Abstract
Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are among the most potent mitogens identified for alveolar type II epithelial cells and may have other important functions in repair of the alveolar epithelium in acute lung injury (ALI). However, neither growth factor has been identified in the distal air spaces or plasma of patients with ALI. The goals of this study were to determine: (1) whether HGF and KGF are present in pulmonary edema fluid from patients with ALI and control patients with hydrostatic pulmonary edema; (2) whether HGF and KGF are biologically active in pulmonary edema; and (3) whether HGF or KGF levels are associated with clinical outcome. Pulmonary edema and plasma samples were obtained within 48 h of onset of acute pulmonary edema requiring mechanical ventilation in 26 patients with ALI and 11 control patients with hydrostatic edema. HGF and KGF concentrations were measured with enzyme-linked immunosorbent assays (ELISAs). The median (25th to 75th percentiles) concentration of HGF in pulmonary edema fluid was 21.4 (8.3 to 41.3) ng/ml in ALI and 6.6 (4.8 to 11.4) ng/ml in hydrostatic edema fluid (p < 0.01). The HGF concentration was 7-fold higher in the edema fluid than in the plasma of patients with ALI. In contrast, KGF was detected in low concentrations in edema fluid of patients with ALI and hydrostatic pulmonary edema; the concentration of KGF did not differ in ALI edema (0.6 [0.3 to 2.1] ng/ml) and hydrostatic edema fluid (0.2 [0.0 to 2.6] ng/ml) (p = NS). HGF and KGF were partly purified from four edema-fluid samples by heparin–Sepharose chromatography. Partly purified edema fluids were potent stimuli of DNA synthesis in cultured rat type II alveolar cells; addition of neutralizing antibodies to HGF and KGF attenuated this increase in DNA synthesis by 66% and 53%, respectively. Interestingly, higher edema-fluid levels of HGF were associated with higher mortality in patients with ALI. These novel results show that HGF and KGF are active in the alveolar space early in ALI, probably mediating early events in lung repair, and that increased levels of HGF in edema fluid may have prognostic value early in ALI.
Several studies have demonstrated morphologic injury to the alveolar epithelial barrier in patients with clinical acute lung injury (ALI) (1-3). In addition, there is evidence of impaired surfactant release and altered alveolar epithelial-fluid transport in many patients with ALI (4-6). The early phase of recovery from ALI requires proliferation and migration of alveolar type II cells in order to restore the integrity of the denuded alveolar epithelium (7). Because hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) have recently been discovered to be the principal mitogens for alveolar type II cells, these growth factors may contribute to alveolar epithelial repair following lung injury. HGF is a unique, heparin-binding, proteolytically activated heterodimer produced primarily by endothelial and mesenchymal cells of the lung, liver, kidney, and adrenal gland (8-11). KGF is the seventh member of the fibroblast growth factor (FGF) family, secreted only by fibroblasts, and mitogenic specifically to epithelial cells (12– 14). Both HGF and KGF are important mediators of interactions between the epithelium and mesenchyme following tissue injury, and are important cell motogens that can regulate wound closure (15-18).
Since establishment of the importance of HGF in experimental models of injury, several studies have reported increased plasma levels of HGF in patients with fulminant hepatitis, acute pancreatitis, hypertension, and inflammatory lung diseases (19-23). Furthermore, higher serum concentrations of HGF correlated with greater severity of illness, and decreasing serum levels of HGF were associated with clinical improvement in patients with interstitial pneumonitis and bacterial pneumonia (20, 22). However, HGF has not been measured in the plasma and alveolar edema fluid of patients with ALI. KGF has not yet been identified in the serum or alveolar fluid of patients with ALI, although several experimental studies have already shown that intratracheal administration of KGF prior to induction of lung injury with hyperoxia, radiation, bleomycin, acid-aspiration, or α-naphthylthiourea attenuates the severity of lung injury (24-28).
On the basis of the experimental data, HGF and KGF may be important in mediating recovery from clinical ALI, and might also have prognostic and even therapeutic value for the care of patients with ALI. However, neither growth factor has yet been identified in the edema fluid or plasma of patients with ALI. Therefore, the first objective of the present study was to determine whether HGF and KGF are present in biologically significant quantities in pulmonary edema fluid and plasma of patients with ALI, and if so, to determine whether HGF and KGF are biologically active. In order to control for the effect of pulmonary edema resulting in the need for positive-pressure ventilation (29), the second objective was to determine whether HGF and KGF concentrations are higher in patients with ALI than in control patients with hydrostatic pulmonary edema. The third objective was to identify whether a relationship exists between the concentration of HGF and KGF in pulmonary edema and mortality in patients with ALI.
