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,
*
Department of Medicine, Section of Pulmonary and Critical Care Medicine,
Department of Physiology, and
Alcohol Research Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112; and
AMGEN, Thousand Oaks, CA 91320
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMN transendothelial migration is a complex process. The presence of chemoattractants is pivotal in guiding the migration of these immune effector cells into infected tissue sites (13, 14). Tissue sites of infection and inflammation contain a variety of inflammatory mediators including cytokines and chemokines, which are responsible for orchestrating PMN recruitment. These mediators also exert other effects within infected tissue sites to modulate the function of recruited PMNs. In addition, the recruited PMNs themselves may affect the local environment by generating and/or scavenging inflammatory mediators. At the present time, relatively little information is known about the changes in pulmonary chemokine profiles that occur during the inflammatory response and how recruited PMNs may modulate these profiles.
In this study, we investigated the effects of PMN recruitment and G-CSF on the pulmonary response of two important C-X-C chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), in rats challenged with intratracheal LPS. The results show that G-CSF significantly increased PMN recruitment into the lung following an LPS challenge, and this response is associated with a marked reduction in soluble MIP-2 and CINC in the alveolar space. The scavenging of chemokines by recruited PMNs in infected tissue sites may serve to modulate the local host defense response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human G-CSF was a gift from Amgen (sp. act. 108 U/mg; Thousand Oaks, CA). Cytoscreen ELISA kits for rat MIP-2 were obtained from BioSource International (Camarillo, CA). CINC, rabbit anti-rat CINC-1 Ab, and biotinylated anti-rat CINC-1 Ab were obtained from R&D Systems (Minneapolis, MN). Escherichia coli (026:B6) LPS was obtained from Difco (Detroit, MI). Streptavidin-HRP was supplied by Jackson ImmunoResearch (West Grove, PA). Tetramethylbenzidine was purchased from Pierce (Rockford, IL). TaqMan Gold RT-PCR kit was obtained from Perkin-Elmer Applied Biosystems (Foster City, CA). RPMI 1640 was obtained from Life Technologies (Grand Island, NY). FCS was purchased from HyClone (Logan, UT). NIM.2 density gradient reagents were obtained from Cardinal Associates (Santa Fe, NM). TRIzol reagent and PBS were obtained from Life Technologies. Complete Protease Inhibitor Cocktail was purchased from Boehringer Mannheim (Mannheim, Germany). Ficoll-Hypaque density gradient reagents and all other chemicals were purchased from Sigma (St. Louis, MO).
Animals
Male virus Ab-free (VAF) Sprague Dawley International Genetic Standard rats (Charles River Breeding Laboratories, Wilmington, MA) with a body weight of 175–200 g were maintained on a standard laboratory diet and housed in a controlled environment with a 12-h light/dark cycle. Rats were pretreated for 2 days with a s.c. injection of recombinant human G-CSF at 50 µg/kg or vehicle (100 µl) twice daily. On the third day, 3 h after the last dose of G-CSF or vehicle, LPS (100 µg in 0.5 ml PBS) was administered intratracheally under ether anesthesia. Control animals were intratracheally injected with an equal volume of PBS. Depending on the experimental protocol, rats were sacrificed at either 90 min or 4 h following the intratracheal injection to obtain a heparinized blood sample from the abdominal aorta and to perform bronchoalveolar lavage (BAL). For some experiments, rats were sacrificed 3 h after the last dose of G-CSF or vehicle to obtain blood PMNs or alveolar macrophages (AMs) for in vitro experiments. Other animals without pretreatment were sacrificed 4 h after intratracheal LPS challenge to collect BAL fluid. A group of naive control rats were sacrificed without prior treatment to perform BAL and isolate AMs.
The experiments described here were performed in adherence to National Institutes of Health guidelines on the use of experimental animals. Approval of the Animal Care and Use Committee of the Louisiana State University Medical Center was obtained before initiating these experiments.
Cell isolation and culture
Circulating PMNs were isolated from the whole blood samples by using NIM.2 gradient reagents based on the protocol supplied by the manufacturer. The viability of the cells was >95% as assessed by trypan blue exclusion. The purity of isolated circulating PMNs was >90% as assessed by morphology using Wright-Giemsa stain.
