Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-β. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.

Both B cells and T cells mediate immune responses, and their function is under the control of APCs. Ag presentation from APCs to B and T cells initiates the immune response. T cells recognize the peptide-MHC complex through the TCR and are then activated and differentiate into the effector T cells. Processed peptides of intracellular Ags bind to MHC class I molecules and then stimulate CTLs. Processed peptides of extracellular Ags are presented by MHC class II molecules to Th cells, and then induce differentiation of Th cells into Th1, Th2, or regulatory T (Treg)3 cells (1). During differentiation of naive Th cells into each phenotype, expression of costimulatory molecules on APCs and cytokine profiles produced from APCs plays a critical role (2). Among APCs, dendritic cells (DCs) are most efficient and crucial. So, DCs would play a central role in controlling immunity and are a logical target for many clinical situations that involve T cells (1).

Allergic asthma is a chronic inflammatory airway disease characterized by airway eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (AHR), and has been recognized as a Th2-mediated disease (3). Its incidence is steadily increasing (4). As the initiation of Ag-induced Th2-type response is under the control of DCs, it is evident that DCs are important in sensitization to Ags in patients with asthma (5). Moreover, one study recently revealed that the removal of DCs during the Ag aerosol challenge period completely eliminated the features of asthma, indicating that DCs are required for generating effector function of memory T cells (6). These data indicate that DCs are crucial in the pathogenesis of asthma, and that interfering with DC function could be of therapeutic benefit even in established asthma (5).

Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes (7, 8) and as a scatter factor (9), targets various cell types (10). HGF has many functions such as induction of angiogenesis, promotion of cell proliferation and migration (10), and inhibition of apoptosis (11, 12). HGF exhibits these functions through its receptor, c-met (10). It is well known that HGF promotes tumor progression (13, 14, 15, 16, 17) and suppresses development of fibrosis after injury (18, 19, 20). In contrast, the role of HGF in immune-mediated disorders such as asthma remains unclear. HGF promotes adhesion and migration of B cells (21, 22) and T cells (23) as well as DC migration (24, 25). HGF frequently counteracts TGF-β, a potent immunosuppressive cytokine (18, 19, 26). These results indicate that HGF might be an immunopotentiator. In contrast, in the mouse model of allogenic heart transplantation, HGF reduces acute and chronic rejection of allograft with the increased expression of TGF-β and IL-10, indicating that HGF might induce allograft tolerance (27).

So far, the role of HGF in Ag-specific immune response has rarely been elucidated. In the present study, we examined the role of HGF in allergic airway inflammation using in vitro studies and a mouse model. Treatment with HGF potently suppressed the Ag-presenting capacity of DCs, thus attenuating not only Th1- and Th2-type immune responses induced by Ag sensitization, but also allergic airway inflammation induced by Ag inhalation. We also confirmed that endogenous HGF played some protective role in the mouse model of allergic airway inflammation.

Male BALB/c mice 6 wk of age were obtained from Charles River Breeding Laboratories. Male BALB/c TCR-transgenic DO11.10 mice age 6–8 wk that recognize the OVA323–339 peptide were purchased from The Jackson Laboratory. They were maintained under conventional animal housing conditions in a specific pathogen-free setting. All of the animal experiments conducted in this study were approved by the Animal Research Ethics Board of the Department of Allergy and Rheumatology (University of Tokyo, Tokyo, Japan).

Concentrations of mouse IL-4, IL-5, IL-10, IL-12p70, IFN-γ, IgE (BD Pharmingen), IgG (Bethyl Laboratories), IL-13, and TGF-β (R&D Systems) were measured using the ELISA kit, following the manufacturer’s protocol. OVA-specific IgE and IgG in sera were measured as reported previously (28). HGF concentration was examined using a rat HGF ELISA kit (Institute of Immunology, Tokyo, Japan). Cell proliferation was measured by BrdU incorporation using a BrdU cell proliferation ELISA kit (Roche). The data were analyzed with Microplate Manager III, version 1.45 (Bio-Rad).

Throughout the present study, complete DMEM was used as the medium for cell incubation as we previously reported (28). Cells were incubated in a 96-well flat-bottom microtiter assay plate in an incubator (37°C, 5% CO2, 90% humidity) for the indicated duration. Some DCs were treated with mitomycin C (10 μg/ml; Sigma-Aldrich) for 35 min in the incubator to inhibit proliferation of DCs themselves.

Single cell suspensions of spleen and lung cells were prepared as reported previously (28).

Mouse splenic CD4+ T cells were obtained using anti-mouse CD4 colloidal superparamagnetic microbeads (Miltenyi Biotec), as previously described (28, 29, 30). The purity of CD4+ cells, confirmed by the flow cytometry, was >95%. To obtain a high purity in DCs, we first isolated spleen or lung DCs by discontinuous density gradient centrifugation using OptiPrep (Miltenyi Biotec) essentially following the manufacturer’s protocol (31). Then, after incubation with anti-mouse CD16/CD32 Ab (BD Pharmingen) to block Fc receptor-mediated magnetic labeling of macrophages, DCs were obtained with anti-mouse CD11c microbeads (Miltenyi Biotec) from the enriched cells obtained by the centrifugation. The purity of spleen and lung CD11c+ cells, confirmed by the flow cytometry, was >90% and ∼85%, respectively. There was no significant difference in the purity among each group of mice in every experiment.

RNA was extracted from CD4+ T cells and DCs obtained from spleens of naive mice. RT-PCR for c-met was conducted, and the products were electrophoresed and were visualized as previously reported (16).

Splenic DCs were positively selected from the spleen cells of naive mice. To examine the effect of HGF on the Ag presentation of DCs, the DCs were incubated with OVA (1000 μg/ml, grade V; Sigma-Aldrich), with or without recombinant human (rh)HGF (Sigma-Aldrich) in the medium, at several concentrations (1, 10, 30, and 100 ng/ml) for 24 h. Some DCs treated with 30 ng/ml rhHGF received additional treatment with anti-human HGF neutralizing Ab (100 μg/ml; R&D Systems). The DCs were washed with HBSS four times, and were treated with mitomycin C. Then, after four times washing with HBSS, DCs (1 × 105 cells/ml) were cocultured with CD4+ T cells obtained from DO11.10 mice (1 × 106 cells/ml). After 3 days of coculture, cell proliferation was measured by BrdU incorporation.

Plasmid pCAGGS-HGF was constructed as previously described (32). The control plasmid was constructed without insertion of the rat HGF cDNA. Plasmids were amplified and purified as reported (32).

We delivered the HGF gene i.v. using hydrodynamic-based naked plasmid delivery as previously reported (33). The HGF expression plasmid pCAGGS-HGF (100 μg), which was dissolved in Ringer’s solution (2.5 ml), was rapidly injected into the tail vein of mice. To examine the effect of pCAGGS-HGF injection, pCAGGS-HGF was injected into the naive mice on day 0, and the sera and lung samples were obtained from the pCAGGS-HGF-treated mice on days 1, 3, 5, and 7. Lung extracts were obtained following the manufacturer’s protocol.

Throughout the present study, BALB/c mice were sensitized with 2 μg of OVA in 2 mg of alum (OVA/alum; Serva) i.p. on days 0 and 11. Saline control mice received i.p. saline injections on days 0 and 11.