The study was approved by the Committee for Human Research at the University of California, San Francisco. A random selection of pulmonary edema fluid and plasma samples was made from patients admitted between 1988 and 1996 to the intensive care units of Moffitt–Long Hospital of the University of California, San Francisco, or to San Francisco General Hospital who had acute respiratory failure and who required mechanical ventilation. Classification of each patient's condition as ALI or hydrostatic pulmonary edema was based on review of medical records, using the North American–European Consensus Conference definitions for ALI (30). Pulmonary edema samples were collected by trained respiratory therapists under the supervision of the authors (M.A.M., G.M.V.), as previously described (6, 31); this method has been well validated experimentally by comparison with transthoracic alveolar aspiration and radionuclide studies (32, 33). All samples were collected within 48 h of development of acute pulmonary edema requiring mechanical ventilation. Briefly, pulmonary edema specimens were obtained by insertion of a soft, size 14 French suction catheter into a wedged position in a distal brochus via the patient's endotracheal tube. Specimens were collected in a Lukens trap and stored at 4° C prior to processing. Pulmonary edema samples were then centrifuged at 14,000 rpm for 20 min, and the supernatant was aspirated and stored at −70° C. Blood samples were collected in ethylenediamine tetraacetic acid (EDTA)-treated tubes within 15 min of pulmonary edema sampling, and centrifuged at 5,000 rpm for 5 min, and the plasma was stored at −70° C.
Concentrations of hepatocyte and keratinoctye growth factors in undiluted pulmonary edema and plasma specimens were measured by standard enzyme-linked immunosorbent assay (ELISA) technique, using antibodies to recombinant human HGF and KGF. HGF was assayed with a commercial ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN); KGF was measured with an ELISA based on standard techniques, with standards and antibodies provided by Amgen, Inc. (Thousand Oaks, CA). The volume of edema fluid assayed by ELISA was 0.5 ml or greater per sample. Concentrations of HGF and KGF were reported in ng/ml; the lower limit of detection was 0.1 ng/ml.
HGF and KGF were partly purified from human pulmonary edema samples by heparin–Sepharose chromatography as described previously (10). Briefly, pulmonary edema-fluid specimens from two patients with ALI and two patients with hydrostatic edema were prepared as outlined earlier. A total of 2.5 to 3.5 ml of edema fluid was added to 1 ml of equilibrated heparin–Sepharose beads (Pharmacia Inc., Piscataway, NJ) containing 0.65 ml/ml of heparin–Sepharose. The mixture was gently agitated for 30 min at 4° C and centrifuged at 200 rpm for 2 min. The supernatant was discarded and the beads were washed four times with 5 ml of heparin–Sepharose buffer (Pharmacia Inc., Piscataway, NJ). HGF and KGF were eluted by washing for 15 min with 1 ml of 2.0 M NaCl in heparin–Sepharose buffer; the eluant was collected following passage through Pasteur pipet columns packed with glass wool, and was dialyzed three times against Dulbecco's modified Eagle's medium (DMEM). Positive and negative control solutions of DMEM with 10% fetal bovine serum (DMEM– FBS) and DMEM–FBS:HGF (91 ng/ml):KGF (143 ng/ml) were prepared identically, using recombinant human HGF (R&D Systems) and KGF (Promega, Madison, WI). The final concentrations of HGF and KGF in the purified samples were 8 to 32 ng/ml and 8 to 9 ng/ml, respectively, as measured by ELISA. Final concentrations of both HGF and KGF in the DMEM–FBS control solution were < 0.1 ng/ml, and in the DMEM–FBS:HGF:KGF solution were 72 ng/ml and > 10 ng/ml, respectively.
