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Centre de Recherche en Infectiologie, Université Laval, Quebec City, Québec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1
, MIP-2, IP-10, monocyte chemoattractant
protein (MCP)-1, T cell activation 3, and KC) within the lungs
during the course of infection. KC and MIP-2 protein expression closely
preceded pulmonary neutrophil recruitment, whereas MCP-1 protein
production coincided more closely than MIP-1 with the kinetics of
macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested
that MCP-1 expression started at peribronchovascular regions and
expanded to alveoli-facing epithelial cells and infiltrated
macrophages. Interestingly, administration of a neutralizing Ab against
MCP-1, RANTES, or MIP-1 alone did not prevent macrophage
infiltration into infected alveoli, whereas combination of the three
Abs significantly reduced macrophage infiltration without affecting
neutrophil recruitment. The use of an antagonist to
N-formyl peptides,
N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe,
reduced both macrophages and neutrophils significantly. These data
demonstrate that a complex chemokine network is activated in response
to pulmonary pneumococcal infection, and also suggest an important role
for fMLP receptor in monocyte/macrophage recruitment in that
model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and RANTES preferentially augment monocyte/macrophage and
lymphocyte recruitment.
The recruitment of monocytes/macrophages at sites of inflammation
generally occurs after neutrophil emigration in vivo, and roles for C-C
chemokines have been explored in various models of lung inflammation.
For example, MCP-1 appears to be involved in the emigration of
monocytes/macrophages in lung allergic inflammation (4)
and pulmonary Cryptococcus neoformans infection
(5) but not in IgG immune complex acute lung injury
(6). Neutralization of RANTES decreases macrophage
infiltration to allergic inflammation (4) but not in IgG
immune complex acute lung injury (6). MIP-1 is
suggested to participate in the alveolar diapedesis of macrophage in
pulmonary C. neoformans infection (7) but not
in lung allergic inflammation (4). Although an increase in
the expression of three C-C chemokines in lung is often observed in
those lung infection/inflammation models, the above studies suggest
that such accumulation of chemokines in lung may not necessarily be
correlated with extravasation of macrophages in alveolar spaces. Thus,
other hierarchical regulation may play a role for appropriate
extravasation of macrophages in alveoli.
With their elegant in vitro works, the group of Butcher proposed that cross-talk between various chemoattractant receptors guides leukocytes to their destinations (8, 9, 10). Particularly interesting are cases of infections where pathogen-derived chemoattractant factors such as formyl peptides are present. Formyl peptides are cleavage products of bacterial or mitochondrial proteins and serve as powerful chemoattractants for both neutrophils and monocytes. Moreover, occupation of formyl peptide receptor with formyl peptides induces heterologous desensitization of receptors for other chemoattractant molecules, including chemokines (8, 11). Whether cross-talk exists between host- and pathogen-derived chemoattractants in in vivo infection models has not yet been established rigorously.
In streptococcal pneumonia, active monocyte/macrophage extravasation
takes place in late stage of infection when pathogens proliferate
(12). It has been suggested that such Streptococcus
pneumoniae growth could result in sustained release of formyl
peptides. Thus, it is likely that high levels of formyl peptide in
infected lungs contribute to macrophage emigration in alveoli.
Moreover, we have previously suggested that an early and sustained
TNF-/IL-1 production takes place in S. pneumoniae
pneumonia (12). As it has been suggested by in vitro and
in vivo studies that TNF- and IL-1 induce chemokine production
(13, 14, 15), we investigated in this study whether chemokines
and formyl peptides are involved in monocyte/macrophage recruitment to
alveolar spaces infected with S. pneumoniae.
We found mRNA expression of eight chemokines, RANTES, MIP-1,
MIP-1, MIP-2, IFN-
-inducible protein 10 (IP-10), MCP-1, T cell
activation 3 (TCA)-3, and KC within the lungs during the course
of murine pneumococcal pneumonia. KC and MIP-2 protein expression in
lungs correlated relatively well with the kinetics of neutrophil
infiltration. Sustained MCP-1 protein production coincided more closely
than MIP-1 with the kinetics of monocyte/macrophage infiltration in
alveoli. In situ hybridization (ISH) of MCP-1 mRNA suggested that MCP-1
expression starts at peribronchovascular regions and expands to
alveoli-facing epithelial cells and infiltrated macrophages.
