* Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, Buenos Aires, Argentina; and
Institute of Medical Research "Alfredo Lanari" and Department of Microbiology, Buenos Aires University School of Medicine, Buenos Aires, Argentina
Correspondence: Silvina Raiden, Laboratorio de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail: geffner{at}mail.retina.ar
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Key Words: chemotaxis • angiotensin • Pseudomonas aeruginosa
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The current study was undertaken to analyze the effect of losartan in vivo in rat models of lung injury triggered by fMLP or Pseudomonas aeruginosa. In both models, we observed that losartan markedly prevents neutrophil recruitment in the lung.
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Preparation of neutrophils
Citrated blood samples were obtained from adults male Wistar
rats, and neutrophils were isolated by dextran sedimentation and
Histopaque gradient centrifugation, as previously described
[10
]. Contaminating erythrocytes were removed by
hypotonic lysis. After washing, the cells (>88% neutrophils on May
Grunwald-Giemsa-stained Cytopreps) were resuspended at the desired
concentration in RPMI 1640 medium (Gibco, Detroit, MI) and supplemented
with 1% heat-inactivated fetal calf serum (FCS; Gibco).
Chemiluminescence (CL) assay
Neutrophils were suspended at 2.5 x 106/ml in
RPMI 1640 medium supplemented with 1% FCS. Luminiscence responses were
measured with a Lumi-aggregometer (Chrono-Log Corp., Haverton, PA) at
1000 revolutions/min and 37°C in the presence of luminol (0.1 µM),
as we previously described [7
]. In all cases, light
emission was registered continuously for 10 min. Data are expressed as
the maximum response observed during this period in relative CL units.
One CL unit was defined as 1-cm shifting of the light-emission signal
on the paper recorder.
Adherence assay
Neutrophils were suspended in RPMI 1640 medium supplemented with
1% FCS and were labeled with Na2CrO4 (1
µCi/106 cells) for 1 h at 37°C. The cells were
then washed four times with saline and resuspended in RPMI 1640 medium
supplemented with 10% FCS to a density of 4 x
106/ml. This suspension (100 µL) was added to each well
in 96-well, flat-bottomed, polystyrene plates. Neutrophils were
incubated in the presence or absence of different stimuli for 30 min at
37°C in 5% CO2/95% humidified air and were washed three
times with culture medium to remove nonadherent neutrophils. Adherent
neutrophils were then lysed with 1 N NH4OH, and the
radioactivity present in the lysates was measured. Cell adherence was
expressed as the number of neutrophils that remained adherent to the
plastic surface after washing.
Assessment of lung myeloperoxidase activity
Neutrophil infiltration into the lung was quantified by
measuring myeloperoxidase (MPO) activity in lungs, as demonstrated
previously [11
]. Briefly, lungs were homogenized and
treated with Triton X-100 in potassium phosphate buffer, pH 6.0. After
centrifugation at 2000 g for 30 min, the supernatant fluids
were reacted with H2O2 (30% stock diluted
1:100; Sigma) in the presence of 0-dianisdine hydrochloride
(1 mg/ml; Sigma), and the MPO content was shown as a change in optical
density (OD) at 460 nm.
Histopathologic studies
Rat lung tissue was fixed with 10% buffered formalin, pH 7.2,
dehydrated in graded alcohols, embedded in paraffin, and cut into 6
µm sections. Mounted sections were stained for light microscopy with
hematoxylin and eosin. Sections were examined for features of lung
injury, including congestion, alveolar edema, and accumulation of
inflammatory cells. All morphologic studies were done by a pathologist
blinded, with respect to the different experimental groups studied.
Animal models
Adults male Wistar rats weighing 
250 g were used in all
experiments. Animals were housed under standard lighting (lights on
from 6.00 to 18.00 h) and temperature (23°C) conditions. Food and
water were available ad libitum. Rats were anesthetized i.p.
with urethane (1.2 g/kg body weight), and the trachea was exposed.
Then, 50 µl fMLP (10-6 M), aIgG (500
µg/ml), IC (100 µg/ml), Zy (200 µg/ml), or human recombinant C5a
(100 µg/ml) was instillated via an intratracheal catheter during
inspiration. The concentration of each stimulus was selected on the
basis of preliminary experiments (unpublished results). Immediately
thereafter, losartan (0.4–20 µg/kg/min), saralasin (20 and 200
µg/kg/min), captopril (20 and 200 µg/kg/min), or saline (controls)
were administered by continuous i.v. infusion. Rats were sacrificed
4 h after challenging, and lung MPO content was evaluated as
described above.
