Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.
acute respiratory distress syndrome; nitric oxide; inducible nitric oxide synthase; induced lung injury; rat
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INTRODUCTION |
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ACUTE RESPIRATORY DISTRESS
SYNDROME (ARDS) is a severe form of acute lung injury
(14) characterized by respiratory failure associated with
abnormal gas exchange, pulmonary infiltrates, and alterations in
surfactant composition and function (13, 32). The
essential precipitating events leading to the cellular and fluid
infiltration that characterize acute lung injury have not been clearly
defined. ARDS can result from direct or indirect insults, resulting in
localized inflammation in the lung (7). Associated with
this inflammatory process is the local production of proinflammatory
mediators (such as cytokines, prostaglandins, and free radicals), and
these mediators appear to further influence lung inflammation and
tissue damage. A number of studies have identified increased levels of
proinflammatory mediators such as tumor necrosis factor- (TNF-
)
(4, 20, 35, 38, 44), interleukin (IL)-8 (56),
IL-1
, and IL-6 (18, 27, 56) in the lungs of ARDS
patients and in animal models of this disease. Importantly, the degree
of polymorphonuclear neutrophil (PMN) activation or infiltration
appears to correlate with the levels of TNF-
, IL-6, and IL-8
contained in the lavage fluid and plasma of ARDS patients, and
bronchoalveolar lavage (BAL) levels are increased compared with
cytokine levels in healthy volunteers (4). Additional
evidence for cytokine participation in lung inflammation includes the
attenuation of lipopolysaccharide (LPS)- or IL-1-induced lung injury by
the administration of IL-1 receptor antagonist in rats
(47). In addition, neutralizing TNF antibodies prevent
lung injury associated with postperfusion syndrome (19, 46), and administration of IL-8 monoclonal antibodies prevents endotoxemia-induced ARDS (56). Other mediators such as
interferon-
(IFN-
) (36, 37) and products of
arachidonic acid metabolism are increased in animal models of acute
lung injury as well as in human studies of ARDS (11,
33).
Recently, attention has focused on the potential role of nitric oxide free radical (NO·) in mediating tissue damage and stimulating lung inflammation during the development of ARDS. Increased levels of NO have been detected in the exhaled gas in a rat model of lung inflammation due to sepsis (43). In addition, NO has been shown to inhibit type II cell metabolism and surfactant synthesis in vitro (15, 28). Importantly, lung injury induced by immune complexes (29), ischemia (21) or paraquat (2) involves NO· or its metabolites as indicated by increased levels of nitrate and nitrite in perfusate or lavage, the attenuation of each of these injuries by NO synthase (NOS) inhibitors, and the reversal of these protective effects by L-arginine. We have recently reported that lung injury induced by N-nitroso-N-methylurethane (NNMU) is dependent on NO· production. Administration of aminoguanidine, a selective inducible NOS (iNOS) inhibitor, significantly attenuates NNMU-induced alterations in gas exchange, decreased surfactant phospholipid protein ratio, elevated surface tension, and neutrophilic infiltration into the alveolar space in rat lungs (5, 6).
While numerous inflammatory mediators appear to participate in lung inflammation and injury, the cellular source(s) of these mediators and the manner by which these mediators induce lung damage have yet to be elucidated. In this study, we describe a novel animal model of lung injury that should allow for the identification of individual components (such as soluble mediators and infiltrating cell types) of the inflammatory response and the determination of how these mediators stimulate lung inflammation. Using an adoptive transfer approach, infiltrating lung cells isolated from LPS-treated rats are transferred to a normal, healthy control rat. The recipient animals show evidence of altered gas exchange and extravasation of plasma 48 h after transfer.