Bioactivity of HGF and KGF in heparin–Sepharose-purified pulmonary edema-fluid specimens was assessed by incorporation of [3H]thymidine into rat alveolar type II epithelial cells, as previously described (34). Alveolar type II cells were isolated from rats by elastase dissociation and density-gradient centrifugation, according to previously described methods (35). Type II cells were seeded into 48-well tissue-culture plates at a density of 105 cells/cm2, incubated at 37° C for 24 h, and washed with DMEM. Pulmonary edema-fluid and control samples were diluted 1:9 in DMEM with 10% fetal bovine serum (DMEM– FBS). Following this, 0.5 ml of sample solution in DMEM–FBS and 0.1 μCi/ml [3H]thymidine were added to each well. The cells were incubated for 48 h at 37° C and the trichloroacetic acid (TCA)-precipitable counts were measured by liquid scintillation counting. Recombinant human HGF and KGF were used as positive controls. All experiments were done in duplicate.
Experiments to assess neutralization of mitogenic activity by specific neutralizing antibodies to HGF and KGF were done similarly. Anti-HGF and anti-KGF antibodies were obtained from R&D Systems. Alveolar edema-fluid specimens were pooled after partial purification by heparin–Sepharose chromatography. [3H]Thymidine incorporation by type II cells was assayed as previously described, using 5% edema fluid in DMEM–FBS, 5% edema fluid with anti-HGF antibody, and 5% edema fluid with anti-KGF antibody. Positive controls containing recombinant human HGF and KGF at 1, 2, and 5 ng/ml were also assayed. Experiments were done in quadruplicate.
The Simplified Acute Physiology Score II (SAPS II) (36) and lung injury score (LIS) (37) were calculated for all patients except three patients for whom complete records were unavailable. Data extraction from archived medical records and chest radiograph scoring was done by a single investigator (G.M.V.). Comparisons of SAPS II scores and LIS scores for ALI and hydrostatic edema patients were done with Student's t test (two-tailed).
Statistical analysis was done with Statview Student (Abacus Concepts, Inc.) and Microsoft Excel 5.0 (Microsoft Corp., Redwood, WA) software, with statistical significance defined at p ⩽ 0.05. HGF and KGF concentrations are presented as median, 25th to 75th percentiles and 10th to 90th percentiles, since the data were not normally distributed. Data were normalized by log transformation, and were analyzed by analysis of variance (ANOVA). Measurements of [3H]thymidine incorporation were normalized relative to the negative control solution; the data were compared by ANOVA, and Bonferroni's t test was used for multiple comparisons.
Twenty-six patients with ALI and 11 patients with hydrostatic pulmonary edema were studied. The patient demographics, etiology of pulmonary edema, and clinical outcomes are detailed in Table 1, with summary statistics in Table 2. The clinical disorders associated with the development of ALI were primary pneumonia (10 of 26 cases), sepsis syndrome (seven of 26 cases), noninfectious pneumonitis (three of 26 cases), neurogenic disorders (two of 26 cases), and other (hemorrhagic shock, postoperative to cardiopulmonary bypass), postoperative to kidney–pancreas transplant, and bowel infarction) (four of 26 cases). The causes of hydrostatic pulmonary edema included acute myocardial infarction and cardiac arrest (four of 11 cases), chronic dilated cardiomyopathy (five of 11 cases), and perioperative volume overload (two of 11 cases). The ratio of protein concentrations in edema fluid and plasma was 0.85 ± 0.27 (mean ± SD) in patients with ALI and 0.56 ± 0.13 in patients with hydrostatic pulmonary edema (p < 0.05). SAPS II scores were 53 ± 21 (mean ± SD) and 50 ± 19 in patients with ALI and hydrostatic edema, respectively (p = 0.65); the LIS was 3.1 ± 0.5 in ALI and 2.9 ± 0.6 in hydrostatic edema (p = 0.54). In-hospital mortality was 73% in the ALI group and 36% in the hydrostatic pulmonary edema group.