In another set of experiments, rats were challenged by intratracheal (i.t.) instillation of live Pseudomonas aeruginosa isolated from a bacteremic patient. Bacteria were maintained in peptone broth containing 25% glycerol. The strain was propagated on tryptone soy agar plates for 24 h at 37°C. Then, one colony was transferred to tryptone soy broth for an additional period of 24 h at 37°C. After this time, the bacteria were centrifuged at 3000 g for 20 min, and the bacterial pellet was washed twice with saline. Finally, the pellet was resuspended in saline at a concentration of 109 colony-forming units (CFU)/ml, and 50 µl of this suspension was i.t. instillated. Immediately thereafter, losartan (20 µg/kg/min) or saline (controls) was administered by continuous i.v. infusion. In both groups of animals (untreated and losartan-treated), we performed two different experiments: 1) Animals were sacrificed 4 h after challenging, and the increase in lung MPO activity was analyzed as described above. 2) The survival of the animals of each group was analyzed at 8 and 18 h after i.t instillation of Pseudomonas aeruginosa.
Hemorrhage index
Anesthetized rats were challenged by i.t. instillation of fMLP
(50 µl, 10-6 M). Immediately thereafter,
they received red blood cells (injected i.v.) from wild-type rats
labeled with 51chromium (51Cr-RBC). Losartan
(20 µg/kg/min) or saline (controls) was then administered by
continuous i.v. infusion. Rats were killed 4 h later, and a blood
sample (1 ml) was obtained from the inferior vena cava. Following
exsanguination, the pulmonary circulation was flushed with 5 ml sterile
saline. The lungs were assayed for 51Cr radioactivity as
was the venuos blood obtained at death. The hemorrhage index was then
determined for each animal as follows: index = lungs cpm/1 ml
blood cpm.
Statistical analysis
Results are expressed as means ± SE.
Statistical significance was determined using Student’s
t-test. A probability level of p < 0.05 was
considered statistically significant.
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Figure 1. Effect of losartan on neutrophil chemiluminescence and adherence
responses triggered by different stimuli. Chemiluminescence emission
(A) and adherence (B) were assessed as described in Materials and
Methods, in the absence (open bars) or presence (cross-hatched bars) of
losartan (10 µg/ml), which was added 5 min before the addition of
stimuli. Results are expressed in relative chemiluminescence units
(RCLU) and number of adherent cells, respectively. The following
stimuli were used: fMLP (25 nM), IC (50 µg/ml), Zy (50 µg/ml), and
Con A (20 µg/ml). Data are expressed as the arithmetic mean ±
SE of four to seven experiments performed in duplicate.
*P < 0.005 vs. neutrophils stimulated by fMLP in the
absence of losartan.
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Figure 2. Losartan inhibits lung-neutrophil recruitment triggered by fMLP.
Neutrophil infiltration was quantified by measuring MPO activity in
lungs 4 h after i.t. instillation of fMLP (50 µl,
10-6 M). Data are expressed as changes in
absorbance at 460 nm. Open bar, saline-treated rats challenged by fMLP;
cross-hatched bars, losartan-treated rats challenged by fMLP. Each bar
represents the arithmetic mean ± SE of five to nine
rats. Statistical significance, *P < 0.01,
**P < 0.05, compared with saline-treated rats
challenged by fMLP.
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Figure 3. Histological examination of lung sections. Four hours after i.t.
instillation of saline or fMLP (50 µl, 10-6
M), lung sections were obtained from: (a) saline-instillated rats
treated with saline, (b) fMLP-instillated rats treated with saline, and
(c) fMLP-instillated rats treated with losartan (20 µg/kg/min).
Sections were stained for light microscopy with hematoxylin and
eosin.
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Figure 4. Time-course study of the inhibitory effect of losartan on
lung-neutrophil recruitment triggered by fMLP. Losartan (10
µg/kg/min) was administered as a continuous i.v. infusion for 1 h. After this time, fMLP (50 µl, 10-6 M) was
i.t. instillated immediately or 2, 4, or 6 h after losartan
infusion was completed. Neutrophil infiltration was quantified by
measuring MPO activity in lungs 4 h after instillation of fMLP.