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MATERIALS AND METHODS |
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Animals. In all experiments, pathogen-free, male Sprague-Dawley rats (250-350 g) obtained from Harlan Sprague Dawley (Indianapolis, IN) were used. At least 1 wk before experiments, heparin-coated polyethylene catheters were inserted into the left carotid artery. The catheter was exteriorized at the base of the skull. The catheter was plugged with a stainless steel pin and used for the collection of blood samples for arterial blood gas (ABG) determinations. The rats were individually housed in plastic cages on self-watering racks in semi-barrier rooms and fed normal rat chow ad libitum. In the studies described, donor rats for the adoptive transfer experiments were placed into one of three groups: untreated, saline instilled, or LPS instilled. Five hours after LPS or vehicle instillation, animals were killed, and cells were collected by BAL (8 × 8 ml). The recovered BAL cells were then either placed in culture as controls and analyzed as described below or instilled into recipient rats. Forty-eight hours after cells were instilled, BAL cells were recovered from the recipient rats by lavage. Analysis of the recovered cells is described below. All common chemical reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted.
Statistics. Statistical significance was calculated using one-way ANOVA with Bonferroni post hoc analysis. The data presented are means ± SE.
Intratracheal LPS instillation. Donor animals were anesthetized by inhalation of halothane (Halocarbon Laboratories, River Edge, NJ). The animals were placed on a surgical board at an angle of ~45° with the head supported by an elastic cord placed over the upper incisors. The vocal chords were visualized with a modified otoscope. Saline or LPS (serotype 0111:B4, 10 mg/ml) was administered using a stainless steel right-angled miniature nebulizer (Penn-Century, Philadelphia, PA) (50, 52). LPS was administered at a dose of 15 mg/kg.
Alveolar-arterial oxygen difference analysis. Arterial blood samples (0.7 ml) were collected directly into a heparin-flushed 1.0-ml syringe via the indwelling arterial catheter from spontaneously breathing, unanesthetized rats. ABGs were measured using a Radiometer ABL 520 (Radiometer, Copenhagen, Denmark) blood microsystem. Oxygen and carbon dioxide partial pressures (PO2 and PCO2, respectively) from ABG determinations were used to calculate the alveolar-arterial oxygen difference. The alveolar PO2 (PAO2) was calculated using the alveolar gas equation, and the arterial PO2 (PaO2) was obtained from ABG determinations. Values are corrected for body temperature determined at the time blood was drawn (22). The respiratory quotient and the inspired O2 fraction were assumed to be constant at 0.8 and 0.21, respectively.
Collection of inflammatory cells. Five hours after LPS or vehicle instillation and after blood samples were drawn and ABGs determined, the lungs were removed, and ~50 ml of BAL were collected from each isolated rat lung in successive 8-ml aliquots. Briefly, rats were anesthetized with pentobarbital sodium (65 mg/kg body wt ip), the trachea was exposed, and a tracheostomy tube was inserted. The lungs were perfused through the right ventricle of the heart with a buffered salt solution (125 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 17 mM HEPES, 10 µg/ml gentamicin, and 1 mg/ml dextrose, pH 7.4) to remove blood cells from the pulmonary vasculature. The intact lungs were then lavaged with 8-ml aliquots of the buffered salt solution. The lavage was centrifuged at 300 g for 15 min at 4°C. The supernatant was reserved for the isolation of a crude surfactant pellet. The cell pellet was resuspended, the total volume was brought to 50 ml with the buffered salt solution, and the suspension was centrifuged again at the same relative centrifugal force. Donor cells were resuspended in a total of 5 ml and the total viable cell number was determined by trypan blue exclusion. The cells were then resuspended in the salt solution such that recipient rats received 38 × 106 inflammatory cells in a total volume of 1 ml while under halothane anesthesia. Aliquots of the cell suspension were centrifuged onto glass slides and stained using a modified Wright's stain procedure (Diff-Quik; Baxter Scientific) for differential cell counts.
