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Thrombospondin stimulates motility of human neutrophils
P J Mansfield1, L A Boxer, S J Suchard
1Department of Pediatrics, Section of Hematology/Oncology, University of Michigan School of Medicine, Ann Arbor 48109.
Abstract:
Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)-mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20-70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.
Insights
Thrombospondin (TSP) influences polymorphonuclear leukocyte (PMN) migration. TSP fragments prime PMNs for chemotaxis and, when bound, significantly enhance random cell locomotion, suggesting a role in inflammation.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) are crucial for inflammatory responses.
- Extracellular matrix (ECM) proteins can modulate PMN behavior at injury sites.
- Thrombospondin (TSP) is an ECM protein secreted by various cells, interacting with PMNs.
Purpose of the Study:
- To investigate the role of Thrombospondin (TSP) and its fragments in polymorphonuclear leukocyte (PMN) migration.
- To determine how TSP influences PMN chemotaxis and random locomotion.
Main Methods:
- Utilized purified TSP, its fragments (heparin-binding domain [HBD] and 140-kD fragment), and monoclonal antibodies.
- Assessed PMN responses to soluble TSP and TSP bound to nitrocellulose filters.
- Measured PMN priming for f-Met-Leu-Phe (fMLP)-mediated chemotaxis and random cell migration.
Main Results:
- Low concentrations of soluble TSP primed PMNs for fMLP-mediated chemotaxis, an effect mediated by the HBD.
- High concentrations of soluble TSP stimulated PMN chemotaxis, requiring both HBD and the 140-kD fragment.
- TSP bound to filters significantly enhanced random PMN locomotion, with activity localized to the 140-kD fragment.
Conclusions:
- TSP plays a dual role in PMN migration: priming for chemotaxis via HBD and promoting random migration when bound via the 140-kD fragment.
- TSP fragments contribute to PMN recruitment at sites of injury, potentially in synergy with chemoattractants.