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Division of Pulmonary Disease, Mount Sinai Medical Center, Miami Beach, Florida 33140
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Pyocyanin (Pyo)
and 1-hydroxyphenazine (1-HP) are extracellular products of
Pseudomonas aeruginosa. To test
whether these products were capable of producing an inflammatory
response in the airways, combinations of Pyo and 1-HP at concentrations
of 10
4 and
105 M were instilled into
sheep airways, and indexes of inflammation were assessed by
bronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazines
caused a significant dose-dependent increase in the number of cells and
neutrophils recovered by BAL. Control challenges produced no such
changes. The lung neutrophilia was accompanied by an increased
concentration of albumin in BAL. The increases in BAL neutrophils and
albumin could be blocked by treating the sheep with the 5-lipoxygenase
inhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophils
when tested in vitro, but when alveolar macrophages (AM) were cultured
in vitro in the presence of both Pyo and 1-HP (1 µM), the
supernatants caused neutrophil chemotaxis. Analysis of AM culture
supernatants incubated with the combination of pigments showed
significant increases in leukotriene
B4 and interleukin-8, and blocking
these mediators separately or together reduced AM supernatant-induced
neutrophil chemotaxis. We conclude that local instillation of Pyo and
1-HP can initiate an inflammatory response in the airways of sheep in
vivo. This effect can be explained, in part, by the release of
chemotactic factors produced by AM.
phenazines; chemotaxis; chemokines; macrophages; neutrophils
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PSEUDOMONAS AERUGINOSA (PA) is a significant
opportunistic pathogen the virulence of which may be increased by the
production of extracellular products, including the blue phenazine
pigment pyocyanin (Pyo) and its metabolite 1-hydroxyphenazine (1-HP). These products have been found at concentrations as high as
104 M in sputum from
patients with cystic fibrosis and from patients with bronchiectasis
(23), with Pyo being the predominant product. The phenazines have
potent biological actions. In vitro, these products can inhibit ciliary
activity (6, 9, 10, 22), increase oxygen radical production, and
degranulate polymorphonuclear leukocytes (PMN) (13, 18, 19), whereas in
vivo they can depress mucociliary transport (14, 16) and produce
bronchoconstriction when nebulized into the airways (4). All of these
effects could compromise host defense mechanisms, thereby increasing
the pathology of PA infection.
Previous studies have implicated low-molecular-weight products of PA in the recruitment of PMN into the airways through the production of interleukin-8 (IL-8) (7, 11). Given the potent biological effects of Pyo and 1-HP, we sought to determine whether these pigments might also contribute to the PA-induced airway inflammation. To test this hypothesis, we instilled a combination of Pyo and 1-HP into subsegmental bronchus of sheep at concentrations found to be present in vivo and then measured the inflammatory response in bronchoalveolar lavage (BAL). Our results showed that these pigments were capable of recruiting neutrophils into the airways. Furthermore, we found that this neutrophilia was associated with an increased concentration of albumin in BAL and that both of these responses could be blocked by treating the sheep with the 5-lipoxygenase (5-LO) inhibitor zileuton. To further study potential mechanisms for this response, we stimulated cultures of alveolar macrophages (AM) with these products and measured the concentration of two potent neutrophil chemotaxins, IL-8 and leukotriene B4 (LTB4), in the culture supernatant. Our results indicate that phenazine pigments can stimulate AM to produce both chemotaxins and that both IL-8 and LTB4 are likely to contribute to the chemotactic response seen.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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All chemicals and reagents were obtained from Sigma Chemical (St.
Louis, MO), unless otherwise indicated. Pyo was synthesized from 1-HP
at Nova Pharmaceuticals (Baltimore, MD) as previously reported (4).
1-HP was obtained from American Tokyo Kasie (Portland, OR). Both agents
were dissolved in ethanol and kept frozen. Dilutions in PBS without
Ca2+ and
Mg2+ were prepared before the
experiment, and the final concentration of ethanol was 0.1%. This
concentration of ethanol in PBS was used as control in all protocols.
Zymosan was prepared by boiling it for 15 min in 0.85%
saline and washing twice by centrifugation. It was opsonized by
incubating 5 mg/ml of sheep serum in a rotatory shaker for 1 h at
37°C, after which the solution was centrifuged and the pellet
washed twice and then resuspended in PBS at a concentration of 100 mg/ml before it was stored in aliquots at 
70°C.