In adoptive transfer experiments, the cells were collected by lavage 48 h after cell instillation. The cells were centrifuged and washed as described above. The cell pellet was resuspended in Ham's F-12 nutrient mixture (GIBCO BRL) culture medium containing 10% FCS, gentamicin (50 µg/ml), streptomycin (1 mg/ml), and penicillin (100 U/ml), and aliquots were taken for cell counting and differential counts.Phospholipid and protein analysis. The BAL cell-free supernatant was centrifuged at 48,000 g for 1 h to obtain a crude surfactant pellet (CSP) that was resuspended in 300 µl of 154 mM NaCl and 5 mM CaCl2 (5, 6, 16). An aliquot was taken and the protein concentration was determined by a modified Lowry assay (25, 31). Phospholipid concentration was determined (16) on an aliquot of CSP extracted according to Bligh and Dyer (3).
Analysis of CSP surface activity. Phospholipid concentration of the CSP was determined as noted above, and the pellet was diluted to a final phospholipid concentration of 1.5 µmol/ml. Surface tension at minimum bubble size was determined on a pulsating bubble surfactometer after 5 min at 37°C, 50% surface area compression, and 20 cycles/min (6, 8, 16). Dynamic surface tension <10 mN/m indicates functional pulmonary surfactant in a crude surfactant pellet.
Analysis of nitrite and nitrate in cell culture media.
Freshly isolated BAL cells were seeded onto a 24-well tissue culture
plate at 300,000 viable cells/well in 500 µl of Ham's F-12 culture
medium containing 10% FCS, gentamicin (50 µg/ml), streptomycin (1 mg/ml), and penicillin (100 U/ml). To designated wells, 1 mM
aminoguanidine, 10 µg/ml LPS, and/or 1 µM actinomycin D was added.
Cells were cultured at 37°C, 85% humidity, and 10% CO2
for 24 h. After 24 h, the culture medium was collected and centrifuged, and the supernatant was stored at 20°C for nitrite and
nitrate determination. The remaining cell pellets were stored at
20°C for Western blot analysis of iNOS protein. Following enzymatic
conversion of nitrate to nitrite by nitrate reductase (12), total nitrite was determined by the Griess assay
(26). Total nitrite concentration was based on a sodium
nitrate standard curve.
Western blot analysis for iNOS.
For culture samples, 25 µl of 5× sample buffer (0.5 M
Tris · HCl, 2.0% -mercaptoethanol, 10% SDS, and 0.5%
bromphenol blue, pH 6.6) were added to 300,000 cells in 100 µl of
saline, and the cells were vortexed and boiled for 10 min. Protein was
separated by SDS gel electrophoresis and transferred to a
nitrocellulose membrane overnight (Bio-Rad Mini Trans-Blot cell).
Detection of iNOS protein was performed by enhanced chemiluminescence
(Amersham) using rabbit anti-mouse iNOS (Cayman Chemicals) at a
dilution of 1:2,000 and horseradish peroxidase-conjugated donkey
anti-rabbit (Jackson Immunological Research) at a dilution of 1:6,000
(5, 6). IL-1
-stimulated (1.0 U/ml, 24 h) RIN-m5F
cells were used as an iNOS-positive control (17).
Extravasation of Evans blue dye into the alveolar space. Untreated animals, animals receiving cells from normal donors, or animals receiving cells from LPS-treated animals were killed 48 h after cell instillation. One hour before death, 20 mg/kg Evans blue dye (EBD) was administered via a tail vein (45). The rats were anesthetized with pentobarbital sodium, a 0.6-ml blood sample was taken from the inferior vena cava, and the rats were exsanguinated as described above. The lungs were instilled with a single 8.0-ml volume of the balanced salt solution, and the lavage was collected. The BAL was centrifuged as described above and the fluid was lyophilized. The dried lavage was resuspended in 0.8 ml of water and extracted according to Bligh and Dyer to partition the dye into the aqueous phase to avoid interference from surfactant in lavage (51). EBD concentration was determined spectrophotometrically on 200-µl aliquots in 96-well microtiter plates at 620 nm using a standard curve prepared in Bligh and Dyer upper phase. Plasma concentrations of the dye were determined using a standard curve prepared in saline. The tissue content of EBD was determined by homogenizing lyophilized lung tissue in 10 ml of formamide. Following overnight extraction, the tissue was centrifuged at 3,000 g. EBD concentration of the supernatant was determined spectrophotometrically at 620 nm using a standard curve prepared in formamide. Lavage content is expressed as a percentage of the plasma concentration, and tissue content of EBD is expressed as micrograms per lung.