| Study No. | Age (yr) | Gender | Diagnosis | Outcome | ||||
|---|---|---|---|---|---|---|---|---|
| Hydrostatic | ||||||||
| H05-90 | 39 | M | Cardiac arrest | Expired | ||||
| H14-93 | 21 | F | SLE, CRT | Survived | ||||
| H24-88 | 60 | M | Acute MI | Expired | ||||
| H12-89 | 81 | F | Dilated CM | Survived | ||||
| H20-89 | 15 | F | Tetralogy of Fallot | Survived | ||||
| H30-96 | 64 | M | Ischemic CM | Survived | ||||
| H34-96 | 45 | M | Acute MI | Survived | ||||
| H50-96 | 64 | M | Ischemic CM | Survived | ||||
| H22-96 | 76 | M | Ischemic CM | Survived | ||||
| H06-93 | 93 | M | Ischemic CM | Expired | ||||
| H24-93 | 77 | F | Acute MI | Expired | ||||
| Acute lung injury | ||||||||
| H10-94 | 85 | F | Pneumonia | Expired | ||||
| H06-88 | 37 | F | Methotrexate | Expired | ||||
| H23-88 | 30 | M | Invasive aspergillosis | Expired | ||||
| H25-88 | 24 | F | Cerebellar hemorrhage | Survived | ||||
| H26-88 | 61 | M | Sepsis versus chemotherapy | Expired | ||||
| H09-89 | 47 | F | Sepsis | Expired | ||||
| H39-90 | 88 | M | Pneumonia | Expired | ||||
| H43-90 | 33 | F | Aspiration | Expired | ||||
| H09-96 | 82 | F | Sepsis | Expired | ||||
| H22-95 | 73 | M | Pneumonia | Expired | ||||
| H11-94 | 40 | F | Pneumonia | Survived | ||||
| H06-93 | 59 | F | Hemorrhagic shock s/p OLT | Expired | ||||
| H12-94 | 27 | F | Pneumonia | Survived | ||||
| H08-94 | 27 | M | Sepsis | Expired | ||||
| H11-93 | 31 | M | Intracranial hemorrhage | Expired | ||||
| H17-96 | 38 | M | s/p kidney-pancreas transplant | Survived | ||||
| H20-93 | 47 | M | Bowel infarct | Expired | ||||
| H04-93 | 29 | M | Pneumonia | Expired | ||||
| H21-93 | 35 | F | Sepsis | Expired | ||||
| H28-96 | 15 | M | s/p CPB | Survived | ||||
| H29-96 | 30 | F | SLE pneumonitis | Survived | ||||
| H58-96 | 56 | F | Sepsis | Expired | ||||
| H62-96 | 48 | M | Invasive aspergillosis | Expired | ||||
| H63-96 | 60 | F | Sepsis | Expired | ||||
| H64-96 | 24 | M | Pneumonia | Expired | ||||
| H54-96 | 77 | M | Pneumonia | Survived |
| n | Age (Range) (yr) | Gender | SAPS II | LIS | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Hydrostatic HGF | 11 | 58 (21–93) | 64% M | 50 ± 19 | 2.9 ± 0.5 | |||||
| Hydrostatic KGF | 6 | 70 (45–93) | 83% M | 61 ± 17 | 3.0 ± 0.6 | |||||
| ALI HGF | 26 | 46 (15–88) | 50% M | 53 ± 21 | 3.1 ± 0.5 | |||||
| ALI KGF | 25 | 47 (15–88) | 52% M | 53 ± 21 | 3.1 ± 0.5 |
HGF concentrations in edema fluids from patients with ALI ranged from 1.8 to 148 ng/ml, with a median of 21.4 ng/ml (8.3 to 41.3 ng/ml) (25th to 75th percentiles); HGF in pulmonary edema fluid from the patients with hydrostatic edema ranged between 2.4 and 34.5 ng/ml, with a median concentration of 6.6 ng/ml (4.8 to 11.4 ng/ml) (25th to 75th percentiles) (Table 3). The concentration of HGF was significantly higher in the pulmonary edema fluids from patients with ALI than in those from the hydrostatic edema group (p < 0.01) (Figure 1A). HGF was detected in plasma from both groups of patients, but there was no significant difference in HGF concentration between the two groups (Figure 1B). In both groups of patients, the level of HGF in pulmonary edema fluid was significantly greater than in plasma (p < 0.01 for ALI; p < 0.01 for hydrostatic edema). The correlation coefficients between edema fluid and plasma HGF levels were 0.56 (p < 0.01) for the ALI group and 0.79 (p < 0.01) for the hydrostatic edema group (data not shown).
| Clinical Condition | HGF (ng/ml ) | KGF (ng/ml ) | ||
|---|---|---|---|---|
| ALI edema | 21.4 (8.3–41.3) | 0.6 (0.3–2.1) | ||
| ALI plasma | 3.1 (1.8–5.0) | 0.3 (0.2–0.7) | ||
| Hydrostatic edema | 6.6 (4.8–11.4) | 0.2 (0.0–2.6) | ||
| Hydrostatic plasma | 1.7 (1.0–4.4) | 0.1 (0.1–0.2) |
Fig. 1. HGF concentrations in undiluted pulmonary edema fluid (A) and plasma (B) samples from patients with ALI or hydrostatic pulmonary edema. HGF measurements were made with an ELISA. Box plots represent the median and 25th to 75th percentiles; error bars span 10th to 90th percentiles. Analysis was done by ANOVA of log-transformed data.