Zymosan-activated serum (ZAS) was obtained in the same fashion, except
that the incubation mixture contained 15 mg of zymosan per milliliter
serum. Zileuton was a gift from Abbott Laboratories (Abbott Park, IL). Mouse anti-ovine IL-8 antibody was obtained from Serotec (Raleigh, NC).
In Vivo Studies
To determine whether segmental challenge with Pyo and 1-HP caused lung inflammation, six ewes weighing between 27 and 41 kg were used. The study was approved by the Mount Sinai Medical Center Animal Research Committee, which is responsible for ensuring the humane care and use of experimental animals. Conscious sheep were restrained in a modified shopping cart, and BAL was performed in a previously described manner (16). Briefly, the distal tip of a specially designed 80-cm fiber-optic bronchoscope was wedged into a subsegmental bronchus of one lung. Lung lavage was performed by slow infusion and gentle aspiration of 30 ml of PBS at 39°C. Once the baseline lavage was completed, 3 ml of either diluent (0.1% ethanol in PBS) or the combination of Pyo-1HP at 104 or
105 M were instilled into a
segmental bronchus in the contralateral lung. Twenty-four hours later,
this bronchus was lavaged with 30 ml of PBS. The volume of the effluent
was recorded, and then the fluid was filtered through a double layer of
gauze and placed immediately on ice for subsequent analysis. The total
number of cells were counted in a hemocytometer from a sample of
unconcentrated lavage by using phase microscopy. The effluent was then
centrifuged at 250 g for 15 min at
4°C, and the pellet was resuspended at a concentration of
106 cells/ml in RPMI-1640. A
cytocentrifuge preparation was made and stained with Diff-Quick, and
the cell differential was determined by counting 300 cells. Six sheep
were challenged with diluent and with the
104 M combination of
pigments. Five of these same sheep were challenged with
105 M combination of
pigments. Challenges were separated by at least 3 wk.
To determine which chemotactic factors might be involved in PMN
recruitment to the airways, an additional 10 sheep weighing between 30 and 50 kg were used. For these studies, a baseline lavage was performed
as described above, then five sheep were treated orally with 500 ml of
1% methyl cellulose containing 20 mg/kg of the 5-LO inhibitor
zileuton, whereas another five sheep were given the equivalent amount
of methylcellulose alone (controls). One hour later, the animals
underwent segmental challenge with the combination of
104 M Pyo and 1-HP as
described above. The challenged segment was lavaged 24 h later, and the
cell responses were determined as described above. The dose of zileuton
used in these studies was based on our previous work with this compound
showing its efficacy against antigen-induced inflammation in allergic
sheep (1).
Albumin Assay
Albumin levels in lavage samples were determined in the five untreated sheep and in the five sheep treated with zileuton before and 24 h after the instillation of the combination of 104 M Pyo and 1-HP by an
ELISA developed in this laboratory. Microtiter plates with high
protein-binding capacity were used. Wells were coated with 200 µl per
well (5 µg/ml in coating buffered with 1.59 g
Na2CO3
and 2.93 g NaHCO3 per liter, pH
9.6) of polyclonal rabbit anti-BSA for 3 h at 37°C in a humidified
incubator. The plates were washed four times with 300 µl of 0.05%
Tween 20 in PBS. For the assay, 200 µl of standards or an aliquot of
BAL were added to the wells, and then the plates were incubated for 1 h at 37°C. The plates were washed four times with Tween 20 in PBS, and 100 µl of a mouse monoclonal antibody to BSA (1/1,000 dilution in
H2O) were added. The plates were
incubated for 1 h at 37°C and washed four times with PBS. Next, 100 µl of a 1/1,000 in H2O dilution
of anti-mouse IgG peroxidase were added to the plates, incubated for 1 h at 37°C, and then washed four times with PBS. One hundred
microliters of substrate, a 50-mg
2,2'-azino-di(3-ethylbenzthiazoline sulfonate) (ABTS) tablet
dissolved in ABTS buffer (Boehringer Mannheim, Indianapolis, IN), were
added to the wells, and the reaction was stopped after 30 min with 100 µl of 5 M
H2SO4.