The OVA-sensitized mice received injections of pCAGGS-HGF, control plasmid, or Ringer’s solution on days −1 and 10. The saline control mice received i.v. administration of Ringer’s solution on days −1 and 10. On day 18, DCs were obtained from spleen cells of each group of mice. To examine cytokine production, DCs (1 × 106 cells/ml) were incubated with LPS (1 μg/ml; Sigma-Aldrich) for 2 days, and IL-10 and IL-12p70 production was measured by ELISA. To examine the Ag-presenting capacity, DCs (1 × 105 cells/ml) from each group of mice were treated with mitomycin C, and then cocultured with CD4+ T cells (1 × 106 cells/ml) obtained from the OVA-sensitized mice under OVA stimulation (1000 μg/ml). We also examined the Ag-presenting capacity of DCs using CD4+ T cells obtained from DO11.10 mice under stimulation with a lower OVA concentration (10 μg/ml), instead of CD4+ T cells isolated from OVA-sensitized mice. On day 3, cell proliferation was measured.

To examine the effect of HGF on the early stage of Ag-induced immune responses, the serum and single cell suspension of spleen from each group of mice were obtained on day 18. The IgG and IgE concentration in the sera was measured. Spleen cells (5 × 106 cells/ml) were cultured with OVA, and cell proliferation after 3 days of incubation with several concentrations of OVA (10, 100, and 1000 μg/ml) and cytokine concentration after 4 days of incubation with OVA (10 μg/ml) in the supernatant were measured.

On day 18, CD4+ T cells were obtained from the OVA-sensitized mice. Then CD4+ T cells (1 × 106 cells/ml) were stimulated with PMA (1 ng/ml; Sigma-Aldrich) and ionomycin (0.1 μg/ml; Sigma-Aldrich) with or without rhHGF (30 ng/ml). Some of the CD4+ T cells treated with rhHGF also received additional treatment with anti-human HGF neutralizing Ab (100 μg/ml). After 2 days of culture, cell proliferation and cytokine concentrations in the supernatants were measured.

Mice were sensitized with OVA/alum and received injection of HGF expression plasmid, control plasmid, or the vehicle on day 17. Mice were then challenged with 3% w/v OVA aerosol in PBS delivered by nebulizer for 10 min every day on days 18–20. Saline control mice received the vehicle injection on day 17 and PBS inhalation on days 18–20. On day 21, AHR to methacholine chloride (Mch; Sigma-Aldrich) was measured by the enhanced pause (Penh; Buxco Electronics) system, as reported previously (28, 34, 35, 36). In brief, physiologic saline and increasing concentrations of Mch were delivered by the nebulizer for 2.5 min. One minute after each inhalation, Penh was recorded for 2.5 min. Recorded Penh was averaged approximately every 5 s, and the cumulative values were averaged and expressed as Penh for each time point. AHR was examined by the change in Penh to increasing concentrations of Mch. We also used the provocation concentration of Mch (PC100Mch, mg/ml), which induced a 100% increase in Penh over the baseline value, as an indicator of AHR as previously described (28, 35). After measuring AHR, bronchoalveolar lavage fluid (BALF) and lung tissues were obtained as previously reported (28, 34, 35). The cell count and cell differentials in BALF were also examined. Cytokine concentrations in BALF were measured by ELISA.

To examine the effect of HGF on DCs in the lung, DCs from the lung were obtained on day 21 using OptiPrep as previously described. After mitomycin C treatment, lung DCs were cocultured (1 × 105 cells/ml) with CD4+ T cells (1 × 106 cells/ml) selected from the spleen of DO11.10 mice. After 3 days of coculture, cell proliferation was measured. To examine the effect of in vivo treatment with pCAGGS-HGF on APC migration, after sensitization with OVA/alum and treatment with the plasmid on day 17, mice received 80 μl of the FITC-OVA solution (10 mg/ml; Molecular Probes) intratracheally on day 18. Paratracheal and parathymic lymph nodes were extracted 24 h after the intratracheal instillation of FITC-OVA and were then examined under fluorescence microscopy (37).

The OVA-sensitized mice were challenged with 3% w/v OVA aerosol on days 18–22 and on day 26. Control saline mice received PBS aerosol on days 18–22 and on day 26. On days 18 (before OVA inhalation challenge), 21 (after 3 days of OVA inhalation challenges), and 27 (after 6 days of OVA inhalation challenges) we obtained BALF, blood samples, and lung tissues. HGF concentrations in the BALF and the sera were examined by ELISA. Immunohistochemical studies for HGF expression in the lung were also performed as previously reported, with a slight modification using anti-mouse HGFα Ab (rabbit polyclonal IgG; Santa Cruz Biotechnology) instead of anti-rhHGF sheep serum (20).

To examine the role of endogenous HGF, some of the OVA-sensitized mice received i.p. injections of 250 μg of anti-rat HGF neutralizing Ab or control normal rabbit IgG just before each OVA aerosol challenge on days 18–20. Anti-rat HGF neutralizing Ab was obtained as previously described (38). We have previously confirmed that the anti-rat HGF neutralizing Ab cross-reacts with mouse HGF and that treatment of mice with this Ab is effective for neutralizing endogenous HGF present in the lung (38, 39). On day 21, after measuring AHR, we obtained BALF samples and lung tissues from each mouse. The effect of neutralization of endogenous HGF on Ag-presenting capacity of DCs in the lung was also examined as previously discussed.

Values are expressed as mean ± SEM. Data were evaluated using one-way ANOVA followed by the Student t test for comparison between two groups. Values of p < 0.05 were considered to be significant.

To investigate the effect of HGF on DC function, we first examined the expression of c-met, a receptor for HGF, in DCs. DCs and CD4+ T cells were positively selected from the spleens of naive mice, and RNA was then also extracted. We confirmed that DCs expressed c-met messenger RNA, which T cells did not express as previously reported (23) (Fig. 1,A). Then we examined the effect of in vitro treatment with HGF on DC function. DCs obtained from naive mice were incubated using OVA with or without rhHGF. After mitomycin treatment, DCs were cocultured with CD4+ T cells positively selected from the spleens of TCR-transgenic DO11.10 mice, which recognize the OVA323–339 peptide presented on APCs. HGF treatment significantly suppressed the Ag-presenting capacity, with the maximal effect at a concentration of 30 ng/ml (Fig. 1,B). This suppressive effect of HGF on the Ag-presenting capacity of DCs was completely reversed with additional treatment by anti-human HGF neutralizing Ab (Fig. 1 C). We confirmed that HGF significantly reduced CD40 expression on DCs, which was also recovered by additional treatment with anti-HGF Ab, whereas HGF did not affect MHC class II, CD80, or CD86 expression on DCs (data not shown). We also examined the effect of HGF on the phagocytosis of DCs using FITC-conjugated dextran, but could not find any effect (data not shown). These results demonstrated that HGF directly suppressed the Ag presentation of DCs after the phagocytosis of an Ag, and that this effect might be partly mediated by down-regulation of CD40 expression. We next examined the effect of in vivo treatment with HGF on DC functions.

FIGURE 1.

