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.


CONCLUSIONS
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.
Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy.
Maurizio
M
Sorice
Agostina
A
Longo
Antonella
A
Capozzi
Tina
T
Garofalo
Roberta
R
Misasi
Cristiano
C
Alessandri
Fabrizio
F
Conti
Brigitta
B
Buttari
Rachele
R
Riganò
Elena
E
Ortona
Guido
G
Valesini
D000328:Adult; D017306:Annexin A2 / Q000032:analysis / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D016736:Antiphospholipid Syndrome / Q000276:immunology; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000096:biosynthesis; D010766:Phosphorylation; D051197:Toll-Like Receptor 4 / Q000032:analysis / Q000378:metabolism; D053482:beta 2-Glycoprotein I / Q000032:analysis / Q000276:immunology*
C472822:ANXA2 protein, human; D017306:Annexin A2; D000911:Antibodies, Monoclonal; D008055:Lipids; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D053482:beta 2-Glycoprotein I; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases