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Keywords:

  • asthma;
  • chemotaxis;
  • eosinophil;
  • hepatocyte growth factor

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

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.

Abbreviations:
HGF

hepatocyte growth factor

PGD2

prostaglandin D2

ERK

extracellular signal-regulated kinase

MAPK

mitogen-activated protein kinase

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.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

Eosinophil purification

Peripheral blood was obtained from subjects with mild eosinophilia. All subjects gave informed consent. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia, Uppsala, Sweden) density gradients as modified from the method of Hansel et al. (20, 21). The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Eosinophils (>99% purity) were then suspended in Hank's balanced salt solution (HBSS; Life Technologies, Grand Island, NY, USA) with 1% fetal calf serum (FCS).

Chemotaxis assay

Chemotaxis of eosinophils was conducted in duplicate using 5-μm polycarbonate, polyvinylpyrrolidone-free membranes in Boyden chambers. Human eotaxin (10 nM; R&D Systems, Minneapolis, MN, USA) or PGD2 (1 μM; Cayman Chemical, Ann Arbor, MI, USA) was diluted in RPMI 1640 (Gibco, Grand Island, NY, USA) with 1% FCS and placed in the lower wells (100 μl). After the incubation of eosinophils with hrHGF (2.0, 20 and 200 ng/ml; R&D Systems) or medium for 1 h at 37°C, the cells were washed twice. Aliquots of 100 μl of the cell suspension at 2.0 × 105 cells/ml were placed in the upper chambers. The loaded chambers were incubated at 37°C in humidified air with 5% CO2 for 1 h. The membrane was then removed, followed by fixation and staining for 3 min in May-Grünwald solution. Cells that migrated and adhered to the lower surface of the membrane were counted from 10 fields by light microscopy.

Horizontal chemotaxis assay

To investigate real-time horizontal PGD2-induced chemotaxis, the KK chamber (Effector Cell Institute, Tokyo, Japan) was used as previously described (22). In the KK chamber, there is an etched silicon substrate and a flat glass plate, both of which form two compartments with a 5-μm-deep microchannel (23). In this study, eosinophils (1 μl of 1 × 106 cells/ml) treated with and without hrHGF were put into the hole holding the device together, and 1 μl of 10 μM eotaxin or 1 μM PGD2 was put into a contra-hole. The gradient of eotaxin or PGD2 was placed on the microchannel. The KK chamber was incubated for 30 min at 37°C. The migration of eosinophils toward the high concentration of PGD2 was recorded using a charge-coupled device (CCD) camera. Moreover, the rate of migration of each eosinophil was analyzed using the continuous pictures for every 30 s taken from the KK chamber.

Measurement of intracellular calcium

For intracellular calcium measurement, purified eosinophils (10 × 106 cells in HBSS/HEPES without Ca2+) in suspension were loaded with 2 μM fura 2-AM (Dojindo, Kumamoto, Japan) for 40 min at room temperature in the dark. The cells were washed in HBSS/HEPES with 4% FCS, and then resuspended at a concentration of 5 × 106 cells/ml in HBSS with Ca2+. Aliquots of cells (50 μl) were dispensed into cuvettes and equilibrated with 1 mM calcium at room temperature for 10 min. Changes in fluorescence were measured using a Fluorescence Drug Screening System 2000 (Hamamatsu Photoics K.K., Hamamatsu, Japan) as previously described (24). Intracellular free calcium was calculated from fluorescence spectra (excitation wavelengths, 340 and 380 nm) in accordance with established methods (25). The eosinophils were incubated with 20 ng/ml hrHGF or medium for 1 h at 37°C, and then 10 nM eotaxin was added 90 s after commencing recording.

Western blot analysis

Purified eosinophils (1 × 106 cells) were treated with and without hrHGF (20 ng/ml) for 1 h at 37°C followed by stimulation with and without eotaxin 10 nM for 3 min. The reaction was terminated by the addition of 15 volumes of ice-cold HBSS containing 1 mM Na3VO4. The cells were pelleted by centrifugation and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM ethylenediamintetraacetic acid, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 1% Triton X-100, 10% glycerol, and 1 μg/ml aprotinin, leupeptin, and pepstain). After 20 min on ice, detergent-insoluble materials were removed by centrifugation at 4°C at 15 000 g. The supernatants were mixed with sodium dodecyl sulfate (SDS) sample buffer and boiled for 4 min. SDS-polyacrylamide (10%) gels (Ready Gel J; Bio-Rad, Hercules, CA, USA) wereobtained from Bio-Rad (Tokyo, Japan). The electrophoresed gel was blotted onto Hybond ECL membranes (Amersham, Arlington Heights, IL, USA). Blots were incubated in blocking buffer containing 5% bovine serum albumin (BSA) in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, and 0.05% Tween-20, pH 7.6) for 30 min followed by incubation in the primary 0.1 μg/ml antibody [Ab; mouse monoclonal antibody (mAb) against phospho-p38 from Cell Signaling Technology (Beverly, MA, USA); mouse mAb against phospho-ERK from Santa Cruz Biotechnology, Santa Cruz, CA, USA] for the phosphorylation state of signaling proteins with gentle agitation overnight at 4°C. After washing three times in TBS-T buffer, blots were incubated for 30 min with a horseradish peroxidase (HRP)-conjugated secondary 0.04 μg/ml Ab directed against the primary Ab. The blots were developed with the ECL substrate according to the manufacturer's instructions. Blots were subsequently reprobed with another Ab (rabbit polyclonal anti-p38 and anti-ERK2; Santa Cruz Biotechnology) for the nonphosphorylation state of signaling proteins after stripping in a buffer of 62.5 nM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS at 56°C for 30 min. HRP-conjugated goat anti-mouse and anti-rabbit Abs were obtained from Santa Cruz Biotechnology.

Analysis of CCR3 and CRTH2 expression

To investigate CCR3 and CRTH2 expression on human eosinophils, a FACScan flow cytometer (Becton-Dickinson, Immunocytometry Systems, San Jose, CA, USA) was used as previously described (26). Purified eosinophils (1 × 106 cells) were incubated with a fluorescein isothiocyanate (FITC)-conjugate anti-human CCR3 mAb (mouse IgG2; DAKO, Glostrup, Denmark; 5.0 μg/ml) or isotype-matched control mAb (Becton-Dickinson; 5.0 μg/ml), and the PE-conjugated anti-human CRTH2 mAb (rat IgG2; Beckman Coulter, Inc., Flluerton, CA, USA; 5.0 μg/ml) or isotype-matched control mAb (Beckman Coulter, Inc.; 5.0 μg/ml). After washing the cells, the stained cells were analyzed by a flow cytometer.

Statistical analysis

Two groups of data were compared using Student's t-test. Other data were analyzed by analysis of variance, using Dunnett's post-test. The P-value for significance was set at P < 0.05. All results were expressed as the mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

Treatment with HGF attenuates eotaxin and PGD2-induced eosinophil chemotaxis

To determine the effects of HGF on eosinophil migration, purified human eosinophils were treated with hrHGF, and then eotaxin or PGD2-induced chemotaxis was measured using Boyden chambers. Compared with the control (vehicle only), these agents induced eosinophil migration. Preincubation of HGF to eosinophils significantly prevented migration in a dose-dependent manner (Fig. 1A,B). On the other hand, HGF itself did not induce eosinophil migration (Fig. 1A). It has reported that HGF increases the number of apoptotic cells on myofibroblasts (27); therefore, we performed an apoptosis detection assay after incubation with HGF for 48 h using a FACScan cytometer (Becton-Dickinson) as previously described (28). However, HGF did not influence cell viability in the concentration range used in this study (data not shown).

image

Figure 1.  Effect of hepatocyte growth factor (HGF) on eosinophil chemotaxis stimulated with 10 nM eotaxin (A) or 10 μM prostaglandin D2 (B). Purified eosinophils preincubated with increasing concentrations of HGF for 1 h. Migration assays were performed using Boyden chambers. Chemotactic response to 10 nM eotaxin agents was considered as 100%. Hepatocyte growth factor itself had no effect on eosinophil migration in (A). Data are expressed as the mean of four experiments (±SEM). *P < 0.05 vs control (medium; eotaxin alone).

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To further evaluate the inhibitory effects of HGF on eosinophil migration, we also used a KK chamber, which is able to detect the real-time horizontal migration of eosinophils. Few eosinophils moved when PGD2 was not added to the KK chamber [Fig. 2A(a)]. After 1 μl of 1 μM PGD2 was put into the chamber, most eosinophils migrated toward the high concentration of PGD2 [Fig. 2A(b)]. In contrast, following the preincubation of HGF, eosinophil migration was hardly observed [Fig. 2A(c)]. The rate of factor-induced migration was quantified (Fig. 2B). The preincubation with HGF significantly suppressed the speed of eosinophil migration induced by eotaxin [Fig. 2B(a)] and PGD2 [Fig. 2B(b)].

image

Figure 2.   (A) Effect of hepatocyte growth factor (HGF) on eosinophil chemotaxis stimulated with prostaglandin D2 (PGD2). Purified eosinophils preincubated with increasing concentrations of HGF for 1 h. Horizontal chemotaxis assay using a KK chamber. Eosinophil migration was recorded with a charge-coupled device camera. Arrows represent one of the migration cells. These images are a part of individual frames. (a) Eosinophils stimulated with medium alone, (b) eosinophils stimulated with PGD2, (c) eosinophils stimulated with PGD2 following the preincubation of 20 ng/ml HGF for 1 h. (B) Effect of HGF on rate of eosinophil migration induced by (a) eotaxin and (b) PGD2. Data are expressed as the mean of four experiments ±SEM. *P < 0.05 vs control (medium; eotaxin alone).