Lungs were surgically removed under anesthesia and lavaged with cold PBS containing 0.1% dextrose. Recovered lavage fluid was centrifuged at 200 x g for 5 min, and the cell pellet was resuspended in PBS. The cell counts were quantified under a light microscope with a hemacytometer. Cell monolayers were prepared by cytocentrifugation, and Wright-Giemsa stain was used to differentiate AMs and pulmonary recruited PMNs. Cells recovered from BAL fluid were subjected to discontinuous Ficoll-Hypaque density gradient centrifugation to separate AMs and PMNs. The isolated AMs and PMNs were washed twice with PBS and resuspended in RPMI 1640 containing 2% FCS. The viability of the cells was >95% as assessed by trypan blue exclusion. The purity of AMs and pulmonary recruited PMNs was >90%.
AMs isolated from rats treated with G-CSF or vehicle were plated in 96-well tissue culture plates (Costar, Cambridge, MA) at a density of 5 x 104/200 µl RPMI 1640 containing 2% FCS/well in the presence or absence of G-CSF (5000 U/ml). Cell were stimulated with various concentrations of LPS and cultured at 37°C in an atmosphere of 5% CO2 for 6 h.
Coculture of AMs from naive rats with pulmonary recruited or circulating PMNs of G-CSF- or vehicle-treated rats were established by plating AMs (5 x 104/100 µl RPMI 1640 containing 2% FCS/well) with PMNs at an AM/PMN ratio of 1:10 or 1:20 in 96-well tissue culture plates. The cells were stimulated with 50 ng/ml of LPS and cultured at 37°C in an atmosphere of 5% CO2 for 6 h.
In another set of experiments, circulating PMNs isolated from G-CSF- or vehicle-treated rats were cultured at a concentration of 2 x 107/ml RPMI 1640 containing 2% FCS and 50 ng/ml of LPS. The cell cultures were terminated at 6 h, and the conditioned media were collected. AMs isolated from naive rats were plated in 96-well tissue culture plates at a density of 5 x 104/100 µl RPMI 1640 containing 2% FCS and 50% conditioned media of PMNs/well. The cells were stimulated with 50 ng/ml of LPS and cultured at 37°C in an atmosphere of 5% CO2 for 6 h.
To determine PMN-associated chemokines, circulating and pulmonary recruited PMNs from rats pretreated with G-CSF or vehicle and challenged with intratracheal LPS for 4 h, as well as circulating PMNs from naive rats, were treated with a lysing buffer (PBS containing 1% Triton X-100 and 1 tablet Complete Protease Inhibitor Cocktail/7 ml of lysing solution). To determine the capacity of chemokines present in BAL fluid to bind to PMNs in vitro, circulating PMNs isolated from G-CSF- or vehicle-treated rats were incubated (at a density of 5 x 105/200 µl/well) in RPMI 1640 containing 2% FCS and 50% BAL fluid of rats challenged with intratracheal LPS or PBS (control) for 4 h. The cell mixtures are incubated at 37°C in an atmosphere of 5% CO2 for 30 min. The cells were washed twice with cold PBS and treated with the lysing buffer. The cell lysates were kept at -70°C before analysis of cell-associated chemokines by ELISA.
Measurement of chemokines
MIP-2 in BAL fluid, tissue culture media, and cell lysates was measured in duplicates using the immunoassay kits for rat MIP-2 and procedures supplied by the manufacturer. MIP-2 levels are expressed as pg/ml of BAL fluid or tissue culture media or pg/107 PMNs. CINC was determined with a specific ELISA. Wells of a 96-well tissue culture plate are coated with anti-rat CINC-1 neutralizing Ab by adding 50 µl of 0.5 µg/ml Ab to each well. The wells were sealed and incubated overnight at 4°C. The plate was washed five times with washing buffer (PBS containing 0.05% Tween 20) and blocked by adding 200 µl/well blocking solution (2% BSA in washing buffer). After incubation at room temperature for 1 h, the plate was washed. Standard CINC (concentrations 31.25–1000 pg/ml) and samples prepared with the same dilution buffer were then added to each well (50 µl per well). The plate was incubated at 37°C for 1 h and then washed. Biotinylated anti-rat CINC-1 Ab was added to each well (0.25 µg/ml, 50 µl per well) and the plate was incubated at 37°C for 1 h. The plate was washed, and streptavidin-HRP was added (100 µl per well). After incubation at room temperature for 1 h, the plate was washed before adding peroxidase substrate tetramethylbenzidine solution (150 µl per well). The mixtures were incubated at room temperature in the dark for color development. The reaction was stopped by adding 3 M sulfuric acid to each well (50 µl per well). The color developed in each well was determined spectrophotometrically at 450 nm. CINC levels are expressed as pg/ml of BAL fluid or tissue culture media or pg/107 PMNs.
