Abstract
Neutrophils cause lung injuries by releasing proteases and active oxygen radicals in patients with acute respiratory distress syndrome (ARDS). Artificial surfactant is used to replace native surfactant whose functions are deteriorated by serum-derived inhibitors in these patients. We investigated potential interactions between exogenous surfactant (Surfactant TA) and neutrophils in in vivo and in vitro experimental models. Neutrophil alveolitis was induced in hamster lungs by the intratracheal administration of bleomycin (5 mg/kg) on Day 0. Some of the animals were followed by replacement with Surfactant TA (5 and 10 mg/100 g body weight) on Day 1. Alveolar cells were harvested by lung lavage on Day 2. The numbers of the neutrophils obtained from the lungs treated with bleomycin and Surfactant TA were unchanged, but the superoxide production from these cells was significantly decreased when compared with control animals (no Surfactant TA). From the in vitro experiments, Surfactant TA was shown to inhibit adherence and superoxide production of human neutrophils. These effects were derived from the heat-resistant components of Surfactant TA and were mimicked by treatment with liposomes of dipalmitoyl phosphatidylcholine. Surfactant-TA-treated neutrophils were demonstrated to have picnotic nuclei and to express Fas antigens, which were characteristic of apoptotic cells. These results suggest that exogenous Surfactant TA may play an important role not only in improving surfactant functions but in preventing neutrophils from further activation, probably through enhancing apoptosis.
Acute respiratory distress syndrome (ARDS) (1) is characterized by neutrophil-mediated acute lung injury and surfactant dysfunction. Lung injury is, in general, induced by the neutrophil-derived tissue injury factors such as proteases and active oxygen species (2). Surfactant dysfunction is thought to be derived from an absolute decrease in its amount, or from serum- derived inhibitors as a result of the increased vascular permeability (3), and from an increased conversion ratio from large aggregates, an active form of natural surfactant, to inactive small aggregates (4, 5).
Recent clinical investigations have demonstrated that replacement therapy with artificial surfactant is effective for some patients with ARDS (6, 7). Among artificial surfactant preparations, Survanta (Abbott Laboratories, North Chicago, IL) was reported to significantly improve a survival rate of ARDS (8). Surfactant TA, an analogue to Survanta, is a mixture of the bovine lung extracts and synthetic lipids and is widely used to treat neonatal respiratory distress syndrome in Japan (9). This preparation was also reported to be therapeutically effective in patients with ARDS (10, 11).
It seems reasonable that exogenous surfactant may improve surfactant function either by supplementing an absolute lack of endogenous surfactant or by neutralizing the surfactant inhibitors, but the mechanisms on the effectiveness of exogenous surfactant on ARDS are not fully elucidated. It is also plausible that, as neutrophils accumulate in the alveolar spaces, exogenous surfactant may modulate functions of these cells and may play significant roles in preventing lung injury.
Bleomycin-induced lung injury in animal models is characterized by accumulation of neutrophils in the early stages and development of pulmonary fibrosis (12). This model can be used as one of the experimental models for ARDS. Changes in components and functions in endogenous surfactant have also been characterized in these animal models (13). Similar to patients with ARDS, total phospholipids, disaturated phosphatidylcholine, and phosphatidylglycerol were decreased in these animals and the surface-active properties were also deteriorated.
In the present study, we induced neutrophil alveolitis in hamster lungs by the intratracheal instillation with bleomycin and investigated the effects of exogenous surfactant (Surfactant TA) on the functions of these neutrophils. In order to further examine the mechanisms modulating neutrophil functions, we also investigated in vitro interactions between Surfactant TA and neutrophils purified from normal human peripheral venous blood.
Surfactant TA was donated by Tokyo-Tanabe Co. Ltd. (Tokyo, Japan). A vial containing 120 mg Surfactant TA was dissolved on the experimental days with 12 ml of sterile saline for in vivo administration with either 6 ml of Hanks' balanced salt solution (HBSS) for superoxide production assays or 6 ml of RPMI-1640 medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% FCS for adherence assays.
Liposomes were prepared, freshly prior to experiments, from dipalmitoyl phosphatidylcholine (DPPC) (Sigma, St. Louis, MO). DPPC (2 mg) in chloroform was dried in a glass tube under a gentle stream of nitrogen and suspended in 2 ml PBS and then sonicated for 10 min using a probe sonicator. Liposome suspension was centrifuged at 480 × g for 5 min to remove titanium, and the supernatant was used for neutrophil function assays.
