/beta-tubulin: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VDAC1/porin: voltage dependent anion channel 1.

LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells.

LDL receptor-related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical ß-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced ß-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-ß-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving ß-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.

A multimolecular signaling complex including PrPC and LRP1 is strictly dependent on lipid rafts and is essential for the function of tissue plasminogen activator.

Prion protein (PrPC ) localizes stably in lipid rafts microdomains and is able to recruit downstream signal transduction pathways by the interaction with promiscuous partners. Other proteins have the ability to occasionally be recruited to these specialized membrane areas, within multimolecular complexes. Among these, we highlight the presence of the low-density lipoprotein receptor-related protein 1 (LRP1), which was found localized transiently in lipid rafts, suggesting a different function of this receptor that through lipid raft becomes able to activate a signal transduction pathway triggered by specific ligands, including Tissue plasminogen activator (tPA). Since it has been reported that PrPC participates in the tPA-mediated plasminogen activation, in this study, we describe the role of lipid rafts in the recruitment and activation of downstream signal transduction pathways mediated by the interaction among tPA, PrPC and LRP1 in human neuroblastoma SK-N-BE2 cell line. Co-immunoprecipitation analysis reveals a consistent association between PrPC and GM1, as well as between LRP1 and GM1, indicating the existence of a glycosphingolipid-enriched multimolecular complex. In our cell model, knocking-down PrPC by siRNA impairs ERK phosphorylation induced by tPA. Moreover the alteration of the lipidic milieu of lipid rafts, perturbing the physical/functional interaction between PrPC and LRP1, inhibits this response. We show that LRP1 and PrPC , following tPA stimulation, may function as a system associated with lipid rafts, involved in receptor-mediated neuritogenic pathway. We suggest this as a multimolecular signaling complex, whose activity depends strictly on the integrity of lipid raft and is involved in the neuritogenic signaling.

Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells.

Several studies demonstrated that cannabinoids reduce tumor growth, inhibit angiogenesis, and decrease cancer cell migration. As these molecules are well tolerated, it would be interesting to investigate the potential benefit of newly synthesized compounds, binding cannabinoid receptors (CBRs). In this study, we describe the synthesis and biological effect of 2-oxo-1,8-naphthyridine-3-carboxamide derivative LV50, a new compound with high CB2 receptor (CB2R) affinity. We demonstrated that it decreases viability of Jurkat leukemia cells, evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), but mainly induces a proapoptotic effect. We observed an increase of a hypodiploid peak by propidium iodide staining and changes in nuclear morphology by Hoechst 33258. These data were confirmed by a significant increase of Annexin V staining, cleavage of the nuclear enzyme poly(ADP-ribose)-polymerase (PARP), and caspases activation. In addition, in order to exclude that LV50 non-specifically triggers death of all normal leukocytes, we tested the new compound on normal peripheral blood lymphocytes, excluding the idea of general cytotoxicity. To characterize the involvement of CB2R in the anti-proliferative and proapoptotic effect of LV50, cells were pretreated with a specific CB2R antagonist and the obtained data showed reverse results. Thus, we suggest a link between inhibition of cell survival and proapoptotic activity of the new compound that elicits this effect as selective CB2R agonist.

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-ß-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes.

Elevated Serum Level of HMGB1 in Patients with the Antiphospholipid Syndrome.

Pregnancy problems are common in patients with rheumatic disease; indeed, autoimmune disorders and autoantibodies can affect pregnancy progress and lead to maternal complications. Recent studies have highlighted a close association between HMGB1, chronic inflammation, and autoimmune diseases. Thus, in this investigation, we analyzed serum levels of HMGB1, an alarmin which plays a pivotal role in inducing and enhancing immune cell function. Sera from 30 patients with antiphospholipid syndrome (11 primary and 19 secondary APS), 35 subjects with pregnancy morbidity, and 30 healthy women were analysed for HMGB1 and its putative receptor RAGE (sRAGE) by Western blot and for TNF-α by ELISA. Results revealed that APS patients showed significantly increased serum levels of HMGB1, sRAGE, and the proinflammatory cytokine TNF-α, as compared to healthy women. However, also, the pregnancy morbidity subjects showed significantly increased levels of HMGB1 and sRAGE as well as TNF-α compared to healthy women. Our findings suggest that in subjects with pregnancy morbidity, including obstetric APS, elevated levels of HMGB1/sRAGE may represent an alarm signal, indicating an increase of proinflammatory triggers. Further studies are needed to evaluate the role of HMGB1/sRAGE as a possible tool to evaluate the risk stratification of adverse pregnancy outcomes.

Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies.

OBJECTIVES: Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). METHODS: For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. RESULTS: Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients. CONCLUSION: These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response.

Celiac Disease (CD) is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly)-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2) activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release) and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease.

Autoantibodies specific to a peptide of ß2-glycoprotein I cross-react with TLR4, inducing a proinflammatory phenotype in endothelial cells and monocytes.