Treatment with HGF in vitro potently suppresses the Ag-presenting capacity of DCs. A, DCs express c-met (receptor for HGF) mRNA, which CD4+ T cells do not express. RNA was extracted from CD4+ T cells and DCs obtained from spleens of naive mice, and RT-PCR for c-met was conducted. B and C, In vitro treatment with HGF significantly suppresses Ag-presenting capacity of DCs. B, DCs obtained from the spleen cells of the naive mice were pulsed with OVA (1000 μg/ml) for 24 h with or without (−) rhHGF at several concentrations. C, Some of the OVA-pulsed DCs treated with HGF (30 ng/ml) were also treated with anti-human HGF neutralizing Ab (100 μg/ml). Then, DCs were cocultured with CD4+ T cells obtained from the DO11.10 mice. After 3 days of coculture, cell proliferation was measured. The DC to CD4+ T cell ratio was 1:10 (B). Data were obtained from four wells per group of mice in B and C. Data are expressed as a percentage of the response compared with that induced by DCs without HGF treatment at the DC to CD4+ T cell ratio of 1:10 (B and C). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs DCs without HGF treatment). +, p < 0.05; ++, p < 0.01; +++, p < 0.001 (vs DCs treated with 30 ng/ml HGF).

FIGURE 1.

Treatment with HGF in vitro potently suppresses the Ag-presenting capacity of DCs. A, DCs express c-met (receptor for HGF) mRNA, which CD4+ T cells do not express. RNA was extracted from CD4+ T cells and DCs obtained from spleens of naive mice, and RT-PCR for c-met was conducted. B and C, In vitro treatment with HGF significantly suppresses Ag-presenting capacity of DCs. B, DCs obtained from the spleen cells of the naive mice were pulsed with OVA (1000 μg/ml) for 24 h with or without (−) rhHGF at several concentrations. C, Some of the OVA-pulsed DCs treated with HGF (30 ng/ml) were also treated with anti-human HGF neutralizing Ab (100 μg/ml). Then, DCs were cocultured with CD4+ T cells obtained from the DO11.10 mice. After 3 days of coculture, cell proliferation was measured. The DC to CD4+ T cell ratio was 1:10 (B). Data were obtained from four wells per group of mice in B and C. Data are expressed as a percentage of the response compared with that induced by DCs without HGF treatment at the DC to CD4+ T cell ratio of 1:10 (B and C). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs DCs without HGF treatment). +, p < 0.05; ++, p < 0.01; +++, p < 0.001 (vs DCs treated with 30 ng/ml HGF).

Close modal

Generally, exogenously administered HGF proteins vanish very rapidly from organs within several hours (40), so to deliver HGF effectively we used a hydrodynamic-based transfer system (33) with the rat HGF expression plasmid pCAGGS-HGF. We confirmed that the up-regulation of HGF protein peaked on day 1 both in the sera (Fig. 2,A) and in the lung (Fig. 2 B). Based on these data, the time schedule of plasmid administration was determined for the following experiments.

FIGURE 2.

Hydrodynamic-based delivery of HGF expression plasmid induces up-regulation of HGF in the serum and lung. Naive mice received hydrodynamic-based injections of HGF expression plasmid or control plasmid on day 0. On the indicated days, mice were sacrificed, and HGF concentration was measured. A, HGF in the serum. B, HGF in lung extracts. Data were obtained from three animals per group of mice. #, p < 0.05; ###, p < 0.001 (vs mice that received control plasmid).

FIGURE 2.

Hydrodynamic-based delivery of HGF expression plasmid induces up-regulation of HGF in the serum and lung. Naive mice received hydrodynamic-based injections of HGF expression plasmid or control plasmid on day 0. On the indicated days, mice were sacrificed, and HGF concentration was measured. A, HGF in the serum. B, HGF in lung extracts. Data were obtained from three animals per group of mice. #, p < 0.05; ###, p < 0.001 (vs mice that received control plasmid).

Close modal

We proceeded to examine the effect of in vivo treatment with HGF on DC functions following a previously reported method using a slight modification (28). It is well known that sensitization with OVA/alum induces allergic immune response (5, 28, 34, 35). Mice were sensitized with OVA/alum on days 0 and 11, and HGF expression plasmid, control plasmid, or the vehicle Ringer’s solution was administered to the mice on days −1 and 10. On day 18, DCs were positively selected from the spleens of each group of mice, and the OVA-Ag-presenting capacity and cytokine production of DCs after LPS stimulation were examined. Treatment with the HGF expression plasmid during sensitization significantly suppressed OVA-Ag presentation of the DCs (Fig. 3, A and B). Treatment with HGF in vivo also suppressed IL-12p70 production of DCs after LPS stimulation (Fig. 3,C), without affecting IL-10 production of DCs (Fig. 3 D). These results clearly demonstrated that HGF treatment in vivo significantly suppressed DC functions that were activated by Ag sensitization. We therefore examined whether in vivo treatment with HGF could suppress B and T cell activation induced by Ag sensitization.

FIGURE 3.

Treatment with HGF in vivo significantly down-regulates DC functions. Mice were sensitized with OVA/alum on days 0 and 11, and treated with HGF expression plasmid (OVA/HGF), control plasmid (OVA/Cont), or the vehicle Ringer’s solution (OVA) on days −1 and 10. Saline control mice received saline injections on days 0 and 11, and Ringer’s solution on days −1 and 10 (saline). On day 18, DCs were positively selected from spleen cells of each group of mice, and their functions were examined. A and B, Ag-presenting capacity of DCs. DCs were cocultured under OVA stimulation with CD4+ T cells from OVA-sensitized mice (A) or those from DO11.10 mice (B) for 3 days, and cell proliferation was measured. Data are expressed as a percentage of the response compared with that induced by DCs from the OVA mice. C and D, Cytokine profile of DCs from each group of mice under LPS stimulation. DCs were stimulated with LPS (1 μg/ml) for 2 days, and IL-12p70 (C) and IL-10 (D) concentrations in the supernatants were measured. Data were obtained from three wells per group of mice. #, p < 0.05; ###, p < 0.001 (vs DCs obtained from saline mice). ∗, p < 0.05; ∗∗, p < 0.01 (vs DCs from OVA/Cont mice).

FIGURE 3.

Treatment with HGF in vivo significantly down-regulates DC functions. Mice were sensitized with OVA/alum on days 0 and 11, and treated with HGF expression plasmid (OVA/HGF), control plasmid (OVA/Cont), or the vehicle Ringer’s solution (OVA) on days −1 and 10. Saline control mice received saline injections on days 0 and 11, and Ringer’s solution on days −1 and 10 (saline). On day 18, DCs were positively selected from spleen cells of each group of mice, and their functions were examined. A and B, Ag-presenting capacity of DCs. DCs were cocultured under OVA stimulation with CD4+ T cells from OVA-sensitized mice (A) or those from DO11.10 mice (B) for 3 days, and cell proliferation was measured. Data are expressed as a percentage of the response compared with that induced by DCs from the OVA mice. C and D, Cytokine profile of DCs from each group of mice under LPS stimulation. DCs were stimulated with LPS (1 μg/ml) for 2 days, and IL-12p70 (C) and IL-10 (D) concentrations in the supernatants were measured. Data were obtained from three wells per group of mice. #, p < 0.05; ###, p < 0.001 (vs DCs obtained from saline mice). ∗, p < 0.05; ∗∗, p < 0.01 (vs DCs from OVA/Cont mice).