Our preparations were tested for endotoxin contamination by the method described by Iwanaga and coworkers (14). The results demonstrated that endotoxin contamination in our preparations was negligible (control, 21.6 pg/ml; Surfactant TA, 15.0 pg/ml; DPPC liposomes, 5.4 pg/ml).
For the binding study, 3H-DPPC liposomes were prepared. 3H-DPPC (1 μCi, 18 ng; DuPont–New England Nuclear Research Products, Boston, MA) was mixed with 2 mg unlabeled DPPC in chloroform prior to sonication. The mixtures were then sonicated in the same manner. Specific activity of 3H-DPPC liposomes varied from 14 to 98 cpm/μg in four different experiments.
To induce neutrophil alveolitis, hamsters were anesthetized with pentobarbital, intubated through the trachea, and then instilled intratracheally with 0.5 mg in 0.5 ml saline per 100 g body weight of bleomycin on Day 0. Twenty-four hours later (Day 1), the animals were reanesthetized and instilled intratracheally with 5 mg (BS5) and 10 mg (BS10) in 0.5 ml saline per 100 g body weight of Surfactant TA. Control animals (B) were instilled with equivalent volumes of saline in the same way. On Day 2, the animals were killed by anesthesia and the lungs were washed with 8 ml PBS until 50 ml of recovery was obtained. Alveolar cells were washed with 50 ml PBS by centrifugation at 240 × g for 5 min and suspended with HBSS. After cell suspensions were adjusted to contain 1 × 106 cells/ml, cell viability and population were examined by trypan blue dye exclusion and Diff-Quik staining, respectively. Cells were finally tested for superoxide production as a marker for their functions. The measurement of superoxide production is described below.
To investigate the in vitro effects of Surfactant TA on neutrophil functions, human neutrophils were purified from venous blood of healthy volunteers using a Ficoll density gradient method (15). Heparinized venous blood was mixed with an equal volume of 6% (wt/vol) dextran 70 (Midori Juji Co. Ltd., Osaka, Japan), the erythrocytes being allowed to sediment for about 60 min at room temperature. The upper fraction, including the buffy-coat, was collected and centrifuged at 240 × g for 5 min and the cell pellet was suspended with PBS. Cell suspension was then applied on a Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ) density gradient and centrifuged at 400 × g for 30 min at 15° C. After centrifugation, the mononuclear-cell-enriched interface layer was carefully aspirated and the cell pellet at the bottom was then used as a neutrophil source. Residual erythrocytes were lysed by treating with 0.2% NaCl for 30 s, with the isotonicity being restored by the rapid addition of an equal volume of 1.6% NaCl. The cells were washed once with PBS and finally suspended with the assay medium as shown below. The cell viability determined by trypan blue dye exclusion was almost 100% in all experiments, and the neutrophil purity of the preparation measured by Diff-Quik staining was consistently more than 93% (others: eosinophils and lymphocytes).
To examine if Surfactant TA has any direct cytotoxic effects on neutrophils, cell viability and lactate dehydrogenase (LDH) release were checked after incubation with Surfactant TA. Neutrophils were incubated with varying concentrations of Surfactant TA at 37° C for 1 h. Cell viability was determined from a trypan blue exclusion test. Cell suspensions were centrifuged at 240 × g for 5 min to separate supernatants from cells, and their LDH activities were measured using the method described by Fanestil and coworkers (16). An LDH release was expressed as percent release (medium LDH activity/cell LDH activity × 100).
The neutrophil adherence assay was based on the method described previously (17). This assay was chosen for evaluating neutrophil functions not only by its simplicity but also by the principle that degrees of adherence to the plastic surfaces were paralleled with those of neutrophil activation (17). Cells, stimuli, and Surfactant TA were prepared in an RPMI-1640 medium supplemented with 10% FCS. In principle, 100 μl of cell suspension (5 × 105/well), 50 μl of stimuli such as phorbol myristate acetate (PMA, 100 ng/ml; Sigma), n-formyl-methionyl-leucyl-phenylalanine (FMLP, 10 μM; Sigma), and tumor necrosis factor-α (TNF-α, 100 U/ml; Asahi Chemical Industry Co. Ltd., Shizuoka, Japan) and 50 μl of varying concentrations of Surfactant TA (as much as 5 mg/ml) were mixed in each well of a 96-well tissue culture plastic plate and incubated for the designated periods at 37° C. At the end of incubation the nonadherent cells were vigorously removed by blotting the plate, and the adherent cells were fixed and stained with 100 μl of a 0.5% crystal violet solution dissolved in 12% neutral formaldehyde and 10% ethanol for 30 min at the room temperature. Adherent cells were then vigorously washed in tap water to remove unincorporated dye. Crystal violet incorporated into adhered cells was extracted by adding a 1% sodium dodecyl sulfate solution and read for determination of absorbance at 570 nm as an indicator of the neutrophil adherence activity.
To examine if aggregates of Surfactant TA physically interfered with neutrophil adherence to the plastic surface, neutrophils were preincubated with Surfactant TA before assays. Neutrophils were mixed with varying concentrations of Surfactant TA in 1 ml of medium in a 50-ml centrifuge tube and incubated at 37° C for 1 h in the shaking water bath. After incubation the mixtures were diluted with 50 ml PBS and centrifuged at 240 × g for 5 min to sediment cells from Surfactant TA. The supernatant was discarded and the cell pellet was suspended with the same volume of PBS. This washing step was repeated three times until the supernatant became clear. The cells, free of Surfactant TA, were finally suspended in the assay medium, adjusted to 5 × 106/ ml, and subjected to the adherence assay. Control neutrophils were processed in parallel for the incubation and the washes.
There was the possibility that the agonists did not act with neutrophils by acting with Surfactant TA. To exclude this possibility, we designed the following experiment. PMA and Surfactant TA were reacted in the absence of neutrophils, then they were centrifuged at 100,000 × g for 20 min to make a Surfactant-TA-free supernatant. The control supernatant that has PMA without Surfactant TA was obtained by conducting the same centrifugation. These supernatants were examined for a neutrophil-stimulating activity. The result demonstrated that both supernatants equally stimulated neutrophil adherence to the plastic plates and clearly suggested that PMA was still stimulative to neutrophils even in the presence of Surfactant TA since PMA would be involved into the surfactant pellet if it was absorbed to Surfactant TA.
This assay employed a cytochrome c reduction method (18). Cells, Surfactant TA, and all other agents were dissolved in HBSS. In the representative assay, 50 μl of neutrophils (1 × 106/ml), 50 μl of varying concentrations of Surfactant TA, 40 μl of cytochrome c (5 mg/ml; Sigma), 40 μl of superoxide dismutase (SOD, 300 μg/ml; Sigma, from E. coli) or its control solution, and 20 μl of PMA (1 mg/ml) or its control solution were mixed in a final volume of 200 μl in each well of a 96-well culture microplate. The reaction mixture was then incubated at 37° C for 30 min. After incubation the reaction was stopped by putting the plate on ice. Cells and Surfactant TA were sedimented by centrifugation at 480 × g for 5 min and 100 μl of the supernatants was carefully transferred for determination of absorbance at 550 nm. The superoxide specific cytochrome c reduction (nmol/106 neutrophils) was determined from the absorbance difference in the presence and absence of SOD.
To determine whether Surfactant TA inhibited the superoxide production from neutrophils or scavenged the superoxide anions produced by neutrophils, the superoxide anions were produced enzymatically with xanthine (XA, 500 μM; Sigma) and xanthine oxidase (XO, 10 mU/ml; Sigma) (19) in the presence or the absence of Surfactant TA. The reaction and the determination of cytochrome c reduction were processed in the same way as described above.
To determine if DPPC, a main lipid component of Surfactant TA, mimicked the effects of Surfactant TA, liposomes made of DPPC were prepared as described above and tested for inhibiting neutrophil superoxide production.
To partially investigate the mechanism for neutrophil inhibition by Surfactant TA, 3H-DPPC liposomes were prepared as described above and their binding to neutrophils was examined. Neutrophils (1 × 106) were incubated with varying concentrations of 3H-DPPC liposomes in microtubes at 37° C for 1 h. After incubation the neutrophils were washed three times with PBS. The final cell pellets were immersed in scintillation liquid (ACS II; Amersham, Arlington Heights, IL) and measured in a scintillation counter. 3H-DPPC liposomes (μg) bound specifically to neutrophils were calculated from a specific activity (cpm/μg) and expressed as μg DPPC/106.