[More] [Minimize]KGF was detectable in pulmonary edema fluid and plasma from patients with ALI and hydrostatic pulmonary edema (Figure 2). The median (25th to 75th percentiles) concentrations of KGF in pulmonary edema fluid were 0.59 ng/ml (0.26 to 2.13 ng/ml) in patients with ALI/ARDS, compared with 0.21 ng/ml (0.00 to 2.60 ng/ml) in patients with hydrostatic pulmonary edema (see Table 3). There were no statistically significant differences between KGF concentrations in pulmonary edema fluid or plasma of patients with ALI and those with hydrostatic edema. There was no significant correlation between the concentrations of KGF detected in edema fluid and plasma in either group (data not shown).
Fig. 2. KGF concentrations in undiluted pulmonary edema (A) and plasma (B) samples from patients with ALI or hydrostatic pulmonary edema. KGF measurements were made with an ELISA. Box plots represent the median and 25th to 75th percentiles; error bars span 10th to 90th percentiles. Analysis was done by ANOVA after normalization of data by logarithmic transformation.
[More] [Minimize]In order to determine whether the growth factors detected by ELISA were biologically active, HGF and KGF in pulmonary edema samples from two patients with ALI and two patients with hydrostatic edema were partly purified by heparin– Sepharose chromatography. The activity of HGF and KGF from these specimens was assessed by their ability to stimulate DNA synthesis in primary cultures of rat alveolar type II cells. DNA synthesis was measured by incorporation of [3H]thymidine into the type II cells during 48 h of incubation with partly purified pulmonary edema fluids. The HGF concentration in the purified samples ranged from 5 to 32 ng/ml; KGF concentrations were between 8.2 and 9.0 ng/ml. As shown in Figure 3, [3H]thymidine incorporation in cells treated with the edema fluids increased by 6.3- to 9.2-fold as compared with the DMEM–FBS control (p = 0.0039 for Bonferroni's t test), which was similar in magnitude to the effect of high concentrations of recombinant human HGF and KGF (72 ng/ml and > 10 ng/ ml, respectively) (p = 0.102 for Bonferroni's t test). Pooled, partly purified edema fluid was diluted in DMEM–FBS to 1%, 2%, 5%, and 10% solutions; a dose–response curve for stimulation of DNA synthesis showed maximum [3H]thymidine incorporation with 5% edema fluid (data not shown). As shown in Figure 4, a 5% edema-fluid solution stimulated [3H]thymidine incorporation by 5.8-fold as compared with the control (p = 0.002). Addition of anti-HGF antibody reduced DNA synthesis by 66% (p = 0.003); addition of anti-KGF antibody diminished DNA synthesis by 53% (p = 0.009). Thus, most of the DNA synthesis stimulated by heparin-binding growth factors in the pulmonary edema fluids was due to HGF and KGF.
Fig. 3. Bioactivity of HGF and KGF in edema fluids. HGF and KGF were partly purified from edema fluids obtained from two patients with ALI and two patients with hydrostatic edema by heparin–Sepharose chromatography and elution with 2.0 M NaCl. The negative and positive controls were DMEM–FBS and recombinant human HGF (72 ng/ml) and recombinant human KGF (10 ng/ml). HGF concentrations ranged from 5 to 32 ng/ml and KGF concentrations from 8.2 to 9.0 ng/ml in the purified samples. Bars represent the relative uptake of [3H]thymidine by primary cultures of rat type II pneumocytes.
[More] [Minimize]
Fig. 4. Antibody neutralization of DNA synthesis stimulated by pulmonary edema fluid. Anti-HGF and anti-KGF, neutralizing antibodies to recombinant human HGF and KGF, were added to a 5% solution of pooled pulmonary edema fluid samples that were partly purified by heparin–Sepharose chromatography. The DNA synthesis index is a measure of [3H]thymidine incorporation by primary cultures of rat type II alveolar epithelial cells treated with edema fluid and relative to [3H]thymidine incorporation by cells of the DMEM-FBS-treated control. †, †† Statistically significant differences compared with the 5% pulmonary edema fluid.
