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Background: Hepatocyte growth factor (HGF) is known to influence a number of cell types, and regulate various biologic activities including cell migration, proliferation, and survival. In a recent study, we found that, in vivo, HGF suppresses allergic airway inflammation, i.e. the infiltration of inflammatory cells including eosinophils into the airway, and further, that HGF reduces Th2 cytokine levels; however, the directly physiologic role of HGF with eosinophils remains unclear. In this study, we investigate the potential of recombinant HGF to regulate the factor-induced chemotaxis of human eosinophils.
Methods: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16-negative selection. After culture with or without recombinant HGF, esoinophil chemotaxis was measured by Boyden chamber and KK chamber.
Results: Treatment with HGF prevented eotaxin or prostaglandin D2 (PGD2)-induced chemotaxis of eosinophils. Moreover, we demonstrated that extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinases as well as the enhancement of Ca2+ influx, which are indispensable for eosinophil chemotaxis, were attenuated by HGF treatment.
Conclusion: Taken together, these data suggest that in allergic diseases, HGF not only mediates eosinophils through the inhibition of Th2 cytokines, but also regulates the function of eosinophils directly, provides further insight into the cellular and molecular pathogenesis of allergic reactions.
Bronchial asthma is a syndrome associated with allergen-induced chronic airway inflammation and airway hyperresponsiveness. Eosinophils play a pivotal role in the mechanism of allergic airway inflammation, and the chemotaxis of eosinophils is one of the most important events in the pathogenesis of allergic inflammation. The chemotactic response of eosinophils is mostly mediated by CCR3 (1), which is the specific receptor of eotaxin (2), a CC chemokine, and is known to transduce signals, eliciting Ca2+ influx (3). This calcium-signaling pathway involves myosin light chain kinase activation by the rearrangement of actin cytoskeleton, and then leads to chemotaxis (4). Moreover, some studies have recently found that the activation of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK) also play an important role in the eosinophil chemotaxis induced by eotaxin (5, 6). Prostaglandin D2 (PGD2), a biologic lipid rapidly synthesized by antigen-activated mast cells is implicated in the pathogenesis of allergic diseases such as asthma (7). The activity of PGD2 is mediated by binding to its receptors such as the D prostanoid receptor 1 (DP) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). In particular, CRTH2 is also expressed on eosinophils, and induces PGD2-dependent eosinophil migration (8).
Hepatocyte growth factor (HGF) was originally identified and cloned as a potent mitogen for mature hepatocytes as well as other cell types (9–11). There is now evidence that HGF plays an essential part in parenchymal repair and protection in various organs. For example, in vivo, human recombinant HGF (hrHGF) prevented the onset and progression of hepatic fibrosis/cirrhosis, renal, lung, and myocardial fibrosis (12–15). Thus, HGF is widely recognized as a multifunctional cytokine and a humoral mediator. Recently, we have found that in a murine model of asthma, HGF attenuated airway hyperresponsiveness and remodeling, and airway inflammation, eosinophil and lymphocyte accumulation in the peribronchial areas of the lung were also suppressed remarkably by HGF treatment (16). In vitro, HGF promotes the proliferation as well as survival of various cells including epithelial cells and carcinoma cells, and regulates the migration of those cells (17–19); however, very little is known about the functional role of HGF in the migration of hematopoietic cells including eosinophils.
In this study, to define the direct effect of HGF in regulating the chemotaxis of eosinophils, we examined whether HGF promotes the migration of nonstimulated eosinophils, and further, whether HGF produces an effect on eotaxin and PGD2-induced migration of human purified eosinophils. We found that HGF treatment attenuated the factor-induced chemotaxis of eosinophils but HGF itself failed to induce migration.
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The pathophysiology of asthma is complex, with allergens triggering a cascade of cellular interactions and the release of cytokines and chemokines in sensitized individuals. Eosinophils are believed to be important effector cells in allergic inflammation including asthma, and the chemotaxis of eosinophils is essential for tissue inflammation. In this study, we investigated the functional role of HGF in human eosinophil chemotaxis, and showed that HGF attenuated eosinophil migration toward both eotaxin and PGD2. To our knowledge, this is the first report showing HGF ability in the function of eosinophils.