Neutrophils were incubated at 37° C for 1 h with Surfactant TA, DPPC liposomes, and their control solution. After incubation, cells were harvested on the slide glass by a cytospin and stained by a Diff-Quik method. Under a microscope, it was determined whether neutrophils had picnotic nuclei, characteristic of apoptosis, by two blinded observers. As evaluation by one observer coincided well with that by the other, the average values were employed.
To further estimate whether neutrophils treated with Surfactant TA had a characteristic of apoptosis, an immunocytochemical analysis using a monoclonal antihuman Fas-antibody (UB2; BML, Nagoya, Japan) was conducted. Bound antibody was detected using a biotin-linked secondary antibody in conjunction with the avidin-peroxidase method (Vectastain; Funakoshi Co. Ltd., Tokyo, Japan).
We also examined if Surfactant TA enhanced neutrophil apoptosis in vitro in the presence of human recombinant granulocyte-colony stimulating factor (rG-CSF; Chyugai Co. Ltd., Tokyo, Japan), which has been shown to inhibit neutrophil apoptosis (20). Neutrophils were incubated for 3 h with or without Surfactant TA (5 mg/ml) in the presence or absence of 1 μg/ml of rG-CSF, and stained by a Diff-Quik method. Apoptotic neutrophils were counted by two blinded observers from 200 cells in each preparation under a microscope and expressed as a percentage of total cells.
Each experiment was carried out in a triplicate assay. The results shown in the figures were the most representative among more than three independent experiments. The values were expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA with p < 0.05 as a significant border.
Bleomycin is known to cause neutrophil alveolitis for several days after intratracheal instillation (12). To examine if exogenous Surfactant TA affected the superoxide production of neutrophils infiltrating the lungs, we targeted Surfactant TA on the cells on Day 1 and recovered the cells on Day 2 (Figure 1). Administration of Surfactant TA did not change the numbers in recovered cells (Figure 1A), the cell populations (neutrophil > 80%) (Figure 1B), and the cell viabilities (B: 97.2 ± 0.5%, BS5: 93.0 ± 2.1%, and BS10: 97.6 ± 0.8%) when compared with control animals. The superoxide production, however, was significantly decreased in the neutrophils treated with Surfactant TA in vivo (Figure 1C). The inhibition was shown depending upon the doses of exogenous Surfactant TA and observed both in nonstimulated and PMA-stimulated production. From these results it was demonstrated that exogenous Surfactant TA reduced superoxide production from neutrophils without changing the degree of neutrophil accumulation.
Fig. 1. Effects of exogenous Surfactant TA on neutrophils induced by bleomycin in hamster lungs. Hamsters were divided into three treatment groups; B, BS5, and BS10. Animals in all groups were intratracheally administrated with 0.5 mg/100 g body weight of bleomycin on Day 0, and then followed by the intratracheal instillation with saline (B) and 5 mg (BS5) and 10 mg/100 g body weight (BS10) of Surfactant TA on Day 1. On Day 2, all animals were killed and subjected to bronchoalveolar lavage (BAL). (A) Total cell yield. (B) Cell population. (C ) Superoxide production from BALF cells. PMN = polymorphonuclear cells; AM = alveolar macrophages; PMA = phorbol myristate acetate. Data are means ± SEM of each treatment group (n = number of animals). *p < 0.05, when compared with B.
[More] [Minimize]Exogenous Surfactant TA was shown to affect neutrophil functions in vivo. We investigated in the next step whether Surfactant TA affected purified human neutrophils in vitro.
Neutrophil adherence has been shown to be induced by the various stimuli, including FMLP, PMA, or cytokines such as TNF (17). The first experiments examined the effects of Surfactant TA on both basal and stimulated neutrophil adherence to plastic surfaces (Figure 2A). Surfactant TA inhibited FMLP (10 μM)-, PMA (100 ng/ml)-, and TNF (100 U/ml)-stimulated adherence as well as basal adherence (No Stimulation). In addition, FMLP-stimulated adhesion was inhibited by Surfactant TA in a dose-dependent manner (Figure 2B). FMLP-induced adherence was inhibited by 50% at 2 mg/ml of Surfactant TA. These results demonstrated that Surfactant TA inhibited neutrophil adherence to the plastic plate.