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RESULTS |
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Total cells recovered after adoptive transfer.
To more fully characterize the role of inflammatory cells in the
development of ARDS, we have developed a novel adoptive transfer model
in which cells from LPS-injured rats are transferred to normal animals
intratracheally. In these experiments, we first determined the total
number of inflammatory cells in recipient lungs 48 h after
adoptive transfer. Each recipient rat received ~38 × 106 donor cells isolated from either control rats or rats
5 h after intratracheal LPS instillation. This number of cells is
comparable to the number recovered from animals 5 h after LPS
administration (data not shown). Figure
1A shows that the
total number of cells recoverable from recipient rats receiving cells
from LPS-treated donors 48 h after instillation was of the same
order of magnitude as the number of cells delivered to each recipient
animal (45.9 ± 11.6 × 106 vs. 37.8 × 106, respectively). Importantly, the number of cells
recovered from recipient rats receiving cells from normal donors was
not different from animals receiving saline (11.0 ± 3.6 × 106; Fig. 1A) or no treatment (7.7 ± 0.8 × 106; data not shown), although these animals
received 38.3 × 106 donor cells. These results show
that rats receiving cells from normal donors were able to clear the
donor cells from the lung over the 48-h period. However, rats receiving
cells from LPS-treated donors did not clear these inflammatory cells.
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Differential cell counts of bronchoalveolar cells after adoptive transfer. To gain a more thorough understanding of the types of cells present in healthy animals and in the injury model, differential cell counts were performed. As shown in Fig. 1B, cells recovered by BAL of healthy animals were almost entirely macrophages. BAL cells recovered from donor rats treated for 5 h with LPS were 24.6% macrophages and 73.4% neutrophils. Importantly, 31.9 ± 7.4% of the BAL cells isolated from recipient rats 48 h after transfer of LPS-treated donor cells were PMNs. In contrast, cells recovered from recipient rats receiving normal donor cells were 81.5 ± 3.2% macrophages and 5.6 ± 1.2% neutrophils, a cellular composition comparable to the populations observed in saline-treated rats and in untreated rats (93.4 and 97% macrophages, respectively). These results indicate that the cell population recovered after adoptive transfer, like that seen after 5 h of LPS exposure, contained a significant percentage of neutrophils in the lung.
Alveolar-arterial oxygen difference after adoptive transfer.
As an indicator of lung function, ABGs of recipient rats 48 h
after adoptive transfer of BAL cells isolated from control and LPS-treated rats were determined, and the alveolar-arterial oxygen difference was calculated. Five hours of LPS treatment produced an
alveolar-arterial oxygen difference indicative of alterations in gas
exchange associated with lung injury (Fig.
2A). The difference observed
in recipient rats receiving 37.8 × 106 cells from
LPS-treated donors was increased 13-fold compared with that in the
control rats and rats receiving donor cells (38.3 × 106) isolated from untreated donors. While this increase
did not achieve statistical significance, the level of impairment in
gas exchange did approach the value used to define significant
deterioration of gas exchange (25 mmHg) (1, 16).
Alveolar-arterial oxygen difference from animals receiving cells from
normal donors was not different from that in saline-treated animals
(Fig. 2A) or untreated animals (data not shown).