Administration of a neutralizing Ab against MCP-1, MIP-1-, or RANTES
alone failed to reduce macrophage recruitment to infected alveoli,
whereas combination of the three Abs reduced it by 33%
(p < 0.05). Injection of an antagonist to
N-formyl peptides,
N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe
(Boc-PLPLP), reduced macrophage recruitment to infected alveoli by 42%
(p < 0.05), suggesting an important role for
bacterial formyl peptides as chemoattractants for macrophages during
streptococcal pneumonia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal goat anti-murine MCP-1, MIP-1, KC, and MIP-2
Abs used in our ELISAs and in neutralization studies were purchased
from R&D Systems (Minneapolis, MN). mAb to mouse MCP-1 used in
neutralization studies was purchased from PharMingen (Mississauga,
Ontario, Canada). The mouse rMCP-1, rMIP-1, rKC, and rMIP-2 were
purchased from R&D Systems. Boc-PLPLP was obtained from Sigma
(Mississauga, Ontario, Canada).
Pneumococcal pneumonia model
A murine model of pneumococcal pneumonia has been developed in our laboratory (12). Briefly, lightly anesthetized female CD1 mice (18–20 g) received an inoculum of 107 CFU of S. pneumoniae in 50 µl of PBS applied at the tip of the nose and involuntarily inhaled. This inoculation induced typical pneumonia symptoms (12) and 100% mortality rate within 4 days. In this experiment, infected animals were sacrificed by decerebration (under anesthesia) at different time points after infection over a 3-day period. Blood, bronchoalveolar lavage (BAL) fluid, and lung tissue were sampled for assessment of leukocyte recruitment, chemokine mRNA expression, and protein level. For plasma sampling, blood was collected in heparinized tubes from the retro-orbital sinus of the left eye, immersed in ice, and centrifuged (1000 x g at 4°C for 15 min). BAL was performed as previously described (12). Briefly, the trachea was exposed and intubated with a catheter, then the lungs were washed two times with 1 ml PBS. BAL fluids were centrifuged at 550 x g for 10 min, and supernatants were stored at -70°C until evaluation of the chemokines by ELISAs. Pellets were resuspended in PBS for total BAL cell count with a hemacytometer and for differentiation of the cell populations with Diff-Quick-stained (Baxter, Quebec, Canada) cytospin preparations. Lungs were also taken for assessment of neutrophil infiltration and chemokine protein expression in tissue. First, the pulmonary vasculature was perfused with 10 ml PBS injected via the right ventricle of the heart, then lungs were homogenized in 50 mM sodium phosphate buffer containing 0.5% Triton X-100 for chemokine analysis or 0.5% hexadecyltrimethylammonium bromide for measuring myeloperoxidase (MPO) activity (pH 7.5). For chemokine analysis, homogenates were incubated on ice for 30 min, then centrifuged for 15 min at 4°C (3000 x g), and supernatants were stored at -70°C until measurements. Neutrophil infiltration in lung tissue was determined by MPO activity, as previously described (12). Briefly, homogenates were sonicated and centrifuged at 3000 x g in a microcentrifuge for 30 min at 4°C. The supernatants were then mixed 1:8 with assay buffer, and absorbance was read at 450 nm against a standard curve made with commercial MPO (Sigma).
Measurement of mRNA expression by Northern blots and RNase protection assay (RPA)
Whole lungs were harvested at specific times after inoculation with S. pneumoniae. They were immediately homogenized in 4 ml of TRIzol reagent (Life Technologies, Grand Island, NY) and stored at -70°C. Total cellular RNA was isolated from lung tissues by acid guanidinium thiocyanate-phenol-chloroform extraction according to the manufacturer’s protocol. KC mRNA expression was determined by Northern blots. For each sample, 10 µg of RNA was separated in a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane (MANDEL Scientific, Quebec, Canada). Blots were then hybridized with a KC cDNA probe labeled by random priming using the T7 QuickPrime kit (Pharmacia Biotech, Uppsala, Sweden). Hybridized probe was visualized by autoradiography.