Close modal

Mice were sensitized with OVA/alum, and the sera and spleen cells were collected from each group of mice on day 18. Treatment with HGF expression plasmid significantly decreased the total (Fig. 4,A) and OVA-specific (Fig. 4,B) IgG and IgE concentration in the sera on day 18, which significantly increased after OVA/alum sensitization, indicating that HGF suppressed Ag-induced B cell activation. To examine the effect of HGF on Ag-induced T cell priming, spleen cells were restimulated with OVA in vitro. Spleen cells obtained from the mice treated with HGF expression plasmid demonstrated significantly reduced cell proliferation (Fig. 4,C) and both Th1- and Th2-type cytokine production, such as IL-5 and IFN-γ (Fig. 4,D) and IL-10 (Fig. 4,E), in response to OVA restimulation. We also confirmed that treatment with HGF expression plasmid exhibited the same suppressive effect on Ag priming even when mice were sensitized with OVA/CFA to induce Th1-biased T cell activation (data not shown). These results clearly demonstrated that HGF treatment significantly suppressed OVA-induced T cell activation. To our knowledge, the c-met-HGF receptor is not expressed in normal T cells (23). We also confirmed that HGF had no direct suppressive effect on the proliferation (Fig. 4,F) and cytokine production (Fig. 4 G) of mouse splenic CD4+ T cells induced by PMA and ionomycin. These results also indicated that HGF would potently suppress Ag-induced T cell activation in an indirect manner.

FIGURE 4.

Treatment with HGF in vivo significantly suppresses Ag-induced Ig production and T cell priming. A–E, Mice were sensitized and treated as described in Fig. 3. On day 18, sera and spleen cells were obtained from each group of mice. A and B, Ig concentrations in the sera. Total (A) and OVA-specific (B) IgG (□) and IgE (▪) concentrations in the sera were examined by ELISA. CE, Spleen cell responses to OVA restimulation in vitro. Spleen cells obtained on day 18 were restimulated in vitro with OVA. C, Cell proliferation was measured after 3 days incubation with indicated concentrations of OVA. C, Data are expressed as a percentage of the response compared with that of spleen cells from OVA mice at OVA (1000 μg/ml). D and E, Cytokine production for IL-5 (□) and IFN-γ (▪) (D), as well as IL-10 (E) was measured by ELISA after 4 days incubation with OVA (10 μg/ml). ##, p < 0.01; ###, p < 0.001 (vs saline mice). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs OVA/Cont mice). F and G, HGF has no direct suppressive effect on CD4+ T cells. CD4+ T cells were obtained from OVA mice on day 18, and were stimulated with PMA and ionomycin with or without rhHGF. Some of CD4+ T cells treated with rhHGF also received additional treatment with anti-human HGF neutralizing Ab. After 2 days of incubation, cell proliferation (F), and IL-5 (□) and IFN-γ (▪) production (G) were measured. F, Data are expressed as a percentage of the response compared with that of CD4+ T cells with PMA and ionomycin stimulation without any treatment. Data were obtained from three to four animals per group of mice described in A and B, and from four wells per group of mice in CG.

FIGURE 4.

Treatment with HGF in vivo significantly suppresses Ag-induced Ig production and T cell priming. A–E, Mice were sensitized and treated as described in Fig. 3. On day 18, sera and spleen cells were obtained from each group of mice. A and B, Ig concentrations in the sera. Total (A) and OVA-specific (B) IgG (□) and IgE (▪) concentrations in the sera were examined by ELISA. CE, Spleen cell responses to OVA restimulation in vitro. Spleen cells obtained on day 18 were restimulated in vitro with OVA. C, Cell proliferation was measured after 3 days incubation with indicated concentrations of OVA. C, Data are expressed as a percentage of the response compared with that of spleen cells from OVA mice at OVA (1000 μg/ml). D and E, Cytokine production for IL-5 (□) and IFN-γ (▪) (D), as well as IL-10 (E) was measured by ELISA after 4 days incubation with OVA (10 μg/ml). ##, p < 0.01; ###, p < 0.001 (vs saline mice). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs OVA/Cont mice). F and G, HGF has no direct suppressive effect on CD4+ T cells. CD4+ T cells were obtained from OVA mice on day 18, and were stimulated with PMA and ionomycin with or without rhHGF. Some of CD4+ T cells treated with rhHGF also received additional treatment with anti-human HGF neutralizing Ab. After 2 days of incubation, cell proliferation (F), and IL-5 (□) and IFN-γ (▪) production (G) were measured. F, Data are expressed as a percentage of the response compared with that of CD4+ T cells with PMA and ionomycin stimulation without any treatment. Data were obtained from three to four animals per group of mice described in A and B, and from four wells per group of mice in CG.

Close modal

We then examined the effect of exogenous administration of HGF during the effector phase in experimental allergic airway inflammation. After sensitization with OVA/alum, mice received injection of HGF expression plasmid, control plasmid, or the vehicle and were challenged with OVA aerosol. Treatment with HGF expression plasmid significantly reduced AHR (Fig. 5,A), eosinophilic lung inflammation (Fig. 5, B and C), and Th2 cytokine production in the lung (Fig. 5,D). It is well known that treatment with HGF in vivo frequently suppresses TGF-β production (18, 19). We also confirmed that treatment with HGF significantly reduced the TGF-β concentration in BALF (Fig. 5 E).

FIGURE 5.

Treatment with HGF in the effector phase significantly attenuates the development of allergic airway inflammation and the Ag-presenting capacity of DCs in the lung. AE, Treatment with HGF significantly attenuates eosinophilic lung inflammation and AHR. Mice were sensitized with OVA on days 0 and 11. On day 17, they received injection of HGF expression plasmid (OVA/HGF/OVA), control plasmid (OVA/Cont/OVA) or the vehicle (OVA/OVA). Then, mice were challenged with OVA aerosol on days 18–20. Saline control mice received saline injections on days 0 and 11, the vehicle injection on day 17, and PBS aerosol challenges on days 18–20 (saline/PBS). On day 21, after measuring AHR, BALF, and lung tissues were obtained. AHR (A) and cell differentials in BALF (B) are shown. C, H&E staining of lung tissues. Scale bars: 100 μm. D, IL-5 (□) and IL-13 (▪) concentrations in BALF. E, TGF-β concentration in BALF. F and G, Treatment with HGF significantly suppresses Ag-presenting capacity of lung DCs, although it does not affect the migration of lung APCs. F, On day 21, DCs in the lung were positively selected, and cocultured with CD4+ T cells from the DO11.10 mice. After 3 days of coculture, cell proliferation was measured. Data are expressed as a percentage of the response compared with that induced by DCs obtained from OVA/OVA mice. G, Mice received the intratracheal instillation of FITC-conjugated OVA instead of OVA aerosol on day 18. One day after, regional lymph nodes were extracted. Cryostat sections (5 μm) were examined under fluorescence microscopy. FITC was expressed as light green (▵). Scale bars: 25 μm. Data were obtained from six animals per group of mice in A, B, D, and E, and from three wells in F. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (vs saline/PBS mice). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs OVA/Cont/OVA mice).

FIGURE 5.

