Pharmacological analysis for mechanisms of GPI-80 release from tumour necrosis factor-alpha-stimulated human neutrophils.
1 GPI-80, a glycosylphosphatidylinositol (GPI)-anchored protein initially identified on human neutrophils, plays a role(s) in the regulation of beta2 integrin function. Previous studies have shown that GPI-80 is sublocated in secretory vesicles. It is also found in soluble form in the synovial fluid of rheumatoid arthritis patients, and in the culture supernatant of formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils. To understand the behaviour of GPI-80 under conditions of stimulation, we investigated the effects of tumour necrosis factor (TNF)-alpha on its expression and release. We also probed the mechanism of its release with various pharmacologic tools. 2 TNF-alpha induced the release of GPI-80 from human neutrophils in a concentration- and time-dependent manner (in the range of 1-100 u ml(-1) and 30-120 min, respectively), but did not affect surface GPI-80 levels. 3 Cytochalasin B, genistein, and SB203580 but not PD98059 inhibited TNF-alpha-stimulated GPI-80 release and neutrophil adherence at the same concentration. In addition, TNF-alpha-induced GPI-80 release was inhibited by blocking monoclonal antibodies specific to components of Mac-1 (CD11b and CD18). 4 Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-L-cysteine) inhibited GPI-80 release by TNF-alpha stimulation, but superoxide dismutase did not. Antioxidants but not superoxide dismutase reduced an intracellular oxidation state. 5 These findings indicate that TNF-alpha-stimulated GPI-80 release from human neutrophils depends upon adherence via beta2 integrins. They also suggest that cytochalasin B, genistein, and SB203580 inhibit GPI-80 release by suppressing signals for cell adherence, rather than by a direct effect on its secretion. Finally, we suggest that GPI-80 release involves an intracellular change in a redox state.
Nitto
Takeaki
T
Department of Immunology and Parasitology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Araki
Yoshihiko
Y
Takeda
Yuji
Y
Sendo
Fujiro
F
D000581:Amidohydrolases; D000975:Antioxidants / Q000494:pharmacology; D018821:CD18 Antigens / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D003571:Cytochalasin B / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D058851:GPI-Linked Proteins; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D006867:Hydrolases; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000975:Antioxidants; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D005419:Flavonoids; D058851:GPI-Linked Proteins; D007093:Imidazoles; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D003571:Cytochalasin B; D019833:Genistein; D006867:Hydrolases; D000581:Amidohydrolases; C121771:VNN2 protein, human; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one