Fig. 2. Effects of Surfactant TA on human neutrophil adherence to the plastic plate in vitro. Purified human neutrophils (5 × 105) were mixed with Surfactant TA (concentrations as indicated) or control solution and incubated at 37° C for designated periods in the presence or absence of stimuli in a final volume of 200 μl in each well of a 96-well culture microplate. The nonadherent cells were vigorously washed and the adherent cells were stained with 0.5% crystal violet solution and subjected to measurements of absorbance at 570 nm. (A) Surfactant TA (5 mg/ml) inhibits both nonstimulated and stimulated neutrophil adherence to the plastic dishes. FMLP (10 μM), PMA (100 ng/ml), and TNF (10 U/ml) were used as stimulators. (B) Surfactant TA inhibits FMLP (10 μM)-stimulated neutrophil adherence in a dose-dependent manner. (C ) Time course of inhibition of neutrophil adherence by Surfactant TA. The neutrophil adherence assay was performed with FMLP (10 μM) as a stimulator in the presence or absence of Surfactant TA (5 mg/ml). At each designated time point, the incubation was terminated and the adherent activity was measured. (D) Effects of Surfactant TA pretreatment on neutrophil adherence. Neutrophils were preincubated for 1 h at 37° C with varying concentrations of Surfactant TA and washed three times with PBS to remove Surfactant TA. The adherence assay was then performed using FMLP (10 μM) as a stimulator. All data are shown as means ± SEM of a triplicate assay. *p < 0.05, when compared with Surfactant TA (−).
[More] [Minimize]To examine the possibility that Surfactant TA might enhance detachment of neutrophils that had adhered to the plastic plate, the time kinetics were examined up to 2 h of incubation (Figure 2C). In this experiment, the FMLP-stimulated adherence showed a biphasic pattern as described previously (17) and Surfactant TA inhibited the neutrophil adherence from the beginning of the incubation, suggesting that Surfactant TA did not cause detachment of the cells. The additional experiments, in which Surfactant TA was added to the neutrophils that had adhered to the plastic by PMA, demonstrated no difference in absorbance (data not shown), supporting that Surfactant TA did not cause detachment of neutrophils.
Aggregates of Surfactant TA might physically interfere with neutrophil adherence to the plastic plate. To exclude this possibility, the neutrophils were incubated with Surfactant TA and washed out free of Surfactant TA prior to the assay. Using these neutrophils, the adherence assay was performed with FMLP as a stimulator (Figure 2D). The result demonstrated that neutrophil adherence was inhibited depending upon the amounts of pretreated Surfactant TA. These results suggested that inhibition of neutrophil adherence by Surfactant TA was unlikely due to physical interference with cell-aggregates interaction.
Surfactant TA is a mixture of lipids and proteins like natural surfactant (21). To partially determine which component was more potent for inhibition of neutrophil adherence, Surfactant TA was pretreated by heating (100° C, 10 min) and used for the neutrophil adherence assay. Heat-treated Surfactant TA, however, still inhibited neutrophil adherence to the plastic plate like untreated Surfactant TA (FMLP-stimulated adherence at OD 570 nm, no Surfactant TA: 0.781 ± 0.037, 5 mg/ ml of untreated Surfactant TA: 0.415 ± 0.016, and 5 mg/ml of heat-treated Surfactant TA: 0.468 ± 0.059, triplicated assay). This result suggested that inhibition was mainly derived from heat-resistant components in Surfactant TA.
We next examined if Surfactant TA modulated production of superoxide anions from neutrophils in vitro. Surfactant TA inhibited PMA-stimulated superoxide production from neutrophils in a dose-dependent manner (Figure 3A). To address whether Surfactant TA scavenged the superoxide anions produced by neutrophils, superoxide anions were enzymatically generated from the reaction with xanthine and xanthine oxidase in the presence or absence of Surfactant TA (Figure 3B). The level of cytochrome c reduction by the enzymatically generated superoxide anions was not altered in the presence of Surfactant TA (5 mg/ml), whereas SOD (60 μg/ml) completely scavenged the produced superoxide anions. These results demonstrated that Surfactant TA acted on the neutrophils to inhibit superoxide production.
