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Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific
S J Suchard1, M J Burton, S J Stoehr
1Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor 48109.
Abstract:
The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.
Insights
Thrombospondin (TSP) receptors on polymorphonuclear leucocytes (PMNs) increase with activation. DIDS, an anion-channel blocker, reveals two specific granule subpopulations in PMNs, differing in TSP receptor content and release mechanisms.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Thrombospondin (TSP) is an extracellular matrix protein that binds to polymorphonuclear leucocyte (PMN) surface receptors, promoting cell adhesion and motility.
- TSP receptor expression on PMNs significantly increases upon activation with chemotactic peptides like N-formylmethionyl-leucylphenylalanine (FMLP) or ionophores like A23187, especially when combined with cytochalasin B.
- This upregulation of TSP receptors correlates with the exocytosis of both specific and azurophil granules, but newly expressed receptors do not originate from easily mobilized specific granules.
Purpose of the Study:
- To investigate the source of newly expressed TSP receptors on PMNs after activation.
- To elucidate the role of anion-channel blockers, specifically 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), in modulating TSP receptor expression and granule release.
Main Methods:
- PMNs were activated using combinations of cytochalasin B with FMLP or A23187, in the presence or absence of DIDS.
- TSP receptor expression was quantified.
- The release of granule components, including vitamin B12-binding protein (from specific granules) and myeloperoxidase (from azurophil granules), was measured.
- The effect of DIDS on the release of granules induced by various agonists (PMA, FMLP, A23187) was assessed.
Main Results:
- DIDS (30-100 microM) treatment during PMN activation with cytochalasin B and FMLP or A23187 led to increased TSP receptor expression.
- This increase in TSP receptors coincided with the release of vitamin B12-binding protein from specific granules but significantly inhibited the release of myeloperoxidase from azurophil granules.
- DIDS inhibited the release of PMA-mobilized specific granules and azurophil granules mobilized by cytochalasin B plus FMLP/A23187, but not the specific granules mobilized by these latter combinations.
Conclusions:
- PMNs possess at least two distinct subpopulations of specific granules.
- One subpopulation is easily mobilized, lacks TSP receptors, and its release is inhibited by DIDS.
- A second, less easily mobilized subpopulation of specific granules contains a substantial pool of TSP receptors, and their release is enhanced by DIDS.