Treatment with HGF in the effector phase significantly attenuates the development of allergic airway inflammation and the Ag-presenting capacity of DCs in the lung. AE, Treatment with HGF significantly attenuates eosinophilic lung inflammation and AHR. Mice were sensitized with OVA on days 0 and 11. On day 17, they received injection of HGF expression plasmid (OVA/HGF/OVA), control plasmid (OVA/Cont/OVA) or the vehicle (OVA/OVA). Then, mice were challenged with OVA aerosol on days 18–20. Saline control mice received saline injections on days 0 and 11, the vehicle injection on day 17, and PBS aerosol challenges on days 18–20 (saline/PBS). On day 21, after measuring AHR, BALF, and lung tissues were obtained. AHR (A) and cell differentials in BALF (B) are shown. C, H&E staining of lung tissues. Scale bars: 100 μm. D, IL-5 (□) and IL-13 (▪) concentrations in BALF. E, TGF-β concentration in BALF. F and G, Treatment with HGF significantly suppresses Ag-presenting capacity of lung DCs, although it does not affect the migration of lung APCs. F, On day 21, DCs in the lung were positively selected, and cocultured with CD4+ T cells from the DO11.10 mice. After 3 days of coculture, cell proliferation was measured. Data are expressed as a percentage of the response compared with that induced by DCs obtained from OVA/OVA mice. G, Mice received the intratracheal instillation of FITC-conjugated OVA instead of OVA aerosol on day 18. One day after, regional lymph nodes were extracted. Cryostat sections (5 μm) were examined under fluorescence microscopy. FITC was expressed as light green (▵). Scale bars: 25 μm. Data were obtained from six animals per group of mice in A, B, D, and E, and from three wells in F. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (vs saline/PBS mice). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (vs OVA/Cont/OVA mice).

Close modal

As we have shown, treatment with HGF significantly suppressed DC functions in the sensitization phase both in vitro and in vivo (Figs. 1 and 3). We then examined the effect of HGF on the Ag presentation of DCs in the effector phase. After 3-day OVA aerosol challenge, DCs were positively selected on day 21 and cocultured with CD4+ T cells obtained from the spleen of naive DO11.10 mice. After 3-day coculture, cell proliferation was measured. Treatment with HGF during OVA aerosol challenges markedly suppressed the OVA-Ag presenting capacity of DCs in the lung (Fig. 5,F). In another experiment, we also confirmed that treatment with HGF exhibited no suppressive effect on APC migration (Fig. 5 G). To clarify whether the suppressive effect of in vivo treatment with HGF on allergic immune response would be exhibited through the cytotoxic effect of HGF on DCs and other immunocytes in the lung, we conducted TUNEL staining of lung tissues of each group obtained on day 21 for detection of apoptotic cells. We did not find any increase in apoptotic cells in lung treated with HGF (data not shown). We also conducted trypan blue staining of incubated cells and confirmed that in vitro treatment with HGF did not increase death of DCs or T cells (data not shown). These results indicated that the suppressive effect of HGF in vivo would not be mediated by the cytotoxic effect.

We next examined whether endogenous HGF actually played some role in the lung of the mouse model of allergic airway inflammation. The OVA-sensitized mice were challenged with OVA aerosol on days 18–22 and day 26. Saline control mice received PBS inhalation. HGF concentrations in BALF and sera after OVA sensitization and inhalation were examined. Saline injections and PBS inhalation did not alter the baseline level of endogenous HGF in either BALF (Fig. 6,A) or sera (Fig. 6,B). In contrast, endogenous HGF production in BALF was slightly increased by OVA sensitization and significantly increased after OVA inhalation (Fig. 6,A), without affecting the serum HGF concentration (Fig. 6,B). Immunohistochemical staining of lung tissues, using an anti-mouse HGFα Ab that recognizes the active form of HGF (10), showed that OVA sensitization alone increased the active form of HGF in the lung interstitium, and that OVA inhalation gradually increased the expression not only in the lung interstitium, but also at the apex of the bronchial epithelium (Fig. 6,C). In peripheral alveolar spaces, positively stained cells were also detected and consisted mainly, morphologically, of monocytes/macrophages (Fig. 6 D).

FIGURE 6.

Endogenous HGF is up-regulated in the lung following OVA sensitization and inhalation. Mice were sensitized with OVA on days 0 and 11, and then received OVA aerosol challenges on days 18–22 and 26. Saline control mice received saline injections on days 0 and 11, and PBS challenges on days 18–22 and 26. On days 18 (before inhalation), 21 (after 3 days of inhalation), and 27 (24 h after the final inhalation), mice were sacrificed, and samples were obtained, respectively. A and B, HGF concentrations were measured using an ELISA kit. A, BALF. B, Serum. Data were obtained from four to six animals per each group of mice. ###, p < 0.001 (vs saline mice). C and D, Immunohistochemical analysis of lung tissues. Lung tissues were stained using anti-mouse HGFα Ab, which recognizes the active form of HGF, and was expressed as blue staining. C, Positive staining was observed in the lung interstitium (▵) on days 18, 21, and 27, and in the apex of the bronchial epithelium (▴) on days 21 and 27. Scale bars, 50 μm. D, A representative lung section (right) from the OVA mice on day 27 is shown. In peripheral alveolar spaces, positively stained cells (←) mainly consisted of, morphologically, monocytes/macrophages. In contrast, positively stained cells were rarely detected in peripheral alveolar spaces of saline control mice (left). Scale bars, 5 μm.

FIGURE 6.

Endogenous HGF is up-regulated in the lung following OVA sensitization and inhalation. Mice were sensitized with OVA on days 0 and 11, and then received OVA aerosol challenges on days 18–22 and 26. Saline control mice received saline injections on days 0 and 11, and PBS challenges on days 18–22 and 26. On days 18 (before inhalation), 21 (after 3 days of inhalation), and 27 (24 h after the final inhalation), mice were sacrificed, and samples were obtained, respectively. A and B, HGF concentrations were measured using an ELISA kit. A, BALF. B, Serum. Data were obtained from four to six animals per each group of mice. ###, p < 0.001 (vs saline mice). C and D, Immunohistochemical analysis of lung tissues. Lung tissues were stained using anti-mouse HGFα Ab, which recognizes the active form of HGF, and was expressed as blue staining. C, Positive staining was observed in the lung interstitium (▵) on days 18, 21, and 27, and in the apex of the bronchial epithelium (▴) on days 21 and 27. Scale bars, 50 μm. D, A representative lung section (right) from the OVA mice on day 27 is shown. In peripheral alveolar spaces, positively stained cells (←) mainly consisted of, morphologically, monocytes/macrophages. In contrast, positively stained cells were rarely detected in peripheral alveolar spaces of saline control mice (left). Scale bars, 5 μm.

Close modal

Finally, we examined the effect of abrogation of endogenous HGF on allergic airway inflammation. In general, deficiency of HGF gene is lethal (10), so we used anti-HGF neutralizing Ab for abrogation of endogenous HGF. Considering that the active form of HGF expression in the lung increased strongly after OVA inhalation (Fig. 6,C), treatment of mice with anti-rat HGF neutralizing Ab was performed on days 18–20. Neutralization of endogenous HGF significantly enhanced AHR (Fig. 7, A and B) and aggravated eosinophilic lung inflammation (Fig. 7, C and D). We also examined the effect of neutralization of endogenous HGF on DC function in the lung. On day 21, lung DCs were obtained, and OVA-presenting capacity of lung DCs was measured. Neutralization of endogenous HGF significantly reinforced the Ag-presenting capacity of DCs in the lung (Fig. 7 E). These results confirmed that HGF actually played an immune-protective role in vivo in the experimental model of allergic airway inflammation with suppression of DC function.

FIGURE 7.