Determination of chemokine mRNA expression
Total RNA was extracted from the AMs by TRIzol reagent following the manufacturer’s procedure. The RNA was quantified spectrophotometrically at 260 nm. mRNA for specific chemokines was analyzed by real time quantitative RT-PCR procedure that was performed on a Gene Amp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). Ribosomal RNA was evaluated as an internal control. Equal amounts of total RNA (2 µg) from each sample were reverse transcribed in a 100-µl reaction volume containing 1 x TaqMan buffer, 5.5 mM MgCl2, 40 U RNase inhibitor, 2.5 µM random hexamer primer, 500 µM dNTPs, 125 U multiscribe reverse transcriptase, and RNase-free water. The mixture was incubated at 25°C for 10 min and then at 48°C for 30 min. The reaction was inactivated by incubating at 95°C for 5 min. PCR was then performed using a 5-µl aliquot of the RT reaction mixtures. cDNA was amplified using specific primers for the cytokines and chemokines as well as the specific probes (rat MIP-2: 5'-CTGTCAATGCCTGACGACCCTACCAAG-3', and CINC: 5'-CCCACTGCACCCAAACCGAAGTCA-3') labeled with a fluorophore and a quencher in a 50-µl reaction volume containing 5 mM MgCl2, 2.5 mM dNTP, 1.25 U Amplitaq Gold DNA polymerase, and 0.5 U AmpErase UNG. The primers used were as follows: rat ribosomal RNA (sense, 5'-CGGCTACCACATCCAAGGAA-3'; antisense, 5'-GCTGGAATTACCGCGGCT-3'), MIP-2 (sense, 5'-GGGTTGTTGTGGCCAGTGA-3'; antisense, 5'-TCAAGCTCTGGATGTTCTTGAAGT-3'), and CINC (sense, 5'-AGTGGCAGGGATTCACTTCAA-3'; antisense, 5'-CAAGCCTCGCGACCATTCT-3'). PCR amplification was performed using 40 thermal cycles of denaturation, annealing, and extension. Negative control PCRs without cDNA and positive ribosomal control PCRs were performed in all experiments to exclude contamination and other potential errors in the processes.
Statistics
Data are given as means ± SEM of the number of experiments indicated in each figure, and table comparisons of data sets were performed with unpaired Student’s t test or one way ANOVA followed by the Student-Newman Keuls test. Differences were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the magnitude of PMN recruitment following
intratracheal challenge, rats were challenged with either saline or LPS
and then their lungs were lavaged at either 90 min or 4 h. In rats
challenged with intratracheal saline, very few PMNs were recovered from
the BAL fluid. G-CSF pretreatment had no effect on the number of PMNs
recovered from the BAL fluid in animals challenged with intratracheal
saline (0.09 ± 0.01 x 106/BAL vs
0.05 ± 0.03 x 106/BAL,
p > 0.05). At 90 min post intratracheal LPS challenge,
very few PMNs were recovered in BAL fluid in rats pretreated with
either vehicle or G-CSF. Greater than 95% of the recovered cells were
AMs in these animals. Intratracheal LPS induced significant PMN
recruitment into the lung by 4 h after the challenge (16.27
x 106/BAL). G-CSF pretreatment augmented this
LPS-induced pulmonary PMN recruitment 
5-fold (Fig. 1
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PMN recruitment into infected tissue sites is a complex process
requiring the presence of chemoattractants. MIP-2 and CINC are potent
C-X-C chemokines accounting for the major chemotactic activity of the
rat lung for PMNs (15, 16). We determined the pulmonary
chemokine responses following an intratracheal challenge with LPS in
rats pretreated with or without G-CSF. As shown in Table I, MIP-2 and CINC levels in BAL fluid of
saline-challenged rats at 4 h after the challenge were very low
and not different between vehicle- and G-CSF-pretreated rats.