The plates were read in a microtiter plate reader (Dinatech
Laboratories model MRX; Chantilly, VA) at a wavelength of 405 nm, and
the concentration of albumin (in µg/ml) was determined from a
standard curve (BSA) by interpolation. All assays were done in triplicate.
In Vitro Studies
Neutrophil (PMN) isolation from peripheral blood. PMN were obtained from venous blood by a method described previously (16). Briefly, cells were collected from blood by differential density centrifugation by using Histopaque-1077 and resuspended at a concentration of 50-100 × 106 cells/ml in Medium 199 for the chemotactic studies.Isolation and culture of AM. AM were
obtained by BAL and resuspended in RPMI-1640 containing 10%
deactivated fetal bovine serum, penicillin (100 µg/ml), and
streptomycin (100 µg/ml) at a concentration of 2.5 × 106 cells/ml. The cells were
placed in 35 × 10-mm plates and then were incubated for 1 h at
38.5°C in a humidified CO2
incubator. The adherent cells were washed with PBS and then incubated
with 1 ml of RPMI-1640 without serum for 2 h. This process was repeated two times at 2-h intervals before the study was started. To determine whether phenazines could stimulate AM to produce chemotactic factors, adherent AM were incubated with 1-µM concentrations of Pyo, 1-HP, or
the combination of Pyo+1-HP for 24 h. Supernatants from AM incubated
with zymosan (0.5 mg/ml) were used as positive controls, and cultures
incubated without any additives were used as negatives controls.
Supernatants were collected after 24 h and centrifuged at 250 g at 4°C to remove the nonadherent
cells and then at 3,000 g to clear the
samples. These supernatants were stored at 70°C until they
were analyzed for chemotactic factors and mediators. After removal of
the supernatants, the viability of the cultures ranged from 80 to 95%,
as assessed by trypan blue exclusion.
Chemotaxis under agarose. The chemotactic activity of the AM supernatants was determined by PMN migration under agarose by the method of Nelson et al. (15). Briefly, 250 mg of agarose (Accurate Chemical, Westbury, NY) were dissolved in 12.5 ml of water, boiled for 10 min, and cooled down to 48°C in a water bath. The agarose solution was mixed with an equal volume (12.5 ml) of media containing 2 ml of 10× Medium 199, 2 ml of deactivated FCS, 0.5 ml of a 1 M HEPES, pH 8, and 8 ml of distilled water. Six milliliters of this agarose solution were added to 60 × 15-mm culture dish, the solution was allowed to solidify, and then the culture dishes were placed in the refrigerator for 30-60 min. Macrophage supernatants to be tested for chemotactic activity and controls, Medium 199-stimulated (negative controls) and ZAS-stimulated (positive controls), were added to the outer wells. PMN (50-100 × 106 cells/ml) were added to the middle wells and Medium 199 to the inner wells in 10-µl volumes. Plates were incubated for 2 h in a humidified incubator at 38°C. They were fixed with 2.5% gluteraldehyde in PBS and stained by Wright-Giemsa to quantitate migration. Chemotaxis was measured with a micrometer by using an inverted microscope. Chemotaxis was defined as the distance traveled toward the outer wells, containing the chemoattractant agents, minus the distance traveled toward media alone, which is due to chemokinesis. Chemotactic activity in macrophage supernatants was expressed as migratory units (1 unit = 27 µm). Determinations were made in quadruplicate.
Because the chemotactic experiments indicated that the phenazines were stimulating PMN migration, we examined the supernatants for LTB4 and IL-8, two potent chemotaxins. Concentrations of LTB4 and IL-8 in the supernatants were determined by enzyme immunoassays, following the manufacturers instructions (Amersham, Arlington Heights, IL).
Inhibition of chemotactic activity in AM
supernatants. To determine whether the chemotactic
factors contained in the AM supernatants could be inhibited by a 5-LO
inhibitor or an antibody to IL-8, the following experiments were
performed. AM cultures were treated with 10 or 50 µg/ml zileuton 15 min before the addition of the combination of 105 M Pyo
and 105 M 1-HP (cultures without zileuton were run as
controls). The AM were cultured for 24 h. The viability of the cultures
after removal of the supernatant ranged from 80 to 95%, as assessed by
trypan blue exclusion. Before chemotactic activity was assessed, supernatants from control and treated AM were incubated at 37°C for
30 min, alone or in the presence of 100 µg/ml (final concentration) of mouse anti-ovine IL-8 antibody (Serotec). All volumes were adjusted
to 50 µl with media, and the chemotactic activity was assessed.
