The principal eosinophil peroxidase product, HOSCN, is a uniquely potent phagocyte oxidant inducer of endothelial cell tissue factor activity: a potential mechanism for thrombosis in eosinophilic inflammatory states

Abstract

In vivo, bromide (Br ؊ ), nitrite (NO 2 ؊ ), and thiocyanate (SCN ؊ ) compete for oxidation by eosinophil peroxidase (EPO) and H 2 O 2 , yielding, respectively, HOBr, NO 2 ⅐ , and HOSCN. We have recently shown that SCN ؊ is the strongly preferred substrate for EPO in vivo and that HOSCN, in contrast with other EPO-generated oxidants and HOCl, is a relatively weak, cellpermeant, sulfhydryl (SH)-reactive oxidant. We here show that HOSCN is a uniquely potent (up to 100-fold) phagocyte oxidant inducer of tissue factor (TF) activity in human umbilical vein endothelial cells (HUVECs). This induction is attributable to transcriptional up-regulation of TF gene expression dependent upon both activation of the p65/c-Rel TF-B transcription factor and activity of the ERK1/2 kinase pathway upstream of Egr-1 and was markedly further enhanced in the presence of wortmannin, an inhibitor of the PI3 kinase/Akt pathway. HOSCN also markedly activates the proinflammatory p65/p50 NF-B pathway. Based on these findings we hypothesize that HOSCN generated by adherent and infiltrating eosinophils may provoke the development of a prothrombotic and proinflammatory endothelial/endocardial phenotype that promotes the pronounced thrombotic diathesis characteristic of the hypereosinophilic syndrome. ( IntroductionEosinophils (EOs) are specialized phagocytes that function to protect the host against metazoan parasites but can also mediate pathologic tissue damage in allergic inflammatory states. Perhaps the most striking example of EO-mediated pathology is the hypereosinophilic syndrome (HES), a systemic hematologic order characterized by multiorgan system involvement, most notably a characteristic, often lethal, form of endocarditis. 1,2 Eosinophilic, or Loeffler, endocarditis is characterized by massive infiltration into the endocardium and myocardium of activated degranulating EOs and mural thrombosis leading to both pulmonary and systemic embolism. HES is also accompanied by a prominent systemic thrombotic diathesis that can manifest as hepatic vein thrombosis, cerebral sinus thrombosis, disseminated intravascular coagulation (DIC), skin microvessel thrombosis, and deep venous thrombosis. [3][4][5][6] The etiology of this thrombotic diathesis is unclear.EO-specific granule proteins are exocytosed and accumulate at sites of EO inflammation. These include the unique eosinophil peroxidase (EPO), a protein that accounts for 40% by weight of the EO-specific granule weight and shares 70% amino acid homology with the better-characterized neutrophil myeloperoxidase (MPO). 7 In addition, EOs are endowed with a highly active NADPH oxidase system capable of sustaining up to 10 times the superoxide anion and H 2 O 2 generation capacity of neutrophils. 8 While it is clear that MPO functions predominantly to utilize H 2 O 2 to oxidize chloride to hypochlorous acid (HOCl), the preferred physiologic substrate for EPO is less certain. Three unusual substrates-bromide (Br Ϫ ), nitrite (NO 2 Ϫ ), and thiocyanate (SCN Ϫ )-compete for oxidation by EPO in physi...

Histone Deacetylase Inhibitors Suppress TF-κB-dependent Agonist-driven Tissue Factor Expression in Endothelial Cells and Monocytes

“…The next day, nonadherent cells were washed away, and adherent macrophages were overlaid with fresh RPMI medium. Macrophages were pretreated with 0.3 or 1 M TSA for 4 h before stimulation with 10 g/ml LPS, 20 ng/ml human TNF-␣ or IL-1␤ for 5 h. Macrophage TF activity in whole cell lysates was detected by a one-stage clotting assay, as described before (5). Relative TF activity was normalized using a standard curve generating using recombinant human TF protein.…”

“…Reverse Transcription-PCR Analysis-Confluent HUVEC monolayers were pretreated with 1 M TSA for 4 h and then stimulated by 1 ng/ml TNF-␣ for 1 or 2 h. Total cellular RNA was isolated, and reverse transcription-PCR was performed as previously described (5). Specific primers were purchased from Integrated DNA Technologies (Coralville, IA): long tissue factor, sense (5Ј-GGCACTACAAATACTGTGGCAGCA-3Ј) and antisense (5Ј-ACTCTTCCGGTTAACTGTTCGGGA-3Ј); fulllength tissue factor, sense (5Ј-CGCCGCCAACTGGTAGAC-3Ј) and antisense (5Ј-TGCAGTAGCTCCAACAGTGC-3Ј); ICAM-1, sense (5Ј-CAGTGACCATCTACAGCTTTCCGG-3Ј) and antisense (5Ј-GCTGCTACCACAGTGATGATGACAA-3Ј).…”

“…One-stage Clotting TF Activity Assay-Confluent HUVEC monolayers or PBMC were pretreated with TSA (0.01-1 M) for 4 h and then stimulated by either 1 g/ml LPS, 1 ng/ml TNF-␣, 1 ng/ml IL-1␤, or 100 M reagent HOSCN for another 4 h. Tissue factor activity was detected by a one-stage clotting assay standardized against recombinant human TF as described before (5). Alternatively, HUVEC were treated with increasing doses of apicidin, MS-275, sodium butyrate, or valporic acid and then stimulated by LPS and TNF-␣ for another 4 h before assaying TF activity.…”

“…Oligonucleotides were end-labeled with [␥-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (Amersham Biosciences). Five g of nuclear extract was loaded per lane (10 g for the TF-B probe), and EMSA was carried out as described (5).…”

“…In monocytes, SP-1 sites contribute to the constitutive expression of base-line TF, whereas the AP-1, Egr-1, and TF-B sites mediate inducible TF expression (6). In endothelial cells, constitutive AP-1 and inducible TF-B activation mediate TF induction by LPS, TNF-␣, IL-1␤ (7), and, as we have recently shown, the principal phagocyte peroxidase-derived oxidant, HOSCN (5).…”