Intratracheal LPS induced a marked increase in MIP-2 and CINC
concentrations in BAL fluid obtained at both 90 min and 4 h after
the challenge. G-CSF pretreatment had no effect on the pulmonary MIP-2
and CINC responses to intratracheal LPS at 90 min after the challenge,
a time before PMN entering into the alveolar compartment. In contrast,
at 4 h after the challenge LPS-induced increases in MIP-2 and CINC
were significantly lower in animals pretreated with G-CSF. Thus, the
observed decreases in MIP-2 and CINC in G-CSF-pretreated rats was
evident when PMN recruitment was increased compared with animals not
given G-CSF.
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Because AMs are essential for the pulmonary production of
chemokines in rats with lung infection and inflammation (17, 18), we determined whether in vivo or in vitro G-CSF would
affect MIP-2 and CINC production by AMs. AMs were isolated from animals
pretreated with vehicle or G-CSF and stimulated with LPS in vitro in
the absence and presence of G-CSF. As shown in Fig. 2, A and B, in
vitro LPS induced a dose-dependent increase in MIP-2 and CINC
production by cultured AMs. AMs of G-CSF-pretreated animals showed
slightly higher MIP-2 and CINC production in vitro in response to each
dose of LPS as compared with AMs of vehicle-pretreated animals. Adding
G-CSF to the in vitro culture systems did not show any direct effect on
LPS-stimulated MIP-2 and CINC production by AMs isolated from either
G-CSF- or vehicle-pretreated rats. Thus, G-CSF does not have a direct
effect on LPS-induced chemokine production by AMs. This suggests that
the reduced chemokine concentration in BAL fluid in G-CSF-pretreated
animals is not due to the direct effects of G-CSF on AMs.
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We determined MIP-2 and CINC mRNA expression by AMs by using
Taqman RT-PCR methods with specific probes for MIP-2 and CINC. Data are
expressed as 1/Ct value where Ct is the cycle number in which the MIP-2
or CINC signal is detected to be significantly above background. Thus
the lower the Ct value or the higher the 1/Ct value, the greater the
content of MIP-2 or CINC transcript in the sample. Ribosomal RNA
content was used as reference RNA in each sample. As shown in Fig. 3A, in vitro LPS (50 ng/ml)
induced a significant increase in MIP-2 mRNA expression by cultured AMs
of vehicle-pretreated rats. AMs from animals pretreated with G-CSF
showed a comparatively lower expression of MIP-2 mRNA in the absence of
LPS stimulation when compared with those of vehicle-pretreated rats,
whereas the increase in MIP-2 mRNA expression was greater in these
cells following LPS stimulation. Adding G-CSF into the in vitro culture
system did not have a direct effect on LPS-stimulated MIP-2 mRNA
expression by AMs of either G-CSF- or vehicle-pretreated animals. Fig. 3B shows CINC mRNA expression by cultured AMs. In vitro LPS
(50 ng/ml) induced significant increases in CINC mRNA in AMs of both
vehicle- and G-CSF-pretreated animals. Neither in vivo G-CSF
pretreatment of rats nor adding G-CSF to the culture systems had an
effect on either baseline or LPS-stimulated expression of CINC mRNA by
cultured AMs.
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G-CSF caused a significant increase in the recruitment of PMNs
into the alveolar space in response to intratracheal LPS. Because G-CSF
did not suppress AM chemokine expression directly, we investigated
whether the PMNs themselves may modulate the LPS-induced chemokine
responses by AMs. AMs isolated from normal rats were cocultured with
different ratios of either circulating or pulmonary recruited PMNs. As
shown in Table II, adding circulating
PMNs to AM cultures significantly decreased MIP-2 and CINC
concentrations in the culture media at 6 h following LPS
stimulation. This decrease in MIP-2 and CINC concentrations in the
culture media was PMN dose dependent. Circulating PMNs isolated from
the vehicle-pretreated rats showed a greater ability to reduce
chemokines in the AM culture media compared with circulating PMNs
isolated from G-CSF-pretreated animals. In contrast to circulating
PMNs, pulmonary recruited PMNs isolated from G-CSF-pretreated rats at
4 h following intratracheal challenge with LPS had no effect on
MIP-2 and CINC concentrations in the media of AM cultures in the
presence of LPS stimulation (Table III).