Effect of phenazines on PMN migration.
To determine whether the pigments themselves affected neutrophil
migration, the following experiment was performed. One milliliter
(107 cells/ml) of PMN
was incubated for 30 min with Pyo
(105 M), with 1-HP
(105 M), or the combination
of Pyo and 1-HP (105 M).
The PMN were then centrifuged at 250 g
for 5 min, and the supernatant was aspirated, leaving the pellet in 0.1 ml to obtain a final concentration ranging from 50 to 100 × 106 cells/ml. The chemotactic
assay was performed as described in MATERIALS AND
METHODS. ZAS was used as the chemoattractant. PMN viability for the different studies ranged between 88 and 99%, as
assessed by trypan blue exclusion.
Statistics
Statistical analysis was performed with statistical programs in SYSTAT for Windows (Version 5; SYSTAT, Evanston, IL). Because of the variability of responses, data were log10 transformed before analysis. The statistical significance of the mediator levels and the chemotaxis of PMN incubated with phenazines was determined by using the ANOVA for repeated measures followed by a paired t-test. Statistical significance of the chemotaxis data, with and without inhibitors, was determined by a one-way ANOVA followed by Tukey's post hoc test. Paired and unpaired t-tests were used for analysis of the remaining data where appropriate. Values are reported as means ± SE. P < 0.05 obtained by using a two-tailed test was considered significant.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of Segmental Bronchial Challenge with Pyo and 1-HP
Instillation of PA pigments caused an inflammatory response in sheep airways. The total cells per milliliter recovered from sheep after challenge with diluent and the 104 and
105 M combinations of Pyo
and 1-HP are illustrated in Fig. 1. At the
105 M concentration, there
was a small but significant (P < 0.05) increase in the total cells per milliliter recovered. However, the inflammatory response was increased 64% after instillation of the
104 M combination of
pigments. Instillation of diluent did not produce an inflammatory
response. Figure 2 shows the change in the
number of PMN per milliliter recovered in BAL 24 h after challenge with the two doses of Pyo+1-HP. Both concentrations of pigments
significantly increased the number of PMN per milliliter recovered in
the BAL fluid. There was no increase in the number of PMN after
instillation of diluent.
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Both pigments appeared to contribute to the inflammatory response
because instillation of each pigment alone produced an increase in BAL
PMN. After challenge with
105 M Pyo
(n = 3), BAL PMN increased to 22.4 ± 1.5 × 103 cells/ml
from a baseline value of 3.4 ± 1.9 × 103 cells/ml
(P < 0.05). Similarly, in these same
three sheep, challenge with
105 M 1-HP increased BAL
PMN to 49.9 ± 9.8 × 103
cells/ml from a baseline value of 1.5 ± 0.7 × 103 cells/ml
(P < 0.05). Interestingly, the
average response of the two agents separately (i.e., 36.4 × 103 cells/ml) is similar to that
obtained with the combination of 105 M Pyo and 1-HP seen in
Fig. 2.
Treatment of the sheep with zileuton significantly affected the PMN response to the combination of Pyo+1-HP (Fig. 3). In the control trial, 24 h after challenge, PMN increased to 850.6 ± 513.5 × 103 cells/ml from a baseline value of 14.1 ± 3.5 × 103 cells/ml (P < 0.05 from baseline and from 24-h zileuton treated); whereas when the animals were treated with zileuton the PMN response after challenge (13.1 ± 6.2 × 103 cells/ml) did not change significantly from before challenge (10.9 ± 1.4 × 103 cells/ml).
