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Regulation of polymorphonuclear neutrophil CD16 and CD11b/CD18 expression by matrix proteins during hypoxia is VLA-5,
1Department of Surgery, School of Medicine, Rhode Island Hospital/Brown University, Providence 02903, USA.
Abstract:
We investigated the effects of hypoxia on matrix protein regulation of polymorphonuclear neutrophil (PMN) CD16 and CD11b/CD18 expression. Adherence of PMN to fibronectin increased CD16 expression during hypoxia over levels seen during normoxia, while adherence of PMN to either fibronectin or laminin increased CD11b/CD18 expression during hypoxia over levels seen during normoxia. Kinetics assays demonstrated a t1/2 approximately 60 min and 30 min of hypoxia for maximal up-regulation of CD16 and CD11b/CD18, respectively. Incubation of fluid-phase PMN with anti-VLA-5 (alpha 5/beta 1) and anti-VLA-6 (alpha 6/beta 1) mAbs blocked the effect of fibronectin and laminin on CD16 and CD11b/CD18 expression. Cross-linking of both fluid-phase and adherent PMN VLA-5 and VLA-6 receptors resulted in a progressive increase in CD16 and CD11b/CD18 expression during hypoxia, but not normoxia. Increases in CD16 and CD11b/CD18 expression resulted in increased E anti-Fc gamma RIII and EC3bi rosetting. Inhibition of GPLC activity and IP3 production with U73122 and cyclopiazonic acid blocked the ability of fibronectin to increase CD16 expression and E anti-Fc gamma RIII rosetting. Inhibition of protein tyrosine kinase with genistein and herbimycin A blocked the ability of laminin to increase CD11b/CD18 expression and EC3bi rosetting. Depletion of intracellular Ca2+ abrogated the effects of fibronectin and laminin on CD16 and CD11b/CD18 expression, while restoration of intracellular Ca2+ restored the ability of fibronectin and laminin to increase CD16 and CD11b/CD18 expression. These results demonstrate that during hypoxia integrin signaling via alpha 5/beta 1 and alpha 6/beta 1 results in the increased expression of CD16 and CD11b/CD18. This increase in receptor expression results in biologically active receptors and is dependent upon intracellular Ca2+, GPLC, and protein tyrosine kinase activity.
Insights
Hypoxia increases polymorphonuclear neutrophil (PMN) expression of CD16 and CD11b/CD18 receptors via integrin signaling. This hypoxia-induced upregulation enhances PMN function and relies on intracellular calcium, GPLC, and tyrosine kinase activity.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Biology
Background:
- Polymorphonuclear neutrophils (PMNs) play a crucial role in immune responses.
- Integrin receptors like CD16 and CD11b/CD18 are vital for PMN adhesion and function.
- The impact of hypoxic conditions on PMN integrin expression and signaling remains incompletely understood.
Purpose of the Study:
- To investigate the effects of hypoxia on matrix protein-mediated regulation of PMN CD16 and CD11b/CD18 expression.
- To elucidate the signaling pathways involved in hypoxia-induced changes in PMN integrin expression.
Main Methods:
- PMN adherence assays to fibronectin and laminin under normoxic and hypoxic conditions.
- Flow cytometry to quantify CD16 and CD11b/CD18 expression.
- Kinetics assays to determine the time course of receptor upregulation.
- Inhibition studies using monoclonal antibodies against VLA-5 and VLA-6 integrins.
- Pharmacological inhibition of GPLC, IP3 production, and protein tyrosine kinase activity.
- Intracellular calcium depletion and restoration experiments.
Main Results:
- Hypoxia significantly increased CD16 expression on PMNs adhering to fibronectin and CD11b/CD18 expression on PMNs adhering to fibronectin or laminin.
- Integrin signaling via VLA-5 (alpha 5/beta 1) and VLA-6 (alpha 6/beta 1) was essential for these hypoxia-induced increases.
- The upregulation of CD16 and CD11b/CD18 resulted in enhanced PMN rosetting.
- Intracellular calcium, GPLC activity, and protein tyrosine kinase activity were critical for the observed effects.
Conclusions:
- Hypoxia induces increased expression of CD16 and CD11b/CD18 on PMNs through integrin signaling pathways.
- This upregulation enhances PMN receptor function and is dependent on intracellular calcium, GPLC, and protein tyrosine kinase signaling.
- These findings highlight a novel mechanism by which hypoxia modulates PMN immune function.