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Effects of adoptive transfer on pulmonary surfactant. To determine the effects of adoptive transfer on pulmonary surfactant, a crude surfactant pellet was isolated from cell-free lavage, and the phospholipid protein ratio of the CSP was determined. Adoptive transfer of cells from control donor or from LPS-treated donor rats had no effect on the phospholipid protein ratio of recipient rat surfactant 48 h after transfer (Fig. 2B). However, 5 h after LPS treatment of LPS donor rats, the phospholipid protein ratio was significantly increased compared with that observed in CSP isolated from control donor rats. Thus adoptive transfer of inflammatory cells does not appear to affect the quality of pulmonary surfactant over the 48 h.
Consistent with normal phospholipid protein ratio, surfactant function is not altered in rats receiving BAL cells isolated from LPS-treated donor rats compared with animals receiving control BAL cells from control rats. As seen in Fig. 2C, the minimum surface tension was well below 10 mN/m in all recipient groups examined, indicative of normal surface activity in each group of rats.Activation state of BAL cells before and after adoptive transfer.
To determine the activation state of BAL cells before and after
adoptive transfer, BAL cell production of NO and expression of iNOS
were examined. For these experiments, BAL cells were isolated from
recipient rats 48 h after the transfer of 38 × 106 cells isolated from control or LPS-treated donor rats.
The cells were then cultured for 24 h in the presence and absence
of LPS or actinomycin D, and iNOS expression and NO· production were determined. Also, samples were prepared for SDS gel electrophoresis immediately after isolation. As shown in Fig.
3, following a 24-h ex vivo culture, BAL
cells isolated from rats treated with LPS for 5 h produced high
levels of NO· (Fig. 3A) and expressed iNOS (Fig.
3B). iNOS expression appears to be stimulated in vivo
because the subsequent addition of LPS during the ex vivo 24-h culture did not further enhance iNOS expression above the level of iNOS expressed immediately after isolation. In addition, the transcriptional inhibitor actinomycin D did not inhibit iNOS expression during the 24-h
ex vivo culture. Cells isolated from saline-treated animals did not
express iNOS (either immediately after isolation or following the 24-h
culture) or produce measurable amounts of NO following a 24-h culture.
However, treatment with LPS (10 µg/ml) during this 24-h culture of
control cells elicited an ~10-fold increase in NO· production. BAL
cells isolated from recipient rats 48 h after adoptive transfer of
BAL cells isolated from LPS-treated donor rats produced NO· at levels
similar in magnitude to those produced by control BAL cells treated for
24 h with LPS or untreated BAL cells isolated from rats treated
with LPS for 5 h as determined by nitrite/nitrate determination.
Consistent with NO· production, these cells expressed high levels of
iNOS following the 24-h culture. Although the BAL cells isolated from
rats treated for 5 h with LPS expressed iNOS, BAL cells isolated
48 h after adoptive transfer no longer expressed iNOS immediately
after isolation (preculture condition). However, BAL cells isolated
48 h after adoptive transfer expressed iNOS following a 24-h
culture, and iNOS expression was prevented by actinomycin D. Importantly, these findings suggest that BAL cells isolated 48 h
after adoptive transfer were activated in vitro, and this is most
likely due to the release of soluble mediators that stimulated iNOS
expression during the ex vivo culture. Consistent with these findings,
BAL cells isolated 48 h after transfer of control donor BAL cells
expressed iNOS and produced NO only when treated in vitro with LPS.
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Effects of adoptive transfer on alveolar plasma transudation.
Forty-eight hours after instillation of BAL cells isolated from normal
donors or from animals treated with intratracheal LPS for 5 h,
recipient rats received 20 mg/kg of EBD. After 1 h, the animals
were killed, a blood sample was obtained, and the lungs were lavaged
one time, with the lavage fluid recovery at 75.8 ± 0.8% for
untreated controls, 70 ± 2.6% for animals receiving cells from
LPS-treated donors, and 75.8 ± 1.4% for animals receiving cells
from normal donors. The lung was then perfused and the plasma, lavage,
and tissue concentrations of EBD were determined. There was no
significant difference in the plasma concentration of EBD in the three
groups (297 ± 9.3, 335 ± 9.6, and 317 ± 15.9, respectively). However, there was a significant increase in the
concentration of EBD, expressed as a percentage of plasma
concentration, in the alveolar lavage of recipient rats receiving donor
cells from LPS-treated animals compared with that in untreated controls
or rats receiving normal donor cells (Fig.