[More] [Minimize]The in-hospital mortality rate among the patients with ALI was 73%. In the ALI group, the median (25th to 75th percentiles) HGF concentration in edema fluid of the seven survivors was 6.5 (4.1 to 13.4) ng/ml, whereas the HGF was 27.3 (11.3 to 44.8) ng/ml in the 19 patients who expired (Figure 5). The patients with ALI who survived had significantly lower levels of HGF in their pulmonary edema fluid than did those who expired (p = 0.03). Similar analysis of the data for patients with hydrostatic pulmonary edema showed no significant differences in the edema-fluid HGF concentration between survivors and nonsurvivors (data not shown).
Fig. 5. Relationship between pulmonary edema-fluid levels of HGF and mortality in patients with ALI. Analysis was done by ANOVA of data normalized by log transformation.
[More] [Minimize]Overall hospital mortality was 76% for the group of patients with ALI in whom pulmonary edema-fluid KGF was measured (n = 25). The median (25th to 75th percentiles) KGF in pulmonary edema fluid sampled from patients with ALI was 0.4 (0.3 to 0.6) ng/ml in survivors and 1.1 (0.1 to 2.8) ng/ml in the nonsurvivors (data not shown). There was no significant difference in edema-fluid KGF level in survivors and nonsurvivors (p = 0.13). Equivalent analysis of the data for the patients with hydrostatic pulmonary edema revealed no significant association between edema-fluid KGF concentration and mortality.
HGF was present in both the pulmonary edema fluid and plasma from patients with severe ALI and those with hydrostatic pulmonary edema. HGF was detected in a significantly higher concentration in the pulmonary edema fluid of patients with ALI than of those with hydrostatic edema. The primary source of HGF in these patients seems to have been the lung, since the HGF level was 7-fold higher in the pulmonary edema fluid than in the plasma. Additionally, the heparin-binding growth factors recovered from these pulmonary edema-fluid specimens, primarily HGF and KGF, were biologically active, as indicated by their ability to stimulate DNA synthesis in isolated rat alveolar type II cells.
The concentrations of HGF in plasma from patients with ALI and acute hydrostatic pulmonary edema were much higher than the concentrations measured in prior studies of patients with other inflammatory lung diseases. Yanagita and colleagues found plasma HGF concentrations ranging from 1.1 to 2.8 ng/ml in seven hospitalized patients with bacterial pneumonia (23), and Maeda and coworkers reported similar findings in patients with active interstitial pneumonitis (1.2 ± 0.2 ng/ml, n = 15) and bacterial pneumonia (1.0 ± 0.3 ng/ml, n = 11) (22). Thus, the median levels of HGF in plasma from these patients with ALI were approximately 3-fold greater than those in plasma from patients with other active pulmonary diseases. The higher levels of HGF most likely reflect a greater degree of injury and inflammation in the ALI patients. Indeed, the high plasma concentrations of HGF are similar to those measured in patients with other types of major organ failure, such as fulminant hepatic failure and severe acute pancreatitis, in whom the mean serum HGF concentrations were 8.1 ± 1.8 ng/ml and 3.2 ± 1.7 ng/ml, respectively (19, 20). The concentrations of HGF measured in plasma and pulmonary edema-fluid specimens are within the range of maximal HGF activity of approximately 1 to 10 ng/ml, on the basis of in vitro studies of DNA synthesis by rat alveolar type II cells (8).
