Suzanne J. Suchard

Clinical and biologic effects of granulocyte colony stimulating factor in the treatment of myelokathexis

Successful treatment of a patient with myelokathexis, a rare form of chronic neutropenia associated with recurrent infections, is described. Rapid mobilization of bone marrow neutrophils and improved myeloid morphologic features were observed after treatment with human granulocyte colony stimulating factor. Transient thrombocytopenia and bone pain were observed during treatment. Although neutrophil chemotaxis, superoxide production, and FcRIII surface expression were reduced, the patient improved clinically after restoration of a normal neutrophil count.

Transforming growth factor-β1 stimulates degranulation and oxidant release by adherent human neutrophils

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 (H2O2) and the specific granule component lactoferrin. Transforming growth factor‐β1 (TGF‐β1) is released by many cells including PMN. It has been reported that TGF‐β1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF‐β1 would activate PMN to release H2O2 when they were adherent to fibrinogen, a response mediated by β2 integrin receptors. In this study, we determined whether TGF‐β1 stimulated H2O2 and lactoferrin release by PMN adherent to fibrinogen. TGF‐β1 stimulated H2O2 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 H2O2 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H2O2 and lactoferrin release. Ethanol (200 mM) blocked both H2O2 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso[3H]phoephatidylcholine, we observed that TGF‐β1 treatment caused an increase in [3H]phoephatidate. Propranolol (150 μM), an inhibitor of phosphatidate phosphohydrolase, blocked both H2O2 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF‐β1. 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. J. Leukoc. Biol. 60: 772–777; 1996.

NEUTROPHIL THROMBOSPONDIN RECEPTORS ARE LINKED TO GTP-BINDING PROTEINS

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP‐binding proteins (G‐proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP‐mediated motility. Both PT and CT inhibited TSP‐mediated chemotaxis and substrate‐associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 μM) nor 8‐bromo‐cAMP (300 μM) significantly affected TSP‐mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 ± 0.3 to 14.2 ± 0.1 pmol/106 cells). Although we could not test the PT sensitivity of TSP priming for FMLP‐mediated chemotaxis (as PT inhibits FMLP‐mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP‐dependent priming of FMLP‐mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G‐proteins was obtained by examining the effect of TSP on α‐subunit ADP‐ribosylation, GTPase activity, and GTPγS binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT‐dependent ribosylation of Giα2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G‐proteins. However, TSP did not block the CT‐dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of Giα2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 ± 2.7% of control values). In addition, GTPγS binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 ± 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G‐proteins, a subpopulation of which also associates with FMLP receptors.

THROMBOSPONDIN (TSP)-DEPENDENT MIGRATION OF GLIOBLASTOMA CELLS: RELATIONSHIP TO INVASIVE POTENTIAL AND INVOLVEMENT OF TSP STRUCTURAL DOMAINS.▴ 913

THROMBOSPONDIN (TSP)-DEPENDENT MIGRATION OF GLIOBLASTOMA CELLS: RELATIONSHIP TO INVASIVE POTENTIAL AND INVOLVEMENT OF TSP STRUCTURAL DOMAINS. ▴ 913