4A). Consistent with lung damage, the concentration of EBD was significantly greater in the
adoptive transfer lung tissue (Fig. 4B) after lavage and
perfusion of the vasculature, indicative of extravasation of the dye.
As shown in Fig. 4C, even after lavage and perfusion, areas
of infiltration of EBD into the air spaces were visible in the adoptive
transfer lung (left lung in Fig. 4C). These
results indicate that adoptive transfer of inflammatory cells from
LPS-injured lungs influenced movement of fluid from the pulmonary
vasculature into the airways.
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DISCUSSION |
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Numerous animal models have been developed to examine the various etiologies that can lead to the development of acute lung injury (ALI) (53). These include pneumonia-induced lung injury (48, 49), acid aspiration (9, 10, 24), endotoxin infusion (34, 38, 39), bacteremia (30), intratracheal endotoxin administration (52, 55), and administration of lung toxins such as NNMU (1, 16) and 3-methylindole (23, 54). One characteristic of human ARDS encompassed into each of these models is the infiltration of inflammatory cells into the interstitium and the alveolar space of the lung (7). These inflammatory cells, as well as resident type II pneumocytes and perhaps other resident cells, are capable of producing a number of proinflammatory mediators. The role of each of these inflammatory molecules in the development of ALI and ARDS, as well as their cellular sources, has long been an area of intense investigation. In applying adoptive transfer of lung disease in this study, we have developed a model system that will allow us to dissect the role of each cell type in the development of ALI and ARDS.
In this study, we show that in rats receiving cells from control donors, the number of cells remaining in the lung 48 h after transfer was similar to the number of cells recovered from untreated rats or rats receiving saline alone. In the control adoptive transfer rats, the majority of the cells were macrophages, with a limited number of infiltrating PMNs. This finding is consistent with the cell population in saline-treated rats and indicates that the majority of the cells that were transferred were cleared from the lung within 48 h. After adoptive transfer of cells from LPS-treated rats, the number of cells remaining in the lung was of the same order of magnitude as the number of cells transferred. This cell population 48 h after transfer of cells from LPS-treated donor rats contained significant numbers of PMNs (31%), the level similar to the percentage of PMNs found in the BAL of rats treated for 5 h with LPS (47.5%). These results indicate that after adoptive transfer, the cell population is comparable to that seen following LPS exposure, as well as demonstrating an increase in the number of neutrophils, consistent with the cellular composition observed in ARDS and in animal models of this syndrome.
In examining indicators of lung function, adoptive transfer had no apparent effect on the quality or function of pulmonary surfactant, as indicated by normal phospholipid protein ratios and normal surface activity. The alveolar-arterial oxygen difference was not significantly different from either saline-treated controls or adoptive transfer controls. However, the difference was increased severalfold above the control groups and was 43% of that seen in LPS-treated animals. These results indicate that there was a trend toward alterations in gas exchange in the adoptive transfer experiments. The lack of significant effect on indicators of lung function suggests that unlike the aerosol delivery of LPS, the liquid bolus delivery of cells does not produce as global an injury because of uneven distribution of these cells. Gas exchange in uninjured areas of the lung may be sufficient to keep the alveolar oxygen difference at levels lower then that seen in the other models of ALI or ARDS. It has been observed that lung injury, as indicated by edema formation, can be dissociated from hypoxemia in ARDS models (40, 41).