Abrogation of endogenous HGF significantly increases AHR and eosinophilic lung inflammation with enhancing Ag-presenting capacity of lung DCs. The OVA-sensitized mice received anti-rat HGF neutralizing Ab (OVA/anti-HGF/OVA) or control normal rabbit IgG (OVA/Cont IgG/OVA) just before each OVA aerosol challenge on days 18–20. Saline control mice received saline injections on days 0 and 11, and PBS challenges on days 18–20. On day 21, after measuring AHR, BALF cells, lung samples, and DCs in the lung were obtained. A and B, AHR was examined using the percentage of Penh compared with the baseline value (A), or using PC100Mch (B) as described in Materials and Methods. C, Cell differentials in BALF. D, H&E staining of lung tissues. Scale bars: 100 μm. E, Ag-presenting capacity of DCs in the lung. OVA Ag-presenting capacity of lung DCs were measured as described in Fig. 5 F. Data are expressed as a percentage of the response compared with that induced by DCs obtained from OVA/OVA mice. Data were obtained from five animals per group of mice in AC, and from three wells in E. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (vs saline/PBS mice). ∗, p < 0.05; ∗∗, p < 0.01 (vs mice treated with control normal rabbit IgG).

FIGURE 7.

Abrogation of endogenous HGF significantly increases AHR and eosinophilic lung inflammation with enhancing Ag-presenting capacity of lung DCs. The OVA-sensitized mice received anti-rat HGF neutralizing Ab (OVA/anti-HGF/OVA) or control normal rabbit IgG (OVA/Cont IgG/OVA) just before each OVA aerosol challenge on days 18–20. Saline control mice received saline injections on days 0 and 11, and PBS challenges on days 18–20. On day 21, after measuring AHR, BALF cells, lung samples, and DCs in the lung were obtained. A and B, AHR was examined using the percentage of Penh compared with the baseline value (A), or using PC100Mch (B) as described in Materials and Methods. C, Cell differentials in BALF. D, H&E staining of lung tissues. Scale bars: 100 μm. E, Ag-presenting capacity of DCs in the lung. OVA Ag-presenting capacity of lung DCs were measured as described in Fig. 5 F. Data are expressed as a percentage of the response compared with that induced by DCs obtained from OVA/OVA mice. Data were obtained from five animals per group of mice in AC, and from three wells in E. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (vs saline/PBS mice). ∗, p < 0.05; ∗∗, p < 0.01 (vs mice treated with control normal rabbit IgG).

Close modal

In this study, we clearly demonstrated a role of HGF as an immune regulator. So far, the role of HGF in immune-mediated disorders such as asthma has never been clarified. The current study clarified that HGF potently suppressed Ag-induced immune response through inhibition of DCs.

Ag presentation from APCs initiates T cell activation and differentiation. Among APCs, DCs are the most potent and professional APCs. Therefore, DCs play a central role and are a logical target in many clinical situations that involve T cells including asthma (1, 2). Recently, some studies emphasized that DCs are crucial in asthma pathogenesis beyond Ag sensitization, and that DCs could be a therapeutic target for asthma (5, 6). In the present study, HGF potently suppressed the Ag-presenting capacity of DCs, thus reducing an Ag-induced immune response. Our results indicate that HGF may be very beneficial for treating asthma through modulating DC function.

Contrary to our results, one report has previously noted that HGF treatment in vitro had no effect on the Ag presentation of DCs (24). The exact reason for the discrepancy between the two reports remains unclear, but possible reasons may include the differences in the origin of DCs, the timing of the HGF treatment, or the incubation time of the cells between the two reports. Moreover, LPS contamination in OVA might also affect the biological effect of HGF. One group reported that LPS increased procoagulant activity of DCs (41), and another that activated procoagulation would increase the biological effect of HGF (42, 43). We measured LPS concentration in 0.1% w/v OVA solved in sterile physiologic saline (1000 μg/ml OVA solution), and found the concentration to be ∼10 ng/ml. The inevitable LPS contamination would play some role in activating HGF and enhancing its biological effect. Further, the LPS contamination in the OVA solution would also increase the proliferation of spleen cells obtained from saline control mice in response to OVA (Fig. 4 C).

In general, HGF exhibits its multiple biological effects through its receptor, c-met (10). However, it has been previously reported that HGF could promote adhesion and migration of T cells that did not express c-met (23). In the present study, we confirmed that T cells did not express c-met and that HGF had no direct, at least suppressive, effect on T cells. Our findings indicate that HGF potently suppressed Ag-induced T cell activation indirectly, probably through down-regulating DC function.

Allergic asthma has been regarded as a Th2-mediated disorder (3). Its incidence is steadily increasing (4). Hygiene hypothesis has been proposed to explain the recent increase in allergic disease including asthma (44). This hypothesis explains that an increase in asthma, a Th2-mediated disorder, is due to decrease in the opportunity of infections that induce Th1-type immune response, and that induction of Th1-type immune response could inhibit allergic asthma. However, this Th1/Th2 balance theory is controversial. In clinical situations, we frequently find that Th1-type immune response such as viral infection induces an exacerbation of asthma. Some studies emphasized the importance of Th1-type cells in the development of Th2-type immune responses (45, 46, 47). Moreover, in asthmatic airways, both Th1 and Th2 cells increased (48). These results indicate that suppression of not only Th2 but also an enhanced Th1-type response would be important for treating asthma. In the present study, HGF potently suppressed Ag-induced Th1-type immune responses (Fig. 4 D and data not shown). Our findings show that HGF might be effective in preventing asthma exacerbation preceded by viral infection as well as suppressing Ag-induced Th2-type immune response. Moreover, our results suggested that HGF could be beneficial to suppress immune-mediated disorders other than asthma. Further study should be conducted to extend these findings.

TGF-β is a multifunctional growth factor. The major roles of TGF-β are suppression of immune responses (49) and promotion of fibrosis (50). It is well recognized that HGF frequently counteracts TGF-β, which may be one mechanism of suppression of fibrosis by HGF (18, 19). However, Yamamura et al. (27) recently reported that HGF suppressed acute and chronic rejection in a mouse model of cardiac allograft transplantation with unexpectedly enhanced mRNA expression of TGF-β and IL-10 in cardiac allograft. IL-10 is also regarded as an immunosuppressive cytokine, and both TGF-β and IL-10 produced from APCs can induce Treg cells (2, 49, 51). Treg cells potently suppress immune responses and play a central role in the maintenance of tolerance (51). Treg cells also produce TGF-β and IL-10 to suppress immune responses (51). So, the results of the recent study suggested that HGF might reduce rejection of allograft heart transplantation through induction of Treg cells and allograft tolerance (27). In contrast, in the present study, we confirmed that treatment with HGF during the sensitization phase did not increase mRNA expression of TGF-β in CD4+ T cells (data not shown) and that treatment with HGF during the effector phase suppressed TGF-β production in the lung (Fig. 5,E). We also found that treatment with HGF did not increase IL-10 production either from spleen cells after in vitro OVA restimulation (Fig. 4 E) or in the lung after OVA aerosol challenges (data not shown). Thus, HGF would exhibit its suppressive effect without induction of TGF-β or IL-10-producing Treg cells at least in the present study, although the possibility of induction of Treg cells and tolerance by HGF could not be denied.

We also examined the role of endogenous HGF in the mouse model of allergic airway inflammation. HGF production in the lung significantly increased following Ag sensitization and inhalation challenges (Fig. 6), and neutralization of endogenous HGF during OVA inhalation significantly increased AHR and eosinophilic lung inflammation with enhanced Ag-presenting capacity of DCs in the lung (Fig. 7). These results suggested that endogenous HGF might play some protective role in the pathophysiology of asthma. HGF is a mesenchymal-derived growth factor (10). The results of immunohistochemical staining revealed that expression of the active form of HGF increased after Ag sensitization and inhalation challenges, and that interstitial cells and macrophages would be a major cell source for production of HGF. HGF is secreted as a single chain (pro-HGF) biologically inert precursor, and under appropriate conditions such as tissue damage, pro-HGF is converted by proteolytic digestion to its bioactive form consisting of α and β chains (10). This proteolytic digestion is mediated by a coagulation system (42, 43). Researchers have recently reported that coagulation system activation in the lung occurred in the mouse model of allergic airway inflammation (52, 53). Activation of HGF might therefore be associated with activation of the coagulation system.

