Hepatocyte growth factor (HGF), a heparin-binding factor, is synthesized as a single-chain inactive precursor (pro-HGF), which is converted by proteolysis to an active heterodimer (mature HGF). HGF has pleiotropic activities and has been implicated in the regulation of mitogenesis, motogenesis, and morphogenesis of epithelial and endothelial cells. As polymorphonuclear neutrophils (PMNs) secrete numerous cytokines involved in the modulation of local inflammation, we investigated their ability to produce HGF. We found that HGF was stored in secretory vesicles and in gelatinase/specific granules. This intracellular stock was rapidly mobilized by degranulation when neutrophils were stimulated with phorbol myristate acetate or N-formylmethionylleucyl-phenylalanine. Cycloheximide did not affect the release of HGF. Moreover, HGF messenger RNA and protein expression was found in bone marrow myeloid cells, suggesting that HGF synthesis likely occurs during PMN maturation. In mature circulating PMNs, intracellular HGF was in the pro-HGF form, whereas the HGF secreted by degranulation was the mature form. Furthermore, PMNs pretreated with diisopropyl fluorophosphate only released the pro-HGF form, suggesting that PMN-derived serine protease(s) are involved in the proteolytic process. We also obtained evidence that secreted mature HGF binds PMN-derived glycosaminoglycans (probably heparan sulfate). These findings suggest that PMNs infiltrating damaged tissues may modulate local wound healing and repair through the production of HGF, a major mediator of tissue regeneration. IntroductionHepatocyte growth factor (HGF), a heparin-binding factor, was originally described as a potent mitogen for the growth of hepatocytes in vitro and was subsequently purified from rat platelets, 1 plasma of a patient with fulminant hepatic failure, 2 and normal human plasma. 3 HGF has mitogenic, motogenic, and morphogenic effects on various epithelial and endothelial cell types. 4,5 Many studies have demonstrated that embryogenesis, angiogenesis, hematopoiesis, as well as wound healing and organ regeneration, are critically controlled by HGF. [5][6][7] HGF is secreted as a 92-kd single-chain pro-HGF that requires endoproteolytic processing. This processing is mediated by a serine protease. The HGF activator, 8 blood coagulation factor XII, 9 urokinase, 10 and tissue-type plasminogen activator 11 are reported to activate HGF. Processing of HGF results in a bioactive form (mature HGF) consisting of a disulfide-linked 69-kd ␣ chain and a 32-kd ␤ chain. 12 The biologic effects of HGF are mediated by the activation of its high-affinity binding site known as the tyrosine kinase receptor c-met. 13 Besides c-met, HGF possesses lower-affinity/highcapacity binding sites corresponding to extracellular matrix molecules (glycosaminoglycans or collagen) or to cell surfaceassociated heparan sulfate 14,15 ; this correspondence creates a molecular reservoir of HGF on the cell surface, whereas HGF transfer to c-met initiates the cellular response. 16 The broad r...

Impaired polymorphonuclear neutrophil (PMN) functions during sickle cell anemia (SCA) may have a pathogenic role in the onset of vasoocclusive events. We used flow cytometry to study, in whole blood, the adhesion molecule expression and respiratory burst of PMNs from children with SCA. Three different clinical groups were studied: (1) patients with no history of vasoocclusive events (n ‫؍‬ 15); (2) patients with a history of vasoocclusive events (n ‫؍‬ 17); and (3) patients receiving hydroxyurea therapy for severe vasoocclusive events (n ‫؍‬ 9). Unstimulated PMNs showed decreased L selectin expression and increased H 2 O 2 production whatever the severity of the disease, reflecting PMN activation. This could contribute to endothelial activation reflected by abnormal plasma levels of soluble adhesion molecules (soluble intercellular adhesion molecule-1, sE selectin, and sL selectin). After stimulation with bacterial N-formyl peptides (N-formyl-methionylleucyl-phenylalanine [fMLP]), PMNs from untreated patients with a history of vasoocclusive events showed dysregulated L selectin shedding and increased H 2 O 2 production. Furthermore, in these patients, tumor necrosis factor priming followed by fMLP stimulation induced an H 2 O 2 production significantly higher than in the other patient groups and controls. These impairments could immobilize PMNs on the endothelium, thereby inducing reduced blood flow and fostering microvascular occlusion and vascular damage. In contrast, children treated with hydroxyurea showed near-normal basal and poststimulation H 2 O 2 production as well as normal L selectin shedding after stimulation but no change in plasma levels of soluble adhesion molecules. To our knowledge, this is the first report showing major qualitative changes of PMN abnormalities upon hydroxyurea treatment in SCA patients. This strongly suggests that PMNs are a primary target of this drug. IntroductionSickle cell anemia (SCA) results from a single point mutation that substitutes valine for glutamic acid at the sixth position in the ␤-globin chain and produces hemoglobin S (Hb S). 1,2 The basic pathophysiologic mechanism, based upon the polymerization of deoxy-Hb S and the ensuing deformation, rigidification, and fragilization of the red cells, explains the 3 hallmarks of the disease: hemolytic anemia, vasoocclusive events, and progressive functional asplenia leading to increased susceptibility to infection. Even though all SCA patients have the same biochemical defect, they show wide variability in the frequency and clinical severity of vasoocclusive crises and remain asymptomatic for long periods, indicating that additional factors must contribute to the pathophysiology of vasoocclusion. 3 Abnormal red blood cell (RBC) adhesion to the endothelium has recently been forwarded as the probable initiating event of vasoocclusion, while variations in the endothelium activation state may modulate the unpredictable occurrence of the major complications. 4 Inflammation plays a major role in this process. 5 To date, hydrox...

IL-10 has a wide range of effects tending to control inflammatory responses. We used flow cytometry to study IL-10 binding at the polymorphonuclear neutrophil (PMN) surface and its modulation by various proinflammatory agents. Little IL-10 bound to the surface of resting PMN. However, binding was strongly increased after stimulation with LPS and proinflammatory cytokines such as TNF and GM-CSF. IL-1 and IL-8 did not significantly modify IL-10 binding. Cycloheximide had no effect on TNF-induced IL-10 binding, strongly suggesting the release of a pre-existing pool of IL-10R rather than de novo receptor synthesis by PMN. This was confirmed by the inhibitory effect of pentoxifylline, an inhibitor of degranulation. The existence of an intracellular pool of IL-10R was shown by flow cytometry, immunocytochemical staining, and Western blotting with several anti-human IL-10R Abs. In subcellular fractions of resting PMN, IL-10R was mainly located in the specific granule fraction, and was absent from azurophil granules and cytosol. We also tested the mobilization of specific granules by measuring the release of lactoferrin, their reference marker. The differential effects of the proinflammatory agents on IL-10 binding matched their effects on lactoferrin release and may therefore be related to differential mobilization of specific granules by these agents. Furthermore, the kinetics of TNF-induced up-regulation of IL-10 binding to PMN ran parallel to the kinetics of the inhibitory effect of IL-10 on the oxidative burst, suggesting a key role of IL-10R mobilization from specific granules to the membranes in optimal regulation of inflammatory responses.