Fig. 3. Effects of Surfactant TA and DPPC liposomes on superoxide production from human neutrophils. The superoxide production from neutrophils was determined from a cytochrome C reduction as described in Methods. (A) Effects of Surfactant TA on both nonstimulated and PMA-stimulated superoxide production from neutrophils. The superoxide production is expressed as nmol/106 neutrophils. Data are shown as means ± SEM of a triplicate assay. (B) Effect of Surfactant TA on enzymatically produced superoxide anions. Xanthine (XA, 500 μM) and xanthine oxidase (XO, 10 mU/ml) were reacted in the presence or absence of Surfactant TA (5 mg/ml) or SOD (60 μg/ml). The superoxide production was expressed as the absorbance at 550 nm. Each value is the average of a duplicate assay in each treatment. (C ) Effect of the liposomes made of dipalmitoyl phosphatidylcholine (DPPC) on superoxide production from neutrophils. Data are shown as means ± SEM of a triplicate assay.
[More] [Minimize]The major surfactant lipid component is dipalmitoyl phosphatidylcholine (DPPC), which is important in reducing alveolar surface tension at an air-water interface (21). To investigate the mechanism(s) by which Surfactant TA modulates neutrophil functions, we prepared liposomes made of DPPC and examined if these liposomes inhibited superoxide production from neutrophils (Figure 3C). The results demonstrated that DPPC liposomes inhibited superoxide production from these cells in a dose-dependent manner.
It seemed reasonable to examine, as the first step for investigating the mechanism(s), if Surfactant TA bound neutrophils. As DPPC liposomes likely mimicked the inhibitory effect of Surfactant TA on superoxide production from neutrophils, we prepared 3H-DPPC liposomes, and binding studies were performed (Figure 4). Specific binding of 3H-DPPC liposomes to neutrophils was saturable by increasing liposome concentrations. This result showed that DPPC liposomes bound neutrophils and, therefore, suggested that such interaction was likely to initiate inhibition of neutrophil adherence or superoxide production seen by Surfactant TA.
Fig. 4. Binding of 3H-dipalmitoyl phosphatidylcholine (3H-DPPC) liposomes to neutrophils. The 3H-DPPC was prepared and binding studies were performed as described in Methods. Values are expressed as specific bound DPPC (μg/106 cells, means ± SEM, n = 4).
[More] [Minimize]Before investigating the mechanism(s) of Surfactant TA on human neutrophil functions in vitro, we examined whether Surfactant TA had direct cytotoxic effects on these cells by checking cell viability and LDH release. Even after a 1-h incubation with varying concentrations with Surfactant TA, there was neither a decrease in cell viability nor an increase in LDH release, suggesting that Surfactant TA was not cytotoxic to neutrophils in vitro (data not shown).
To further investigate the inhibitory mechanism(s) by Surfactant TA, human neutrophils after treatment with Surfactant TA were observed under a microscope (Figure 5). Unlike the control cells (Figure 5A), the neutrophils having picnotic nuclei were increased after Surfactant TA treatment (Figure 5B). This observation suggested Surfactant TA enhanced neutrophil apoptosis (20). The similar result was obtained by treating neutrophils with DPPC liposomes (Figure 5C), which mimicked inhibition of neutrophil superoxide production of Surfactant TA.
Fig. 5. Morphology of neutrophils incubated with Surfactant TA and DPPC liposomes. Isolated human neutrophils were incubated with or without Surfactant TA and DPPC liposomes and observed under a microscope (original magnification: ×400). Neutrophils having picnotic nuclei are indicated with closed triangles. (A) No Surfactant TA. (B) Surfactant TA (5 mg/ml). (C ) DPPC liposomes (0.5 mg/ml).
To further examine that Surfactant TA enhances neutrophil apoptosis, an immunocytochemical study was performed by targeting Fas antigen as one of its markers (Figure 6). The result showed that neutrophils after treatment with Surfactant TA expressed more Fas antigens on cell surfaces (Figure 6A) when compared with control cells (Figure 6B).
Fig. 6. Fas antigen presentation on neutrophils treated with Surfactant TA. Isolated human neutrophils were incubated at 37° C for 1 h with or without Surfactant TA and stained with a monoclonal antihuman Fas antibody (original magnification ×400). (A) No Surfactant TA. (B) Surfactant TA (5 mg/ml).