The conclusion that the high levels of HGF found in these patients with severe lung injury is a marker for severe inflammation is supported by clinical studies of interstitial pneumonitis and bacterial pneumonia, fulminant hepatic failure, and acute pancreatitis, in all of which a strong positive correlation was shown between the serum HGF concentration and physiologic markers of organ damage (19, 20, 22). This explanation is also supported by in vitro and in vivo studies of the regulation and cellular effects of HGF. Expression of HGF is upregulated by several proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1β, and tumor necrosis factor-α (TNF-α) (38), which are important in the neutrophilic infiltration occurring in ALI and are present in the alveolar space early in lung injury (39-42). HGF is a potent mitogen for alveolar type II cells in vitro and in vivo, and is upregulated within 6 h of acid-instillation into rat lungs (23). Furthermore, intravenous administration of HGF to mice after induction of ALI by acid instillation stimulated DNA synthesis in alveolar type II cells (43). These experimental results suggest that expression of HGF, probably by pulmonary interstitial fibroblasts (10) and endothelial cells (44), is upregulated by proinflammatory cytokines early in ALI, with the potential benefit of stimulating proliferation of type II cells and restoring the alveolar epithelial barrier. This mechanistic explanation is further strengthened by our findings of physiologic concentrations of bioactive HGF in pulmonary edema fluid from patients with ALI. Other possible contributors to the HGF measured in the plasma of these critically ill patients are nonpulmonary sources, including the liver and kidney in patients with multiple organ damage, or increased pulmonary expression and release of HGF in response to injury of distant organs, as reported in studies of partial hepatectomy and nephrectomy in rats (45). However, these are probably relatively minor factors in view of our findings that the level of HGF in pulmonary edema fluid was 7-fold higher than in the plasma of patients with ALI, and that pulmonary edema and plasma concentrations of HGF were positively correlated, both of which findings suggest that the lung is the major source of HGF in these patients.
In this study, the concentration of HGF in pulmonary edema fluid from patients with ALI was significantly higher in patients who expired prior to hospital discharge than in those who survived. This finding concurs with those in studies of fulminant hepatitis, acute pancreatitis, and inflammatory lung diseases not including ALI, in which progressively increasing levels of serum HGF were found in patients who expired (19, 20, 22). The relationship between mortality and higher HGF levels in pulmonary edema fluid probably represents an intense reparative response to severe lung injury. Although these data should be confirmed in a larger prospective study using predetermined thresholds of HGF concentrations, the correlation between pulmonary edema fluid HGF concentrations and mortality may be a clinically useful marker of prognosis in ALI for several reasons. Most importantly, the HGF concentration measured very early in the course of ALI correlates with mortality; additionally, pulmonary edema fluid is easily sampled from mechanically ventilated patients, and HGF can be measured with ELISA techniques available to most clinical laboratories. This measurement may also be a useful adjunct for identifying those patients at highest risk for nonsurvival, for the purposes of clinical studies of ALI, and perhaps when assessing the relative benefit of providing experimental therapies for ALI.
In contrast to HGF, KGF was present only in small quantities in the edema fluid and plasma of patients with ALI or hydrostatic pulmonary edema. Furthermore, there was no significant difference between the edema fluid or plasma levels of KGF in ALI and hydrostatic edema (Figure 2). Nevertheless, the present study is the first in which KGF was detected in biologic fluids from patients. Are these levels of KGF biologically significant? Large quantities of exogenously administered KGF (1 to 5 mg/kg) are necessary to induce type II-cell hyperplasia and to attentuate lung injury in vivo in normal rats (24-27). However, on the basis of in vitro data, KGF concentrations of only 10 ng/ml are required to maximally stimulate alveolar type II-cell proliferation and surfactant protein gene expression (10, 46). Thus, the concentrations of KGF found in pulmonary edema fluid may be biologically active. Other factors, such as KGF-receptor activation, will also determine the biologic effects of these concentrations of KGF.
Although pretreatment of rats with intratracheal KGF diminishes the severity of ALI caused by a variety of experimental insults, the role of endogenous KGF in recovery from ALI is not understood. In addition to its mitogenic activity, KGF stimulates expression of surfactant proteins and production of lamellar bodies in type II pneumocytes in vitro and in vivo, indicating that KGF may promote differentiation of these cells (46, 47). These data lead to the hypothesis that KGF may be an important mediator of type II-cell hyperplasia and maturation early in the response to ALI, resulting in restoration of the denuded alveolar epithelium and replenishment of surfactant. This would represent an effect similar to that in dermal wound healing, in which KGF is induced in fibroblasts at the wound site, and stimulates both proliferation and migration of keratinocytes at the wound edge (48, 49). Expression of KGF by fibroblasts is upregulated by IL-1α, IL-1β, TNF-α, IL-6, and transforming growth factor-α (TGF-α) (50, 51), cytokines that have been identified in bronchoalveolar lavage fluid (BALF) and pulmonary edema specimens from patients with ARDS (39-42, 52). Furthermore, KGF is known to be secreted by human pulmonary fibroblasts (10). Our findings of detectable levels of bioactive KGF in pulmonary edema fluid from patients early in the course of ALI is consistent with this proposed sequence of events, lending further support to a role for KGF in recovery of the alveolar epithelial barrier following lung injury.