In a clinical situation, one major problem of chronic asthma is irreversible fibrotic changes in the airway tissue induced by repeated immune responses in the lung (54). Considering our results showing that HGF potently suppressed the Ag-specific airway immune response and that HGF has a well-established antifibrotic property, HGF may be very beneficial for treating chronic asthma. Moreover, HGF could be beneficial in suppressing many immune-mediated disorders with tissue inflammation and subsequent fibrosis beyond asthma. In contrast, with clinical use of HGF, the possibility of promoting tumor progression should be considered. Therefore, for practical use of HGF in clinical situations, further studies should be performed.

We thank I. Makino and K. Kurosaki for technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported in part by research Grant No. 13670592 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

3

Abbreviations used in this paper: Treg, regulatory T; AHR, airway hyperresponsiveness; BALF, bronchoalveolar lavage fluid; DC, dendritic cell; HGF, hepatocyte growth factor; Mch, methacholine chloride; Penh, enhanced pause; rh, recombinant human.

1
Banchereau, J., R. M. Steinman.
1998
. Dendritic cells and the control of immunity.
Nature
392
:
245
.-252.
2
Kapsenberg, M. L..
2003
. Dendritic-cell control of pathogen-driven T-cell polarization.
Nat. Rev. Immunol.
3
:
984
.-993.
3
Holt, P. G., C. Macubas, P. A. Stumbles, P. D. Sly.
1999
. The role of allergy in the development of asthma.
Nature
402
:
B12
.-B17.
4
World Health Organization.
2000
.
Bronchial Asthma Fact Sheet no. 206
WHO, Geneva.
5
Lambrecht, B. N., H. Hammad.
2003
. Taking our breath away: dendritic cells in the pathogenesis of asthma.
Nat. Rev. Immunol.
3
:
994
.-1003.
6
Lambrecht, B. N., B. Salomon, D. Klatzmann, R. A. Pauwels.
1998
. Dendritic cells are required for the development of chronic eosinophilic airway inflammation in response to inhaled antigen in sensitized mice.
J. Immunol.
160
:
4090
.-4097.
7
Nakamura, T., T. Nishizawa, M. Hagiya, T. Seki, M. Shimonishi, A. Sugimura, K. Tashiro, S. Shimizu.
1989
. Molecular cloning and expression of human hepatocyte growth factor.
Nature
342
:
440
.-443.
8
Miyazawa, K., H. Tsubouchi, D. Naka, K. Takahashi, M. Okigaki, N. Arakaki, H. Nakayama, S. Hirono, O. Sakiyama, K. Takahashi, et al
1989
. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor.
Biochem. Biophys. Res. Commun.
163
:
967
.-973.
9
Stoker, M., E. Gherardi, M. Perryman, J. Gray.
1987
. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility.
Nature
327
:
239
.-242.
10
Zarnegar, R., G. K. Michalopoulos.
1995
. The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis.
J. Cell Biol.
129
:
1177
.-1180.
11
Bardelli, A., P. Longati, D. Albero, S. Goruppi, C. Schneider, C. Ponzetto, P. M. Comoglio.
1996
. HGF receptor associates with the anti-apoptotic protein BAG-1 and prevents cell death.
EMBO J.
15
:
6205
.-6212.
12
Huh, C. G., V. M. Factor, A. Sanchez, K. Uchida, E. A. Conner, S. S. Thorgeirsson.
2004
. Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair.
Proc. Natl. Acad. Sci. USA
101
:
4477
.-4482.
13
Trusolino, L., P. M. Comoglio.
2002
. Scatter-factor and semaphoring receptors: cell signaling for invasive growth.
Nat. Rev. Cancer
2
:
289
.-300.
14
Imaizumi, Y., H. Murota, S. Kanda, Y. Hishikawa, T. Koji, T. Taguchi, Y. Tanaka, Y. Yamada, S. Ikeda, T. Kohno, et al
2003
. Expression of the c-Met proto-oncogene and its possible involvement in liver invasion in adult T-cell leukemia.
Clin. Cancer Res.
9
:
181
.-187.
15
Siegfried, J. M., L. A. Weissfeld, P. Singh-Kaw, R. J. Weyant, J. R. Testa, R. J. Landreneau.
1997
. Association of immunoreactive hepatocyte growth factor with poor survival in resectable non-small cell lung cancer.
Cancer Res.
57
:
433
.-439.
16
To, Y., M. Dohi, K. Matsumoto, R. Tanaka, A. Sato, K. Nakagome, T. Nakamura, K. Yamamoto.
2002
. A two-way interaction between hepatocyte growth factor and interleukin-6 in tissue invasion of lung cancer cell line.
Am. J. Respir. Cell Mol. Biol.
27
:
220
.-226.
17
Wislez, M., N. Rabbe, J. Marchal, B. Milleron, B. Crestani, C. Mayaud, M. Antoine, P. Soler, J. Cadranel.
2003
. Hepatocyte growth factor production by neutrophils infiltrating bronchoalveolar subtype pulmonary adenocarcinoma: role in tumor progression and death.
Cancer Res.
63
:
1405
.-1412.
18
Mizuno, S., T. Kurosawa, K. Matsumoto, Y. Mizuno-Horikawa, M. Okamoto, T. Nakamura.
1998
. Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease.
J. Clin. Invest.
101
:
1827
.-1834.
19
Ueki, T., Y. Kaneda, H. Tsutsui, K. Nakanishi, Y. Sawa, R. Morishita, K. Matsumoto, T. Nakamura, H. Takahashi, E. Okamoto, J. Fujimoto.
1999
. Hepatocyte growth factor gene therapy of liver cirrhosis in rats.
Nat. Med.
5
:
226
.-230.
20
Dohi, M., T. Hasegawa, K. Yamamoto, B. C. Marshall.
2000
. Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary fibrosis.
Am. J. Respir. Crit. Care Med.
162
:
2302
.-2307.
21
van der Voort, R., T. E. I. Taher, R. M. J. Keehnen, L. Smit, M. Groenink, S. T. Pals.
1997
. Paracrine regulation of germinal center B cell adhesion through the c-Met-hepatocyte growth factor/scatter factor pathway.
J. Exp. Med.
185
:
2121
.-2131.
22
Weimar, I. S., D. de Jong, E. J. Muller, T. Nakamura, J. M. van Gorp, G. C. de Gast, W. R. Gerritsen.
1997
. Hepatocyte growth factor/scatter factor promotes adhesion of lymphoma cells to extracellular matrix molecules via α4β1 and α5β1 integrins.
Blood
89
:
990
.-1000.
23
Adams, D. H., L. Harvath, D. P. Bottaro, R. Interrante, G. Catalano, Y. Tanaka, A. Strain, S. G. Hubscher, S. Shaw.
1994
. Hepatocyte growth factor and macrophage inflammatory protein 1b: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.
Proc. Natl. Acad. Sci. USA
91
:
7144
.-7148.
24