[More] [Minimize]The time course experiments revealed that, after Surfactant TA treatment, apoptotic neutrophils were significantly increased in a time-dependent manner when compared with control neutrophils (Figure 7). We also examined if Surfactant TA enhanced neutrophil apoptosis in vitro in the presence of human recombinant G-CSF, which has been shown to inhibit neutrophil apoptosis (20). The number of apoptotic neutrophils was reduced by rG-CSF, but Surfactant TA increased apoptotic neutrophils even in the presence of this cytokine (Figure 7). These results demonstrated that Surfactant TA could enhance neutrophil apoptosis in vitro even in the presence of the cytokine that inhibits apoptosis.
Fig. 7. Appearance of apoptotic neutrophils after incubation with Surfactant TA. Isolated human neutrophils were incubated for 1 and 3 h with or without Surfactant TA (S-TA, 5 mg/ml). Two hundred neutrophils were observed, and the cells with picnotic nuclei, as shown in Figure 5B, were defined as apoptotic and expressed as percent apoptotic cells. Human recombinant granulocyte-colony-stimulating factor (rG-CSF, 1 μg/ml) was used as a control for preventing apoptosis. Data are expressed as means ± SEM. *p < 0.05, when compared with S-TA (−).
[More] [Minimize]Surfactant TA was originally developed as a therapeutic material for neonatal respiratory distress syndrome (9). Surfactant TA has also been used for treatment of ARDS, and several successful outcomes have been reported (10, 11). Surfactant TA possibly supplements surfactant deficiency occurring in the lungs of patients with ARDS. Although neutrophil accumulation in the alveoli is considered as an important part in the pathogenesis of ARDS, the effects of administrated Surfactant TA on the neutrophil functions are poorly understood. Our present study investigated, as the first step, if exogenous Surfactant TA affected the function of neutrophils infiltrating hamster lungs after intratracheal administration of bleomycin. Although the numbers and the viability of the neutrophils obtained from bleomycin-treated lungs were unchanged, the superoxide production by these cells from Surfactant-TA-treated animals was significantly decreased, when compared with those from control animals (no Surfactant TA). The in vitro studies, as the second step, demonstrated that Surfactant TA inhibited the activities of adherence and superoxide production of human neutrophils purified from peripheral venous blood.
There are two ways of cell death; apoptosis and necrosis. As we obtained the data showing neither a decrease in cell viability nor an increase in LDH release soon after treatment with Surfactant TA, it was suggested that Surfactant TA did not induce neutrophil necrosis in vitro. In addition, morphologic changes of neutrophils after Surfactant TA treatment were noticeable. Neutrophils treated with Surfactant TA had picnotic nuclei (Figure 5B) and expressed more Fas antigens on cell surfaces (Figure 6B). As these characteristics were seen in apoptotic neutrophils (18), Surfactant TA was suggested to enhance apoptosis of these cells. As, in general, dying cells lose their particular functions, this phenomenon was likely related, at least in part, to the inhibitory mechanism(s) for neutrophil functions by Surfactant TA.
The average life span of neutrophils is approximately 24 h and neutrophils infiltrating inflammatory milieu are programmed to die (20). Neutrophils, however, are activated by many kinds of cytokines existing also in the inflammatory milieu. This is considered as one of the reasons for continuous neutrophil accumulation in lung injuries. Our in vitro observation demonstrated that Surfactant TA enhanced neutrophil apoptosis even in the presence of cytokines such as rG-CSF (Figure 7). Therefore, Surfactant TA was suggested to be a potential but strong inducer for neutrophil apoptosis.
From hamster lungs after Surfactant TA treatment, we could not obtain, by lavage, apoptotic neutrophils such as seen in the in vitro experiments. Bleomycin binds to DNA and destroys it by a free radical mechanism, leading to rampant cell death and tissue damage. It is a considerable leap from this model to adding Surfactant TA to normal neutrophils in a plastic dish; it is difficult to decide, at this point, which observation is more relevant. However, Cox and coworkers (22) showed that apoptotic neutrophils were rarely seen in LPS- administered rat lungs, probably because of rapid engulfment by alveolar macrophages. Although the data were not shown, alveolar macrophages engulfing apoptotic neutrophils were really obtained from bleomycin-administrated hamster lungs. One of the reasons for our conflicting observations between in vivo and in vitro treatments with Surfactant TA might be ascribed, in part, to this reason.