Why was mortality 73% in the 26 ALI patients with pulmonary edema fluid sampled in the present study, as compared with 58% in our recently published study of 123 consecutive patients with ALI treated in the same intensive care unit (53)? The most likely explanation is that the quantity of edema fluid is greater in patients with more severe lung injury, and that the method of obtaining direct aspirates of alveolar edema fluid therefore selects a group of patients with more severe ALI. In support of this hypothesis, we have recently reported a mortality of 64% in ALI patients from whom edema fluid was sampled for another study (54). Thus, the results of these studies should not be generalized to all patients with ALI.
An interesting and unexpected finding in the present study was the presence of both HGF and KGF in pulmonary edema fluid and plasma from patients with severe hydrostatic pulmonary edema. The median plasma (25th to 75th percentiles) HGF concentration in this group was 1.7 (1.0 to 4.4 ng/ml), which was much higher than the average of approximately 0.3 ng/ml reported in the literature for normal control subjects (20, 22, 23). Three of these patients had very high levels of HGF measured in pulmonary edema-fluid samples, ranging from 24 to 35 ng/ml, which were more consistent with the values found in the patients with ALI. One explanation for the presence of high levels of HGF and detectable KGF in plasma and pulmonary edema fluid from these patients with hydrostatic edema is the increased production of cytokines in congestive heart failure (CHF). Other possibilities include stimulation of cytokine production by plasma leaking into the interstitial space, and stretching of alveolar structures by positive-pressure ventilation (55). Several clinical studies have found increased concentrations of TNF-α and IL-6 in plasma, which correlated with the severity of CHF (56, 57), although Ferrari and associates also found that the concentration of soluble TNF receptor was increased, and that the overall cytotoxic activity of TNF was no different than in controls (58). Hennein and colleagues (59) have reported that TNF-α, IL-6, and IL-8 can be increased after uncomplicated coronary artery bypass grafting in correlation with the duration of aortic cross-clamping, and that increasing levels of IL-6 and IL-8 were associated with postoperative left ventricular dysfunction. These findings show that the cytokine milieu is enhanced in the serum of patients with left ventricular dysfunction or injury, an effect that may lead to release of HGF and KGF through the regulatory pathways discussed earlier. These mechanisms may even be important mediators of the alveolar epithelial type II-cell hyperplasia observed morphologically in chronic, severe, CHF (60); this response could increase the capacity for alveolar-fluid transport in the presence of chronic heart failure (61).
The need to include control patients with hydrostatic pulmonary edema in measuring levels of HGF and KGF in ALI is evident from the present study. First, the presence of HGF and KGF in alveolar edema fluid is not specific for ALI, but is common to pulmonary edema of all sources. Second, significant quantities of HGF are released in some patients with hydrostatic pulmonary edema, indicating that some patients with hydrostatic pulmonary edema may have a modest proinflammatory environment. These findings underscore the importance of studying hydrostatic pulmonary edema in addition to ALI when investigating potential biomarkers of injury, in order to control for the presence of pulmonary edema and the effects of positive-pressure ventilation. Importantly, the SAPS II and LIS were similar in patients with pulmonary edema resulting from increased permeability or hydrostatic mechanisms (Table 2).
In summary, HGF and KGF are present and biologically active in the pulmonary edema fluid of patients with ALI. Furthermore, higher concentrations of HGF are associated with mortality in patients with ALI. Combined with experimental data, the findings of this study strengthen the hypothesis that both HGF and KGF are induced by proinflammatory cytokines early in lung injury, and may work in concert to restore damaged alveolar epithelium by promoting the proliferation and differentiation of alveolar type II epithelial cells. Of clinical importance is that the degree of increase of HGF in edema fluid may reflect the severity of injury, and may therefore be a useful prognostic marker for poor outcome in ALI.
The authors would like to thank Dr. Gunnard Modin for assistance with the statistical analysis and Clinton D. Wakefield for his assistance in obtaining medical records.
Supported in part by Grants HL52856 (M.A.M., G.M.V.), HL29891, and HL56556 (R.J.M., K.M.) from the National Institutes of Health.
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