Kurz, S. M., S. S. Diebold, T. Hieronymus, T. C. Gust, P. Bartunek, M. Sachs, W. Birchmeier, M. Zenke.
2002
. The impact of c-met/scatter factor receptor on dendritic cell migration.
Eur. J. Immunol.
32
:
1832
.-1838.
25
Scarpino, S., A. Stoppacciaro, F. Ballerini, M. Marchesi, M. Prat, M. C. Stella, S. Sozzani, P. Allavena, A. Mantovani, L. P. Ruco.
2000
. Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells.
Am. J. Pathol.
156
:
831
.-837.
26
Kretzschmar, M., J. Doody, J. Massague.
1997
. Opposing BMP and EGF signaling pathways converge on the TGF-β family mediator Smad1.
Nature
389
:
618
.-622.
27
Yamamura, K., K. Ito, K. Tsukioka, Y. Wada, A. Makiuchi, M. Sakaguchi, T. Akashima, M. Fujimori, Y. Sawa, R. Morishita, et al
2004
. Suppression of acute and chronic rejection by hepatocyte growth factor in a murine model of cardiac transplantation.
Circulation
110
:
1650
.-1657.
28
Okunishi, K., M. Dohi, K. Nakagome, R. Tanaka, K. Yamamoto.
2004
. A novel role of cysteinyl leukotrienes to promote dendritic cell activation in the antigen-induced immune responses in the lung.
J. Immunol.
173
:
6393
.-6402.
29
Kuroda, E., U. Yamashita.
2003
. Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice.
J. Immunol.
170
:
757
.-764.
30
Dabbagh, K., M. E. Dahl, P. Stepick-Biek, D. B. Lewis.
2002
. Toll-like receptor 4 is required for optimal development of Th2 immune responses: role of dendritic cells.
J. Immunol.
168
:
4524
.-4530.
31
Hayashi, T., L. Beck, C. Rossetto, X. Gong, O. Takizawa, K. Takabayashi, D. H. Broide, D. A. Carson, E. Raz.
2004
. Inhibition of experimental asthma by indoleamine 2, 3-dioxygenase.
J. Clin. Invest.
114
:
270
.-279.
32
Komamura, K., R. Tatsumi, J. Miyazaki, K. Matsumoto, E. Yamato, T. Nakamura, Y. Shimizu, T. Nakatani, S. Kitamura, H. Tomoike, et al
2004
. Treatment of dilated cardiomyopathy with electroporation of hepatocyte growth factor gene into skeletal muscle.
Hypertension
44
:
365
.-371.
33
Liu, F., Y. K. Song, D. Liu.
1999
. Hydrodynamic-based transfection in animals by systemic administration of plasmid DNA.
Gene Ther.
6
:
1258
.-1266.
34
Dohi, M., S. Tsukamoto, T. Nagahori, K. Shinagawa, K. Saitoh, Y. Tanaka, S. Kobayashi, R. Tanaka, Y. To, K. Yamamoto.
1999
. Noninvasive system for evaluating the allergen-specific airway response in a murine model of asthma.
Lab. Invest.
79
:
1559
.-1571.
35
To, Y., M. Dohi, R. Tanaka, A. Sato, K. Nakagome, K. Yamamoto.
2001
. Early interleukin 4-dependent response can induce airway hyperreactivity before development of airway inflammation in a mouse model of asthma.
Lab. Invest.
81
:
1385
.-1396.
36
Hansen, G., G. Berry, R. H. DeKruyff, D. T. Umetsu.
1999
. Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.
J. Clin. Invest.
103
:
175
.-183.
37
Vermaelen, K. Y., I. Carro-Muino, B. N. Lambrecht, R. A. Pauwels.
2001
. Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.
J. Exp. Med.
193
:
51
.-60.
38
Ohmichi, H., U. Koshimizu, K. Matsumoto, T. Nakamura.
1998
. Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development.
Development
125
:
1315
.-1324.
39
Hattori, N., S. Mizuno, Y. Yoshida, K. Chin, M. Mishima, T. H. Sisson, R. H. Simon, T. Nakamura, M. Miyake.
2004
. The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism.
Am. J. Pathol.
164
:
1091
.-1098.
40
Michalopoulos, G. K., R. Appasamy.
1993
. Metabolism of HGF-SF and its role in liver regeneration.
EXS
65
:
275
.-283.
41
Carrasco, C. P., R. C. Rigden, R. Schaffner, H. Gerber, V. Neuhaus, S. Inumaru, H. Takamatsu, G. Bertoni, K. C. Mccullough, A. Summerfield.
2001
. Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties.
Immunology
104
:
175
.-184.
42
Miyazawa, K., T. Shimomura, A. Kitamura, J. Kondo, Y. Morimoto, N. Kitamura.
1993
. Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor: structural similarity of the protease precursor to blood coagulation factor XII.
J. Biol. Chem.
268
:
10024
.-10028.
43
Shimomura, T., J. Kondo, M. Ochiai, D. Naka, K. Miyazawa, Y. Morimoto, N. Kitamura.
1993
. Activation of the zymogen of hepatocyte growth factor activator by thrombin.
J. Biol. Chem.
268
:
22927
.-22932.
44
Umetsu, D. T..
2004
. Flu strikes the hygiene hypothesis.
Nat. Med.
10
:
232
.-234.
45
Randolph, D. A., R. Stephens, C. J. L. Carruthers, D. D. Chaplin.
1999
. Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation.
J. Clin. Invest.
104
:
1021
.-1029.
46
Stephens, R., D. A. Randolph, G. Huang, M. J. Holtzman, D. D. Chaplin.
2002
. Antigen-nonspecific recruitment of Th2 cells to the lung as a mechanism for viral infection-induced allergic asthma.
J. Immunol.
169
:
5458
.-5467.
47
Dahl, M. E., K. Dabbagh, D. Liggitt, S. Kim, D. B. Lewis.
2004
. Viral-induced T helper type 1 responses enhance allergic disease by effects on lung dendritic cells.
Nat. Immunol.
5
:
337
.-343.
48
Salvi, S. S., K. S. Babu, S. T. Holgate.
2001
. Is asthma really due to a polarized T cell response toward a helper T cell type 2 phenotype?.
Am. J. Respir. Crit. Care Med.
164
:
1343
.-1346.
49
Gorelik, L., R. A. Flavell.
2002
. Transforming growth factor-β in T-cell biology.
Nat. Rev. Immunol.
2
:
46
.-53.
50
Border, W. A., N. A. Noble.
1994
. Transforming growth factor β in tissue fibrosis.
N. Engl. J. Med.
331
:
1286
.-1292.
51
Fehérvari, Z., S. Sakaguchi.
2004
. CD4+ Tregs and immune control.
J. Clin. Invest.
114
:
1209
.-1217.
52
Wagers, S. S., R. J. Norton, L. M. Rinaldi, J. H. T. Bates, B. E. Sobel, C. G. Irvin.
2004
. Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness.
J. Clin. Invest.
114
:
104
.-111.
53
Yuda, H., Y. Adachi, O. Taguchi, E. C. Gabazza, O. Hataji, H. Fujimoto, S. Tamaki, K. Nishikubo, K. Fukudome, C. N. D’Alessandro-Gabazza, et al
2004
. Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice.
Blood
103
:
2196
.-2204.
54
Elias, J. A., Z. Zhu, G. Chupp, R. J. Homer.
1999
. Airway remodeling in asthma.
J. Clin. Invest.
104
:
1001
.-1006.