Data are expressed as changes in absorbance at 460 nm. Open bar,
saline-treated rats challenged by fMLP; cross-hatched bars, losartan
(10 µg/kg/min)-treated rats challenged by fMLP. Each bar represents
the arithmetic mean ± SE of four to six rats.
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Losartan does not inhibit neutrophil recruitment in the lung
induced by i.t. instillation of aIgG, IC, Zy, or C5a
To determine if losartan was able to inhibit lung-neutrophil
recruitment triggered by other stimuli, in another set of experiments,
we examined the effect of losartan on lung inflammatory responses
triggered by i.t. instillation of aIgG, IC, Zy, and the chemotactic
peptide C5a. As expected, losartan did not prevent the increase in MPO
content induced by these stimuli (Fig. 5
).
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Figure 5. Losartan does not inhibit lung-neutrophil recruitment triggered by
aIgG, IC, Zy, or C5a. Neutrophil infiltration was quantified by
measuring MPO activity in lungs 4 h after i.t. instillation (50
µl) of aIgG (500 µg/ml), IC (100 µg/ml), Zy (200 µg/ml), or C5a
(100 µg/ml). Data are expressed as changes in absorbance at 460 nm.
Open bars, saline-treated rats; cross-hatched bars, losartan (20
µg/kg/min)-treated rats. Each bar represents the arithmetic mean ± SE of four to eight rats.
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Figure 6. Captopril and saralasin do not inhibit lung-neutrophil recruitment
triggered by fMLP. Neutrophil infiltration was quantified by measuring
MPO activity in lungs 4 h after i.t. instillation of fMLP (50
µl, 10-6 M). Data are expressed as changes
in absorbance at 460 nm. Open bars, saline-treated rats; hatched bars,
captopril-treated rats; cross-hatched bars, saralasin-treated rats.
Each bar represents the arithmetic mean ± SE of four
to five rats.
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Figure 7. Losartan inhibits lung-neutrophil recruitment triggered by
Pseudomonas aeruginosa. Neutrophil infiltration was
quantified by measuring MPO activity in lungs 4 h after i.t.
instillation of Pseudomonas aeruginosa (50 µl,
109 CFU/ml). Data are expressed as changes in absorbance at
460 nm. Open bars, saline-treated rats; cross-hatched bars, losartan
(20 µg/kg/min)-treated rats. Each bar represents the arithmetic
mean ± SE of five rats. Statistical significance,
*P < 0.01, compared with saline-treated rats
challenged by Pseudomonas aeruginosa.
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Figure 8. Losartan improves survival of Pseudomonas
aeruginosa-instillated rats. Animal survival was analyzed at
8 h and 18 h postinstillation with Pseudomonas
aeruginosa. Open bars, saline-treated rats; cross-hatched bars,
losartan (20 µg/kg/min)-treated rats. Data are expressed as
percentage of death (n=10 for each group).
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B (NF-B) in
phagocytes [24
]. Further observations indicated that
inhibition of AII activity prevents monocyte chemoattractant protein-1
expression and macrophage infiltration in a rabbit model of early
accelerated atherosclerosis [25
], as well as the
development of renal injury induced by immune complexes in mice
[26
, 27
]. Taken together, these findings
suggest that AII plays an important role in certain inflammatory
responses and supports the possibility that losartan and other
inhibitors of AT1 receptors represent a useful tool in the treatment of
these processes. In the present work, we show that losartan inhibits efficiently neutrophil recruitment in the lungs triggered by i.t. instillation of fMLP. Histological evaluation of lungs and the analysis of hemorrhage indices showed that losartan prevented the induction of lung injury associated with neutrophil infiltration. It seems likely that the mechanism(s) through which losartan exerts these anti-inflammatory effects depends on its ability to inhibit FPR, a property that we have recently described [7 ]. In support of this hypothesis, we found that: 1) losartan inhibited rat neutrophil activation triggered by fMLP markedly, without affecting the responses induced by other stimuli, such as IC, Zy, and Con A; and 2) losartan did not prevent lung-neutrophil recruitment induced by aIgG, IC, Zy, or C5a. Conversely, the fact that neither captopril, an ACE inhibitor, nor saralasin, a peptidic inhibitor of AT1 receptors, were able to prevent lung-neutrophil recruitment in rats challenged with fMLP strongly suggests that the anti-inflammatory activity of losartan cannot be attributed to its ability to antagonize AT1 receptors for AII expressed on inflammatory cells.