These data indicate that after recruitment into an inflammatory site,
the ability of PMNs to modulate chemokine concentrations is
decreased.
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To understand the mechanisms whereby PMNs reduce MIP-2 and CINC in
AM cultures, circulating PMNs isolated from either G-CSF- or
vehicle-pretreated rats were cultured with LPS (50 ng/ml) for 6 h
to generate conditioned media. The conditioned media of PMNs were added
to the AM cultures to evaluate the effects of PMN-released factors on
AM chemokine responses to in vitro LPS stimulation. As shown in Fig. 4, A and B,
LPS-induced MIP-2 and CINC production by AMs tended to be lower when
AMs were cultured with conditioned media of PMNs isolated from rats
pretreated with or without G-CSF. However, these reductions in
chemokine production did not attain statistical significance.
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, A and B,
conditioned media of circulating PMNs isolated from rats pretreated
with vehicle showed a statistically significant inhibition on
LPS-induced MIP-2 but not CINC mRNA expression by cultured AMs.
Conditioned media of PMNs from rats pretreated with G-CSF showed a
statistically significant inhibition of both LPS-induced MIP-2 and CINC
mRNA expressions by cultured AMs.
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Both MIP-2 and CINC have been shown to bind to PMNs
(19). Therefore, we determined cell-associated chemokine
content after incubating PMNs with BAL fluid. Following a 30-min
incubation with BAL fluid from animals challenged with intratracheal
LPS, the circulating PMNs of vehicle-treated rats showed a significant
increase in cell-associated MIP-2 (6634 ± 573 vs 617 ± 54
pg/107 cells, p < 0.05) and CINC
(956 ± 184 vs 124 ± 12 pg/107 cells,
p < 0.05) compared with PMNs incubated with control
BAL fluid. The chemokine binding activity of circulating PMNs isolated
from G-CSF-treated rats was lower (for MIP-2, 3548 ± 361 vs
585 ± 30 pg/107 cells, p <
0.05; for CINC, 492 ± 68 vs 36 ± 3
pg/107 cells, p < 0.05) compared
with circulating PMNs isolated from the vehicle-treated animal (Fig. 6, A and B).
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A). Pulmonary recruited PMNs
showed a marked increase in cell-associated MIP-2 (15,220 ± 1,740
vs 414 ± 58 pg/107 cells, p
< 0.05) compared with PMNs remaining in the blood. This increase in
cell-associated MIP-2 of the recruited PMNs was lower in G-CSF-treated
animals (11,593 ± 1,250 vs 158 ± 16
pg/107 cells, p < 0.05). In
contrast to MIP-2, cell-associated CINC of circulating PMNs isolated
from rats challenged with intratracheal LPS 4 h earlier was
significantly increased when compared with those of circulating PMNs
isolated from native rats (928 ± 30 vs 36 ± 3
pg/107 cells, p < 0.05) (Fig. 7B). Pulmonary recruited PMNs showed a similar increase in
cell-associated CINC (1089 ± 102 pg/107)
compared with circulating PMNs in these animals. Likewise, G-CSF
pretreatment attenuated this increase in cell-associated CINC of both
circulating (525 ± 50 pg/107) and pulmonary
recruited PMNs (248 ± 57 pg/107) in rats
challenged with intratracheal LPS for 4 h. These data suggest that
binding of chemokines to PMNs as these cells are recruited from the
peripheral circulation into the alveolar space is, in part, responsible
for decreasing chemokine concentrations within the lower respiratory
tract.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrins is known to be a
critical step for PMN transendothelial migration (23).
Little is known concerning the effect of G-CSF on the chemoattractants
that guide the migration of PMNs from the vasculature into the infected
tissue site. MIP-2 and CINC, two members from the C-X-C chemokine
family, have been shown to be responsible for the major chemotactic
activity for PMNs in the alveolar space in rats (15, 16).