The cells recovered from animals 48 h after receiving cells from normal donors produced basal levels of NO· in culture, and this level was enhanced sixfold following LPS treatment in vitro. Cells from LPS-treated animals produced much higher levels of NO·, which resulted from iNOS expression in vivo, as indicated by iNOS expression detected immediately after isolation. Importantly, the activation state of the BAL cells isolated from rats treated for 5 h with LPS was significantly different from the activation state of BAL cells isolated from rats 48 h after adoptive transfer of donor cells isolated from LPS-treated rats. This was reflected in the in vivo stimulation of iNOS expression in BAL cells isolated 5 h after LPS administration, while BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation. These cells required a 24-h ex vivo culture period to express iNOS. These findings suggest 1) that donor BAL cells from LPS-treated rats are not the same as the cells isolated 48 h after adoptive transfer or 2) that BAL cells isolated 48 h after adoptive transfer of donor cells from LPS-treated rats are the same cells, but these cells no longer express iNOS. While both these hypotheses are possible, we favor the hypothesis that the adoptively transferred BAL cells (isolated from LPS-treated donors) stimulated additional inflammatory cells to migrate to the lung, and it may be that this influx of inflammatory cells mediates lung injury. However, future studies using vital dyes will be required to determine whether the new inflammatory cells migrate to the lung and whether these cells mediate tissue damage in the adoptive transfer model of lung injury.
Of concern in these studies is that the lung damage observed in the adoptive transfer experiments could result from LPS carryover in the lavage. However, experimental evidence does not support this conclusion. First, iNOS expression differs under the various conditions. Following adoptive transfer, BAL cells do not express iNOS protein immediately after lavage but do after 24 h of ex vivo culture. Cells isolated 5 h after tracheal instillation of LPS express iNOS immediately following isolation as well as after a 24-h ex vivo culture. Similarly, immediately following isolation, cells obtained 48 h after LPS instillation also express iNOS (data not shown). Second, based on the results of Stamme and Wright (42), we calculated that the number of cells that were instilled would bind ~250 ng of LPS, which could become available in the lung. However, BAL cells isolated 48 h after the administration of 250 ng of LPS did not express iNOS either before or after a 24-h ex vivo culture or produce significant amounts of NO· unless exposed to LPS in vitro. Third, treatment of RAW 264.7 cells in culture with increasing concentrations of the lavage supernatant did not result in iNOS expression or significant NO· production following a 24-h incubation. Furthermore, using the RAW 264.7 cells as a biological assay for LPS, we determined that the lavage supernatant obtained 5 h after LPS administration contained 200 ng/ml of LPS in the lavage supernatant. This is <250 ng/ml, which we showed did not induce iNOS expression or NO· production in cells isolated 48 h after LPS instillation. Taken together, these data make it unlikely that lung injury induced by adoptive transfer is due to LPS that may be carried over during transfer.
The results presented in this study indicate that it is possible by adoptive transfer of inflammatory cells to induce lung injury in the rat. Using this new animal model, it should be possible to independently determine the contribution of each of the inflammatory cell types in lung injury by the selective transfer of macrophages and PMNs, either from injured animals or activated in vitro. Using this approach, it will be possible to isolate inflammatory cells from lungs injured by different etiologies as well as from specific transgenic or knockout mice and adoptively transfer these cells to normal control animals, enabling us to assess the contributions of these cell types and/or inflammatory mediators to the development of lung injury.
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ACKNOWLEDGEMENTS |
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We acknowledge the helpful support and discussion of Dr. William J. Longmore. We also acknowledge Dr. Randy Sprague for his helpful discussion of this manuscript.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants DK-52194, AI-44458, and HL-13405.
Address for reprint requests and other correspondence: M. A. Moxley, 1402 South Grand Boulevard, Saint Louis Univ. School of Medicine, Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis, MO 63104-1079 (E-mail: moxleyma{at}slu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2000; accepted in final form 22 May 2000.
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