ß(2)-glycoprotein I (ß(2)GPI) is the major antigenic target for antiphospholipid Abs. Anti-ß(2)GPI Abs are a heterogeneous population of Igs targeting all domains of the molecule. Abs specific to ß(2)GPI domain I are strongly associated with thrombosis and obstetric complications. In the present study, we sought to understand the possible pathogenic mechanism for this subset of anti-ß(2)GPI Abs, investigating their potential cross-reactivity with other self-proteins involved in inflammatory or coagulant events. We compared the amino acid sequence of the ß(2)GPI domain I with human proteins in a protein databank and identified a peptide sharing 88% identity with an epitope of human TLR4. A high percentage of patients with antiphospholipid syndrome (41%) and systemic lupus erythematosus (50%) presented serum IgG specific to this peptide. Anti-ß(2)GPI peptide Abs binding the TLR4 were able to induce NF-κB activation in HEK293 cells that were stably transfected with the TLR4 gene. Anti-ß(2)GPI peptide Abs induced activation of TLR4 and triggered interleukin-1 receptor-associated kinase phosphorylation and NF-κB translocation, promoting VCAM expression on endothelial cells and TNF-α release by monocytes. In conclusion, our observations suggest a novel pathogenic mechanism in the TLR4 stimulation by anti-ß(2)GPI peptide Abs that links adaptive immune responses with innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

A new 4-phenyl-1,8-naphthyridine derivative affects carcinoma cell proliferation by impairing cell cycle progression and inducing apoptosis.

In targeted cancer therapy the search for novel molecules led to the discovery of a plethora of small organic molecules inhibiting cancer cell proliferation. Among these, quinazoline and derivatives, such as quinolines and naphthyridines, have been considered as of particular interest. One of these, the naphthyridine derivative 4-phenyl-2,7-di(piperazin-1-yl)-1,8-naphthyridine, has been analyzed in detail in the present work. We found that this compound elicited a powerful anti-proliferative activity on carcinoma cells, with IC50 values comparable with paradigmatic microtubule-deranging drugs. The mechanisms underlying this effect were seemingly due to a framework of cellular alterations that include peculiar alterations of mitochondria, i.e. an increase of mitochondrial membrane potential (MMP), followed by the typical MMP loss leading to the release of apoptogenic factors, and cell death by apoptosis. Furthermore, the analysis of cell cycle revealed that a significant percentage of treated cells was in G2/M phase. This block was seemingly due to a target effect of the naphthyridine derivative on microtubular network dynamic instability, which impaired mitotic spindle formation, possibly leading to mitotic catastrophy. Since the dual effects of naphthyridine derivative on cell cycle and mitotic spindle were obtained at very low concentrations, i.e. micromolar concentrations, we hypothesize that this compound could represent a new promising tool in the control of cancer cell proliferation.

Anti-beta2-glycoprotein I antibodies induce monocyte release of tumor necrosis factor alpha and tissue factor by signal transduction pathways involving lipid rafts.

OBJECTIVE: To investigate the association of beta(2)-glycoprotein I (beta(2)GPI) with lipid rafts in monocytic cells and to evaluate the proinflammatory and procoagulant effects of anti-beta(2)GPI binding to its target antigen on the monocyte plasma membrane. METHODS: Human monocytes were fractionated by sucrose density-gradient centrifugation and analyzed by Western blotting. Immunoprecipitation experiments were performed to analyze the association of beta(2)GPI with lipid rafts and the possible interaction of beta(2)GPI with annexin A2 and Toll-like receptor 4 (TLR-4). Monocytes were then stimulated with affinity-purified anti-beta(2)GPI antibodies from patients with the antiphospholipid syndrome (APS). Interleukin-1 receptor-associated kinase (IRAK) phosphorylation and NF-kappaB activation were evaluated by immunoprecipitation and transcription factor assay, respectively. Supernatants from monocytes were tested for tumor necrosis factor alpha (TNFalpha) and tissue factor (TF) levels by enzyme-linked immunosorbent assay. RESULTS: We found beta(2)GPI and its putative receptor annexin A2 in lipid raft fractions of human monocytes. Moreover, there was an association between beta(2)GPI and TLR-4, suggesting that it was partially dependent on raft integrity. Triggering with anti-beta(2)GPI antibodies induced IRAK phosphorylation and consequent NF-kappaB activation, which led to the release of TNFalpha and TF. CONCLUSION: Anti-beta(2)GPI antibodies react with their target antigen, likely in association with annexin A2 and TLR-4, in lipid rafts in the monocyte plasma membrane. Anti-beta(2)GPI binding triggers IRAK phosphorylation and NF-kappaB translocation, leading to a proinflammatory and procoagulant monocyte phenotype characterized by the release of TNFalpha and TF, respectively. These findings provide new insight into the pathogenesis of APS, improving our knowledge of valuable therapeutic targets.