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Transforming growth factor-beta1 stimulates degranulation and oxidant release by adherent human neutrophils
K J Balazovich1, R Fernandez, V Hinkovska-Galcheva
1Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor 48109-0238, USA.
Abstract:
The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H202) and the specific granule component lactoferrin. Transforming growth factor-beta1 (TGF-beta1) is released by many cells including PMN. It has been reported that TGF-beta1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF-beta1 would activate PMN to release H202 when they were adherent to fibrinogen, a response mediated by beta2++integrin receptors. In this study, we determined whether TGF-beta1 stimulated H202 and lactoferrin release by PMN adherent to fibrinogen. TGF-beta1 stimulated H202 and lactoferrin release from adherent PMN in a concentration-dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H202 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H202 and lactoferrin release. Ethanol (200 mM) blocked both H202 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso-[3H]phosphatidylcholine, we observed that TGF-beta1 treatment caused an increase in [3H]phosphatidate. Propranolol (150 microM), an inhibitor of phosphatidate phosphohydrolase, blocked both H202 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF-beta1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation.
Insights
Transforming growth factor-beta1 (TGF-beta1) activates human neutrophils (PMN) to release hydrogen peroxide and lactoferrin when they are adherent to fibrinogen. This response involves beta2 integrin receptors and a phospholipase D pathway, similar to chemoattractant activation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Signal transduction pathways in human neutrophils (PMN) activated by cytokines and growth factors are not well understood.
- Adherent PMN stimulated with cytokines or N-formyl-methionyl-leucyl-phenylalanine (fMLP) release hydrogen peroxide (H2O2) and lactoferrin.
- Transforming growth factor-beta1 (TGF-beta1) is known to stimulate PMN chemotaxis but not exocytosis or superoxide production in suspension.
Purpose of the Study:
- To investigate whether TGF-beta1 stimulates H2O2 and lactoferrin release from adherent PMN.
- To explore the role of beta2 integrin receptors in TGF-beta1-mediated PMN activation.
- To elucidate the signal transduction pathway involved in TGF-beta1-induced PMN activation.
Main Methods:
- Human neutrophils were plated onto fibrinogen-coated surfaces.
- Cells were stimulated with varying concentrations of TGF-beta1.
- Hydrogen peroxide and lactoferrin release were measured.
- PMN adhesion and spreading were assessed.
- The involvement of phospholipase D and phosphatidate phosphohydrolase pathways was investigated using ethanol and propranolol, respectively.
- Intracellular signaling molecules were tracked using radiolabeled precursors.
Main Results:
- TGF-beta1 stimulated H202 and lactoferrin release from adherent PMN in a concentration-dependent manner (0.1-100 pg/mL).
- Release of H202 and lactoferrin was observed within 60 minutes and continued for at least 180 minutes.
- PMN adhesion and spreading paralleled the release of H202 and lactoferrin.
- Ethanol and propranolol inhibited TGF-beta1-induced H202 and lactoferrin release, indicating the involvement of phospholipase D and phosphatidate phosphohydrolase.
- TGF-beta1 treatment increased [3H]phosphatidate levels in PMN labeled with lyso-[3H]phosphatidylcholine.
Conclusions:
- TGF-beta1 activates adherent human neutrophils to release H202 and lactoferrin.
- This activation is mediated by beta2 integrin receptors and involves the phospholipase D pathway.
- The findings suggest a common signaling pathway for both chemoattractant and cytokine activation of adherent PMN.