It is well known that neutrophil recruitment in response to
gram-negative infection involves bacterial-derived chemotactic factors
such as N-formylpeptides, as well as endogenous mediators
produced in response to lipopolysaccharide (LPS) and other bacterial
products, such as C5a, LB4, platelet-activating factor (PAF), and
interleukin (IL)-8 [16
, 17
,
28
29
30
31
]. A key finding in our study is that
lung-neutrophil recruitment triggered by i.t instillation of
Pseudomonas aeruginosa is inhibited markedly in
losartan-treated rats. Moreover, we found that losartan improves
survival of rats challenged with Pseudomonas aeruginosa. The
most likely explanation for these results is that
N-formylpeptides account, at least in part, for
lung-neutrophil recruitment triggered by Pseudomonas
aeruginosa. In this regard, it is important to note that although
pulmonary infections with Pseudomonas aeruginosa remain a
serious problem in patients with a variety of underlying diseases as a
result of their ability to induce a vigorous and ultimately toxic
neutrophil-inflammatory response [12
13
14
], the
chemotactic factors responsible for lung-neutrophil recruitment remain
undefined. Studies performed in C5a receptor-deficient mice challenged
with Pseudomonas aeruginosa showed not only a lack of
inhibition but rather a marked increase in lung-neutrophil influx
compared with their wild-type littermates [32
].
Conversely, recent results published by Skerrett et al.
[33
] showed that mice lacking type 1 receptors for tumor
necrosis factor 
(TNF-) exhibited an impaired pulmonary
inflammatory response to inhaled LPS but an augmented response to live
Pseudomonas aeruginosa, despite impaired chemokine responses
to both stimuli. Support for the involvement of
N-formylpeptides in the development of acute lung
inflammation triggered by Pseudomonas aeruginosa has been
provided by two previous findings showing that: 1) supernatants of
Pseudomonas aeruginosa cultures exhibit chemotactic activity
for neutrophils, and this activity was because of, almost exclusively,
the presence of N-formylmethionyl peptides
[17
]; and 2) N-formylpeptides constitute one
of the major sources of chemotactic activity found in bronchial
secretions, which were colonized with Pseudomonas aeruginosa
usually [34
], of cystic fibrosis patients
[35
]. Studies in FPR-deficient mice will provide clear
insights into the role of N-formylpeptides in
lung-neutrophil recruitment triggered by Pseudomonas
aeruginosa infection.
The FPR is a high-affinity receptor that mediates phagocyte-chemotactic responses to N-formylpeptides. Recently, Hartt et al. [36 ] showed that the gene Fpr-rs2 encodes a second mouse neutrophil FPR, FPR2, which recognizes N-formylpeptides with low affinity [the dissociation constant (Kd) of FPR2 is >100x higher compared with FPR]. The expression of both receptors is consistent with the ability of N-formylpeptides to induce two distinct concentrations optimum for chemotaxis of mouse neutrophils. Whether rat phagocytes express high- and low-affinity receptors for N-formylpeptides also remains to be determined. However, our results showing that losartan inhibits in vitro activation of rat neutrophils triggered by nanomolar concentrations of fMLP strongly suggest that in vivo anti-inflammatory effect of losartan is mediated, at least in part, by the inhibition of high-affinity FPR.
Pharmacological modulation of lung inflammation aimed at decreasing the recruitment of potentially harmful cells, without impairing antibacterial lung defenses, is an alternative to be considered in patients with bacterial pneumonia. Our results support the notion that losartan may be useful in treating certain bacterial pulmonary infections in which inflammatory responses mediated by neutrophils exert deleterious effects. In this regard, it is noteworthy that low doses of losartan are able to prevent the development of respiratory distress in septic rats (unpublished results). Further studies are being undertaken currently to define whether the anti-inflammatory effects of losartan observed in our experimental models could be explained solely by its ability to inhibit FPR.
Received January 18, 2000; revised May 30, 2000; accepted June 1, 2000.
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4 integrin Circ. Res. 81,196-201B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis Circulation 95,1532-1539This article has been cited by other articles:
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