Surfactant TA contains lipids and proteins like natural surfactant (9). Thomassen and coworkers (23) demonstrated that synthetic surfactant (Exosurf), which did not contain any protein components, inhibited endotoxin-stimulated cytokine secretion by human alveolar macrophages. In our studies, heat inactivation of Surfactant TA did not alter its inhibitory activity on neutrophil adherence. In addition, DPPC liposomes, which represented a lipid component of surfactant, inhibited neutrophil superoxide production and induced apoptosis. These results suggested attribution of a lipid component to regulatory mechanisms for immune cells. We further prepared 3H-DPPC liposomes to investigate the inhibitory mechanism(s) on neutrophils. 3H-DPPC liposomes were shown to bind neutrophils in a saturable process. This interaction would be enough in initiating the neutrophil inhibitions seen by Surfactant TA.
Chao and coworkers (24) have demonstrated the inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. They further showed that this inhibitory effect by surfactant was specific for respiratory burst of neutrophils since neither calcium mobilization nor lactoferrin release was altered by the same treatment (25). Our results were somehow different from their observations. Firstly, Surfactant TA inhibited not only superoxide production but adherence. In addition, our preliminary data demonstrated that Surfactant TA also inhibited cell aggregation, chemotaxis, and elastase release of human neutrophils (Suwabe and coworkers, unpublished data). Secondly, our DPPC liposomes mimicked the inhibitory effect of Surfactant TA on superoxide production from neutrophils (Figure 3C), although their preparation of DPPC liposomes had no effects on respiratory burst from these cells. These differences may be ascribed to the difference in surfactant preparations or in ways of preparing lipid liposomes.
Surfactant is known to impair lung alveolar macrophage bactericidal activity (26). The present study also demonstrated that Surfactant TA inhibited neutrophil superoxide production. Our preliminary experiment demonstrated that a neutrophil MPO activity, detected from chemiluminescence, was also decreased by Surfactant TA treatment (Suwabe and coworkers, unpublished data). Both superoxides and MPO are involved in intracellular bacterial killing by neutrophils. Although we did not test the in vitro neutrophil bactericidal activities, these results imply that one must be mindful of opportunistic infections in the patients who receive intratracheal administration of Surfactant TA.
Immunomodulatory effects of surfactant and its components have been reported and some of them are contradictory. Wilsher and coworkers (27) reported that surfactant purified from humans, pigs, and rabbits suppressed human peripheral lymphocyte proliferation. Hayakawa and coworkers (28) demonstrated that rabbit natural surfactant inhibited the oxidative burst of alveolar macrophages in infant rabbits. Hoffman and colleagues (29) reported that the extracted rat lung surfactant proteins augmented alveolar macrophage migration, and Tenner and coworkers (30) and van Iwaarden and colleagues (31) found that surfactant-specific protein (SP-A) enhanced the phagocytotic activity of macrophages. In contrast, Weber and coworkers (32) reported that canine SP-A suppressed the respiratory burst of alveolar macrophages or neutrophils. Katsura and colleagues (33) demonstrated that human and rat SP-A inhibited superoxide production from rat alveolar macrophages. Although Surfactant TA is different from natural surfactant, our present study supported regulatory modulations of surfactant on neutrophil functions.
In conclusion, in vivo and in vitro treatments of neutrophils with Surfactant TA inhibited adherence and superoxide production of these cells. The lipid components were considered to be mainly involved in the inhibitory mechanisms. The inhibition was suggested to be mediated, at least in part, by enhancing apoptosis of neutrophils. These experimental results suggested that exogenous Surfactant TA might play an important role not only in improving surfactant functions but in preventing neutrophils from further activation in injured lungs.
The writers thank Dennis R. Voelker, Ph.D., Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO, for his kind review of the manuscript and significant comments. They also thank Tokyo Tanabe Co. Ltd. for the kind donation of Surfactant TA, and Chyugai Co. Ltd. for the kind donation of human recombinant granulocyte-colony-stimulating factor.
Supported by Tokyo Tanabe Co. Ltd., Tokyo, Japan.
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Presented in part at the Annual Meeting of the American Thoracic Society, Miami, Florida, 1992.