Hepatocyte growth factor, now considered to be a mesenchyme-derived pleiotropic factor, is secreted by several cell types such as fibroblasts and macrophages in the lung, and stimulates cell growth, cell survival, mitogenesis, motogenesis, morphogenesis, and organogenesis in a wide range of tissue and organs (10, 17). Regarding cell motility, HGF promotes the invasion of carcinoma cells (29) as well as the migration of epithelial cells (17, 18) and endothelia (30) in the repair and regeneration stage of various injured organs. Furthermore, in hematopoietic cells, HGF also has a promotive role in the progression of diseases such as B-cell lymphoma, leukemia, and myeloma (31–33). However, in this study, we showed that HGF itself did not induce eosinophil migration, and furthermore, that preincubation with HGF significantly attenuated eotaxin and PGD2-induced chemotaxis (Fig. 1). The divergent results seen between these studies may be related to the different cells types, for example, non-neoplastic vs neoplastic cells, or culture conditions. Consistent with our results, in the apoptosis of cells, Mizuno and co-workers reported that HGF induced the apoptosis of myofibroblasts in lung fibrosis (27), whereas HGF inhibited the apoptosis of cancer cells (34).
Recently, we have found that HGF administration suppressed eosinophil and lymphocyte accumulation in bronchoalveolar lavage fluid and lung tissue, and increased Th2 cytokines [interleukin (IL)-4, IL-5, and IL-13] in a murine model of asthma (16). Interleukin-4 and IL-5 regulate eosinophil recruitment and function, orchestrating the allergic inflammatory response and leading to airway hyperresponsiveness (35, 36). These findings may suggest that HGF suppresses eosinophil accumulation in the airway through the inhibition of Th2 cytokine release. On the other hand, in this study, HGF attenuated the factor-induced chemotaxis of human eosinophils in the absence of IL-4, IL-5, or granulocyte-macrophage colony-stimulating factor. Moreover, HGF did not affect the viability of eosinophils although it inhibits the survival of myofibroblasts (27; data not shown). These data may indicate that HGF negatively regulates allergic airway inflammation by eosinophils through not only the suppression of Th2 cytokine, but also direct action against eosinophil migration. To further evaluate eosinophil migration, we also demonstrated a horizontal chemotaxis assay using visually accessible chemotactic apparatus named a KK chamber (23). Although Boyden's chamber technique is used most widely to measure chemotaxis in vitro (37), it is possible to observe cell migration in real-time and obtain sufficient information on the nature of gradients established using the KK chamber. In this way, the inhibitory effects of HGF on factor-induced migration and the rate of migration were evaluated more visually and objectively (Fig. 2).
The specific receptor of eotaxin is CCR3, and the upregulation of CCR3 expression might be associated with the augmentative effect on eotaxin (38). On the other hand, PGD2 had stimulatory effects on eosinophils including calcium mobilization, CD11b expression, and cell migration through CRTH2 (8, 39). In this study, HGF had no effect on the expression of CCR3 and CRTH2 in eosinophils (Fig. 5). Regarding the eotaxin/CCR3 signaling pathway of the eosinophil chemotaxis, cell migration is regulated by various signaling pathways including calcium influx signaling, and the ERK1/2 and p38 MAPK pathways (3, 5, 6). In this study, HGF treatment suppressed the eotaxin-induced Ca2+ response (Fig. 3) as well as the ERK1/2 and p38 MAPK pathways activated by the agent in purified human eosinophils (Fig. 4). These results may suggest that HGF modulates the downstream signaling of CCR3 or CRTH2 to attenuate the response to eotaxin or PGD2. However, some studies found that HGF stimulates the migration of various cells such as hepatic carcinoma cells, endothelial cells, and epithelial cells of the kidney, and that, in HGF-induced migration, the ERK pathway, which is activated by Ras, plays an essential role in inducting the motility response of cells to HGF (40–42). This conflict between the findings may also be related to the different cells types or stimulated conditions. Thus, further studies are necessary to clarify the physiologic role of HGF.
In conclusion, HGF suppresses the factor-induced chemotaxis of human eosinophils, and furthermore, the effect of HGF may be caused by the inhibition of Ca2+ influx and the MAPK signal pathway, which are important in eosinophil chemotaxis. However, these are novel findings different from other reports that HGF promotes the migration of other cells via activation of the MAPK pathway. Although HGF is also recognized as a homeostatic mediator that restores abnormal to normal conditions in the organism, the biologic potential of HGF is extremely obscure in the immune response in particular. Thus, further studies are necessary to elucidate the detailed mechanism of HGF activity in allergic diseases; however, this result may provide new further insights into the role of HGF in immune and allergic reactions.