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In addition to showing an increased PMN response, lavage samples from untreated sheep in the previous experiment showed a significant increase in albumin from baseline after stimulation with phenazines. Albumin levels were seen to increase in all five untreated sheep after challenge, whereas only one of the five zileuton-treated sheep showed an increase after phenazine provocation. Before challenge in the control group, albumin concentration was 0.16 ± 0.04 µg/ml and increased to 0.36 ± 0.08 µg/ml after challenge (P < 0.05). In the zileuton-treated animals, there was no such increase after challenge. The baseline values for the zileuton-treated animals was 0.37 ± 0.12 µg/ml but fell to 0.21 ± 0.05 µg/ml after challenge. Because of differences in baseline values, the comparison between groups was made on the post- to prechallenge ratio. This analysis showed that albumin increased 2.5 ± 0.8-fold in the control trial compared with 0.9 ± 0.4-fold in the zileuton trial (P < 0.05), indicating that zileuton blocked the postchallenge increase in albumin.
Chemotactic Activity Produced by AM Incubated for 24 h with Pyo and 1-HP
AM incubated in the presence of either Pyo or 1-HP alone or the combination of both at 106
M produced chemotactic activity (Fig. 4).
Supernatants from the combination of 1-HP+Pyo produced slightly greater
chemotactic activity than either pigment alone. The response to all
pigments was less than that seen with the positive-control zymosan. PMN did not migrate if 1-HP or Pyo (at doses ranging from 1 to 100 µM)
was used as the chemoattractant, indicating that these agents had no
direct chemotactic activity themselves (data not shown). Figure
5 shows that the supernatants from these
24-h cultures contained both IL-8 and
LTB4. The concentration of these
agents in the supernatants of AM stimulated with the combination of
pigments was approximately the same. This response is different from
the zymosan-stimulated cultures where
LTB4 appeared to be the major chemoattractant.
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The data illustrated in Fig. 5 suggest that blocking IL-8, LTB4, or the combination should reduce the chemotactic activity of AM supernatants. Figure 6 shows that chemotaxis was reduced between 67 and 70% (P < 0.05) when AM were treated with zileuton before stimulation with the phenazines. Incubation of the supernatants with an antibody to IL-8 alone gave similar protection (54% inhibition) (P < 0.05). The level of inhibition was increased when the IL-8 antibody was added to supernatants from zileuton-treated AM, with almost complete inhibition achieved when the antibody was added to AM treated with 50 µg zileuton (99% inhibition).
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Effect of Phenazines on PMN Chemotaxis
Although we found that PMN did not migrate if phenazines were used as the chemoattractants, we determined whether incubation of PMN with these agents affected their ability to migrate. Incubation of PMN with 105 M Pyo or the
combination of 105 M
Pyo+1-HP decreased chemotaxis to ZAS by 48-55%
(P < 0.05). Incubation of PMN with 1-HP alone
before exposure to ZAS had no effect on migration (Fig.
7).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The results of this study show that instillation of Pyo and 1-HP, extracellular products of PA, either alone or together can cause an inflammatory response characterized by an increase in neutrophils and albumin recovered in BAL in normal airways. Furthermore, our results suggest that this inflammatory response appears to be mediated, in part, by the generation of LTB4 and IL-8 and that AM are a likely source of these chemotaxins. These inflammatory changes occurred at concentrations found in the airways of patients with cystic fibrosis and bronchiectasis.
Our interest in Pyo and 1-HP stemmed from previous studies by our group in which we found that these phenazines had potent biological effects (4, 9, 20). We reasoned that these PA products might also have the potential to recruit inflammatory cells to the airways. Studies by Massion et al. (11) showed that a non-lipid- extractable substance of a PA product stimulates IL-8 expression in airway epithelial cells, which results in an inflammatory response. Because the phenazines we studied are lipid extractable, they are different from the substance isolated by Massion and co-workers. Our results support our hypothesis and show, for the first time, that these pigments have the ability to stimulate production of chemotactic factors from AM.
Albumin has often been used as a marker of increased permeability in the airways, which is equated with lung injury (2, 3, 8, 17, 21). The lung neutrophilia following instillation of these phenazines was associated with lung permeability changes, as reflected by an increase in BAL albumin concentration. These data suggest that the phenazines appear to cause an active inflammatory response, rather than just cell recruitment.