In this study, we determined the effects of G-CSF pretreatment on the
expression of these chemokines in the lung following intratracheal
challenge with LPS. Interestingly, LPS-induced increases in MIP-2 and
CINC in the alveolar space were significantly lower in rats pretreated
with G-CSF. Because these chemokines exert their effects on PMNs
through surface receptors and the number of PMNs in the alveolar
compartment was increased at this time in rats pretreated with G-CSF,
we explored the possibility that the increased number of PMNs in
G-CSF-treated rats were responsible for this decrease in chemokines.
Pulmonary recruitment of PMNs typically begins 2 h following
intratracheal LPS challenge. The results of this study show that the
chemokine response to LPS in the lung was not decreased by G-CSF
pretreatment at a time (90 min following intratracheal LPS) before PMN
entering the alveolar space. AMs, the resident phagocytes of the lung, produce chemokines in response to infectious stimuli (24, 25). In addition, monocyte/macrophages have been reported to possess G-CSF receptors (26). We further explored the effects of in vivo and in vitro G-CSF on LPS-induced MIP-2 and CINC production by AMs. The results show that the production of both MIP-2 and CINC by AMs isolated from either vehicle- or G-CSF-pretreated animals was unaffected by adding G-CSF into the in vitro culture system. Similarly, LPS-induced MIP-2 and CINC mRNA expression by this macrophage population was not suppressed by in vitro G-CSF. AMs of G-CSF-pretreated rats exhibited a higher, not lower, MIP-2 and CINC production in response to in vitro LPS compared with AMs from vehicle-pretreated animals. In vitro LPS-induced MIP-2 mRNA expression by AMs of G-CSF-pretreated animals was also higher in comparison to those of vehicle-pretreated controls. Thus, G-CSF does not appear to be directly responsible for the lower chemokine values observed following intratracheal LPS challenge despite reports suggesting an immunosuppressive role for G-CSF (27). The mechanisms underlying the up-regulated chemokine response of AMs from G-CSF-pretreated animals observed in these experiments remain unclear.
To investigate the role of PMNs in attenuating the LPS-induced chemokine response in the alveolar space, AMs isolated from naive rats were cocultured with different ratios of circulating or pulmonary recruited PMNs. The results show that circulating PMNs isolated from either vehicle- or G-CSF-treated rats decrease LPS-induced MIP-2 and CINC in the culture media in a dose-dependent manner. These data suggest that as PMNs migrate into an inflammatory locus, chemoattractants, such as MIP-2 and CINC, are subsequently reduced by their interaction with these cells. Thus, the recruitment of these immune effector cells into an inflammatory site may serve to turn off chemotactic signals by lowering the concentration gradient of chemokines in the surrounding space. G-CSF augments pulmonary PMN recruitment in response to intratracheal LPS challenge. Our data suggest that the increased influx of PMNs in the alveolar space resulting from G-CSF-induced neutrophilia should be limited by the scavenging effect of these cells on intrapulmonary chemokines. In contrast to the effects of circulating PMNs, pulmonary recruited PMNs did not decrease MIP-2 or CINC concentrations in the media of the coculture system following in vitro LPS stimulation. One possible explanation for this is that the capacity of these recruited PMNs to modulate chemokines has been exhausted during their migration into the alveolar space and exposure to chemokines in the process.
To investigate the mechanisms underlying the effects of PMNs on LPS-induced chemokine production by AMs, circulating PMNs isolated from either vehicle- or G-CSF-treated rats were cultured with LPS, and the conditioned media were collected at 6 h. Several studies have previously shown that in vitro LPS stimulates peripheral blood PMNs to produce various mediators (28, 29). After these conditioned media were added into the AM cultures, LPS-induced MIP-2 and CINC production in the supernatant of the AM cultures tended to be reduced, but statistical significance was not reached. LPS-induced MIP-2 mRNA expression by AMs was inhibited by conditioned media of PMNs isolated from both vehicle- and G-CSF-treated rats. LPS-induced CINC mRNA expression by AMs was also inhibited by the conditioned medium of PMNs isolated from G-CSF-treated animals. These results suggest that certain soluble substances released from PMNs can themselves inhibit LPS-induced chemokine expression by AMs. The nature of these inhibitory substances remains to be further defined.