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Treatment with HGF inhibits eotaxin-induced calcium influx

Further, we investigated chemotactic signaling pathways that might be affected by HGF. Calcium influx is crucial for eosinophils to allow CCR3-mediated transmigration (4). Therefore, we studied the effects of HGF in the calcium influx on eotaxin-induced eosinophil chemotaxis. No change in calcium mobilization was found when eosinophils were stimulated with vehicle instead of eotaxin (Fig. 3A). On the other hand, incubation with HGF medium resulted in a marked increase in eotaxin-induced calcium mobilization (Fig. 3B). Preincubation with HGF for 1 h significantly suppressed the increase of calcium mobilization (Fig. 3C).

image

Figure 3.  Effect of hepatocyte growth factor (HGF) on eotaxin-induced intracellular calcium flux and the mitogen-activated protein kinase signaling pathway. Purified eosinophils loaded with 2 μM of fura-2 were pre-equilibrated with Ca2+. Eosinophil preparations were then stimulated with medium or 20 ng/ml HGF. After 1 h of incubation, eotaxin (10 nM) or vehicle was added (closed arrows). Changes in intracellular free calcium levels were detected as the increase in fluorescence intensity of calcium-sensitive dye fura-2. (A) Negative control, (B) eosinophils treated with medium, (C) eosinophils treated with HGF. The results are representative of experiments with similar results using eosinophils from three different donors.

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Treatment with HGF inhibits the MAPK signaling pathway in eotaxin-induced eosinophil chemotaxis

The MAPK signaling pathway was studied to further explore the inhibition mechanisms by HGF in agent-induced migration. To investigate MAPK pathway activity, eosinophils were stimulated with eotaxin and PGD2 followed by electrophoresis and Western blotting with the anti-p-ERK or anti-p-p38 Ab. Treatment with 20 ng/ml HGF partially inhibited eotaxin-induced phosphorylation of ERK1/2 as well as p38 (Fig. 4A,B). The membranes were reprobed with anti-ERK2 or anti-p38 Ab incubated with the same amounts of proteins loaded on the gels.

image

Figure 4.  Examination of the effect of the intracellular signaling pathway on the inhibitory effect of hepatocyte growth factor (HGF). Purified eosinophils were incubated with or without 20 ng/ml HGF for 1 h. After incubation, eosinophils were stimulated with eotaxin (10 nM) for 1 min [for extracellular signal-regulated kinase (ERK) 1/2] or 3 min (for p38). Phosphorylated ERK1/2 (p-ERK) and total ERK2 (ERK2), and phosphorylated p38 mitogen-activated protein kinase (MAPK; p-p38) and total p38 MAPK (p38) were determined by Western blot analysis. Hepatocyte growth factor partially abrogated the eotaxin-induced phosphorylation of ERK (A) and p38 (B). Experiments were repeated at least three times with a different cell preparation.

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Treatment with HGF has no effect on CCR3 and CRTH2 expression

A change of CCR3 and CRTH2 expression is important for eosinophil chemotaxis. Therefore, we also examined whether HGF modulates the surface expression CCR3 and CRTH2 on eosinophils; however, flow cytometric study revealed that HGF had no effect on CCR3 or CRTH2 surface expression (Fig. 5A,B).

image

Figure 5.  Examination of the effect of hepatocyte growth factor (HGF) on CCR3 (A) and CRTH2 (B) surface expression on eosinophils. Purified eosinophils were incubated with 20 ng/ml HGF or medium alone for 1 h. After incubation, eosinophils were treated with monoclonal antibody directed against CCR3 or CRTH2. Representative histograms are shown. Cells stained with isotype-matched antibody were used for negative control staining. No significant difference in surface expression of CCR3 and CRTH2 was observed between eosinophils stimulated with HGF and medium [89.3 ± 7.8%vs 91.1 ± 5.8% difference in mean fluorescence intensity from CCR3-positive cells; 87.2 ± 3.1%vs 85.4 ± 6.1% CRTH2-positive cells]. The results are representative of experiments with similar results using eosinophils from three different donors.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References

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.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
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