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Neutrophils from chronic granulomatous disease fail to increase endothelial permeability
R A Kaslovsky1, L Gibbs, A B Malik
1Department of Pediatrics, Albany Medical College of Union University, New York 12208.
Abstract:
Studies of neutrophil-dependent endothelial injury were made using normal neutrophils (nl-PMN, i.e., normal polymorphonuclear neutrophils) and PMN obtained from a patient with X-linked chronic granulomatous disease (CGD-PMN). Layering of nl-PMN on bovine pulmonary microvessel endothelial monolayers (ratio 10:1) followed by challenge with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) resulted in a 190% increase in 125I-labeled albumin permeability across the monolayers. Pretreatment of endothelial monolayers with tumor necrosis factor-alpha (TNF-alpha; 200 or 1,000 U/ml) further enhanced the increase in permeability after stimulation of nl-PMN with PMA. In contrast, challenge of CGD-PMN layered on endothelial cells with PMA did not increase permeability, and the effect was not enhanced by pretreating the endothelial cells with TNF-alpha. Nl-PMN, but not CGD-PMN, generated superoxide anion on stimulation with PMA (42.5 +/- 0.7 vs. 6.5 +/- 0.6 nM.10(6) PMN-1.h-1). PMA also induced degranulation of nl-PMN as measured by release of elastase (2.32 +/- 0.12 micrograms/10(6) PMN) and myeloperoxidase (173.8 +/- 1.9 U/10(6) PMN) into the medium, whereas release by CGD-PMN was markedly reduced (elastase release of 0.88 +/- 0.04 microgram/10(6) PMN and myeloperoxidase release of 82.6 +/- 19.4 U/10(6) PMN). The adherence response of nl-PMN and CGD-PMN to the endothelial cells after PMA stimulation was similar (79.5 +/- 11.8% nl-PMN and 64.0 +/- 1.9% CGD-PMN). Therefore, the PMN respiratory burst, with the resultant generation of oxidants and PMN-derived proteases, is required to increase endothelial permeability even in the face of increased endothelial adhesivity.
Insights
Neutrophil respiratory burst and proteases are essential for endothelial injury, as shown by studies comparing normal neutrophils (nl-PMN) to those from chronic granulomatous disease (CGD-PMN). CGD-PMN, lacking superoxide generation, did not increase endothelial permeability, highlighting the role of oxidants and proteases.
Area of Science:
- Immunology
- Cell Biology
- Vascular Biology
Background:
- Neutrophils play a critical role in inflammatory responses and tissue injury.
- Endothelial cells form the inner lining of blood vessels and are crucial for vascular integrity.
- Chronic granulomatous disease (CGD) is a genetic disorder affecting neutrophil function.
Purpose of the Study:
- To investigate the role of neutrophil respiratory burst and derived proteases in neutrophil-dependent endothelial injury.
- To compare the effects of normal neutrophils (nl-PMN) and neutrophils from patients with CGD (CGD-PMN) on endothelial permeability.
Main Methods:
- Utilized bovine pulmonary microvessel endothelial monolayers.
- Co-cultured endothelial cells with either nl-PMN or CGD-PMN.
- Stimulated neutrophils with phorbol 12-myristate 13-acetate (PMA) and assessed endothelial permeability.
- Pretreated endothelial cells with tumor necrosis factor-alpha (TNF-alpha).
- Measured superoxide anion generation, elastase, and myeloperoxidase release from neutrophils.
Main Results:
- Normal neutrophils (nl-PMN) significantly increased endothelial permeability (190%) upon PMA stimulation, an effect further enhanced by TNF-alpha.
- Neutrophils from CGD patients (CGD-PMN) did not increase endothelial permeability, even with PMA stimulation and TNF-alpha pretreatment.
- PMA induced superoxide anion generation and degranulation (elastase, myeloperoxidase release) in nl-PMN but not in CGD-PMN.
- Neutrophil adherence to endothelial cells was similar for both nl-PMN and CGD-PMN.
Conclusions:
- The neutrophil respiratory burst, leading to the generation of oxidants and proteases, is a critical requirement for increasing endothelial permeability.
- Endothelial injury mediated by neutrophils is dependent on neutrophil oxidative and enzymatic activity, not solely on adherence.