Because the inhibitory effects of PMN-conditioned media on AM chemokine production were relatively small compared with the in vivo suppression of LPS-induced pulmonary chemokine response in animals pretreated with G-CSF and the in vitro inhibition of circulating PMNs on LPS-induced chemokine production in the supernatant of AM cultures, it appears that other mechanisms are involved. To explore this possibility further, the capacity of PMNs to bind MIP-2 and CINC in the BAL fluid of LPS-challenged lungs was determined. The results show that a brief exposure of circulating PMNs to the BAL fluid of rats challenged with intratracheal LPS resulted in a marked increase in cell-associated MIP-2 and CINC in these PMNs. Pulmonary recruited PMNs isolated from either vehicle- or G-CSF-pretreated rats at 4 h after intratracheal LPS challenge showed a dramatic increase in cell-associated MIP-2 in comparison to their counterparts remaining in the peripheral circulation and the circulating PMNs from naive control rats. This likely explains why pulmonary recruited PMNs were not effective at decreasing MIP-2 and CINC concentrations in the media of LPS-stimulated AMs. Neither circulating nor recruited PMNs appear to be active producers of MIP-2 or CINC in response to LPS. In this study, circulating PMNs and pulmonary recruited PMNs were cultured in vitro for 6 h in the presence of LPS (50 ng/ml). Only trace amounts of MIP-2 and CINC could be detected in the supernatant of the cultures.
PMNs are key components of the pulmonary host defense system against invading pathogens (1, 2). G-CSF augments the pulmonary recruitment of PMNs, which has been shown to decrease the morbidity and mortality of respiratory infections in several animal models (5, 30, 31). However, PMNs have also been implicated as mediators of tissue injury in a variety of inflammatory disorders (32, 33). Thus, inflammation evoked by the host response to infections should be restrained within a certain magnitude and duration. Clinical observations of patients with acute respiratory distress syndrome (ARDS) and experimental studies on different animal models of lung injury have shown that sustained increases in pulmonary chemokines including IL-8, MIP-2, and CINC is a risk factor for the development of acute lung injury (17, 34, 35, 36, 37). Suppression of the ELR+ C-X-C chemokine (including MIP-2 and CINC) response has been shown to be associated with a significant decrease in lung injury irrespective of PMN influx in the lung in a rat model of hepatic ischemia and reperfusion (38). Although the inciting infection triggers the inflammatory cascade which serves to initiate an immune response, scavenging of soluble chemokines by the responding PMNs may be important in modulating the local host response. With the reduction of chemokines in the alveolar space, chemotactic activity for PMNs would be diminished. This would then limit the influx of PMNs into the tissue sites and signal the resolution of the inflammatory response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steve Nelson, Department of Medicine, Section of Pulmonary and Critical Care Medicine, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112.
3 Abbreviations used in this paper: PMNs, polymorphonuclear leukocytes; MIP-2, macrophage inflammatory protein-2; CINC, cytokine-induced neutrophil chemoattractant; AMs, alveolar macrophages; BAL, bronchoalveolar lavage.
Received for publication June 5, 2000. Accepted for publication September 13, 2000.
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X.-p. Gao, T. J. Standiford, A. Rahman, M. Newstead, S. M. Holland, M. C. Dinauer, Q.-h. Liu, and A. B. Malik Role of NADPH Oxidase in the Mechanism of Lung Neutrophil Sequestration and Microvessel Injury Induced by Gram-Negative Sepsis: Studies in p47phox-/- and gp91phox-/- Mice J. Immunol., April 15, 2002; 168(8): 3974 - 3982. [Abstract] [Full Text] [PDF]
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 R. A. Bowden, Z.-M. Ding, E. M. Donnachie, T. K. Petersen, L. H. Michael, C. M. Ballantyne, and A. R. Burns Role of {alpha}4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium Circ. Res., March 22, 2002; 90(5): 562 - 569. [Abstract] [Full Text] [PDF]
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R. A. Bowden, Z.-M. Ding, E. M. Donnachie, T. K. Petersen, L. H. Michael, C. M. Ballantyne, and A. R. Burns Role of {alpha}4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium Circ. Res., March 22, 2002; 90(5): 562 - 569. [Abstract] [Full Text] [PDF]
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