Our in vitro studies indicate that the inflammatory response induced by a combination of phenazines was not a direct effect, but, rather, the effect was mediated through the production of chemotactic factors. Our data suggest that AM are one source of these chemoattractants. We chose to study AM because they are resident cells in the airway that participate in host defense and, hence, could be a key cell in the recruitment of neutrophils during the initial stages of infection. We showed that AM taken from normal sheep, when incubated in vitro with either Pyo, 1-HP, or the combination, induced chemotaxis of PMN. This chemotatic response resulted from the production of IL-8 and LTB4 by these cells, as evidenced by the increase in these mediators in AM supernatants. Furthermore, when AM were incubated with the 10 µg/ml of the 5-LO inhibitor zileuton, before stimulation with the phenazine combination or when the supernatants were incubated with an antibody to ovine IL-8, PMN chemotaxis was reduced by 50-70%. An increase in the zileuton concentration to 50 µg/ml provided even greater protection (83% inhibition). When both agents were combined, chemotaxis was almost completely abolished. These in vitro findings are consistent with the ability of a high dose of the 5-LO inhibitor zileuton to block the inflammatory response in vivo. It should be noted that we used a 5-LO inhibitor for these studies because we did not have an LTB4 antagonist available to us.
Although the mediator measurements in conjunction with the inhibitor
studies provide good evidence that IL-8 and
LTB4 contribute to PMN chemotaxis,
there is one caveat that should be addressed. When AM were incubated
with the individual phenazines at
106 M, the individual
increases in concentrations of IL-8 and
LTB4 did not reach statistical
significance, although Fig. 4 indicates that these supernatants caused
PMN chemotaxis. One possible explanation for this may be that, although
the individual increases in mediator concentrations were not
statistically increased, the combined levels of these agents were
sufficient to cause chemotaxis. In support of this, we found that the
combined concentration of IL-8 and
LTB4 in the Pyo-stimulated AM was
104 pg/ml compared with 92 pg/ml in the 1-HP-stimulated cells. This
small difference might explain the slight difference in mean
chemotactic response between the two agents seen in Fig. 4. As might be
expected using this line of reasoning, the combination of the two
mediators gave the largest total mean mediator level (135 pg/ml) and
the largest mean chemotactic response. Again, we should emphasize that,
although this explanation may be logical, it is speculative at best.
Our results are consistent with and compliment those of Hashimoto and
co-workers (5), who showed decreased PMN chemotaxis to PA infections in
rats depleted of AM. BAL obtained from these AM-depleted rats showed
decreased levels of tumor necrosis factor-
and the chemokines
macrophage inflammatory protein 2 and CINC/gro (cytokine-induced PMN chemoattractant) in response to PA challenge (5).
Thus AM, through the production of cytokines and chemotactic factors,
appear to play a significant role in the recruitment of inflammatory
cells to the site of PA infection. The findings in this study are also
consistent with data obtained from patients with established airway
disease. Sputum from these patients was shown to contain IL-8 and
LTB4, with the levels of these
mediators varying depending on the clinical state of the patient and
the pulmonary inflammation. As in the present study, PMN chemotaxis to
these sputum samples could be inhibited by a monoclonal antibody to
IL-8 and the LTB4 antagonist
LY-293111. The source of these mediators, however, was not identified
(12). Our findings indicate that AM may provide one such source.
One interesting result of this study was the finding that incubation of PMN with Pyo or the combination of Pyo and 1-HP, but not 1-HP alone, reduced PMN chemotaxis to ZAS. Taken in the context of the present study, and in conjunction with the identification of high levels of Pyo in the sputum from patients with cystic fibrosis and bronchiectasis (23), this observation suggests that, although the initial recruitment of PMN to the airway is normal, subsequent PMN function may be compromised. Such a mechanism could contribute to a state of persistent infection.
We conclude that local challenge with the combination of extracellular products of PA, Pyo and 1-HP, can initiate an inflammatory response in the airways of sheep in vivo. This effect appears to result from an increase in the production of LTB4 and IL-8 by AM. The ability of these pigments to increase cell recruitment and alter lung permeability may contribute to an enhanced PA infection in the airway.
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FOOTNOTES |
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Address for reprint requests: W. M. Abraham, Dept. of Research, Mount Sinai Medical Center, 4300 Alton Rd., Miami Beach, FL 33140 (E-mail: abraham{at}msmc.com).
Received 17 November 1997; accepted in final form 20 August 1998.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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