Chemokine mRNA expression was also determined by multiprobe RPA using the RiboQuant RPA kit (PharMingen) as recommended by the supplier. The specific chemokine bands were identified on the basis of their individual migration patterns in comparison with the undigested probes. The sample loading was normalized by the housekeeping genes L32 and GAPDH.
Determination of chemokine protein expression
Sandwich ELISAs were established for KC, MIP-2, MIP-1, and
MCP-1 to detect these chemokines in the supernatant of BAL fluid, in
the supernatant of lung homogenates, and in plasma. Briefly, 96-well
plates (Immunoplate; Nunc, Naperville, IL) were coated with
anti-MCP-1, anti-MIP-2, anti-MIP-1, or anti-KC in
100 µl/well of PBS, pH 7.4. Plates were incubated for 16 h at
4°C. Each plate was blocked with 5% BSA-PBS, and an appropriately
diluted sample (100 µl) was incubated for 2 h at room
temperature. The biotinylated polyclonal goat anti-chemokine Ab
(100 µl/well) was added and incubated for 1 h in 2% BSA-PBS.
Bound biotinylated anti-chemokine Ab was detected by
streptavidin-peroxidase conjugate (Research Diagnostics,
Flanders, NJ). The limit of detection for each chemokine was MCP-1,
20–500 pg/ml; MIP-1, 20–500 pg/ml; KC, 20–1000 pg/ml; and MIP-2,
20–500 pg/ml.
Preparation of murine JE (MCP-1) digoxigenin (DIG) cRNA probes
Murine JE (MCP-1) DIG-cRNA probes were synthesized by in vitro transcription of a JE cDNA fragment of 600 bp subcloned into the pGEM-1 vector. After linearization of the vector with HindIII, the antisense probe was transcribed according to the manufacturer’s protocol using T7 RNA polymerase (Boehringer Mannheim, Mannheim, Germany) and nucleoside triphosphates including digoxigenin-uridine triphosphate (Dig-11-dUTP; Boehringer); the control sense probe was prepared by linearization with PvuII and transcription with Sp6 RNA polymerase (Boehringer Mannheim).
ISH for MCP-1
The ISH protocol was established from a combination of published procedures (16, 17). All reactions were conducted with RNase-free materials and solutions. Fresh left lungs were perfused and fixed overnight at 4°C in 0.1 M phosphate-buffered 4% paraformaldehyde. Tissues were then dehydrated in solvent and embedded in paraffin. Five-micrometer sections were collected on positively charged slides (BioGenex Laboratories, San Ramon, CA) and dried overnight at 37°C.
Tissue sections were incubated in Tissue Clear III (Fisher Scientific, Pittsburgh, PA) and rehydrated by passage through graded ethanol and RNase-free water, followed by enzymatic treatment with 10 µg/ml proteinase K (Boehringer Mannheim) for 15 min at 37°C. Sections were then acetylated with freshly prepared 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8.0, for 10 min.
Lung tissue sections were then hybridized overnight at 44°C with 25 µl heat-denatured antisense or sense DIG-MCP-1 cRNA probes at a concentration of 100 ng/ml in hybridization solution (50% formamide, 4x SSC, 1x Denhardt’s solution, 1 mg/ml salmon sperm, and 10x dextran sulfate). Hybridization reactions were detected by immunostaining with alkaline phosphatase-conjugated anti-DIG Abs (Boehringer Mannheim). Nonspecific staining was blocked by incubation for 30 min with 1% lamb serum in TBS buffer containing 0.1% Triton X-100. After an incubation of 1 h with anti-DIG Ab diluted in blocking buffer, the hybridization signal was visualized with the substrates nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt. Levamisole (Sigma) was added to the reaction to inhibit endogenous alkaline phosphatase. Sections were mounted with AquaPerm (Fisher Scientific).
Passive immunization with neutralizing Abs against chemokines
Groups of infected mice received daily i.v. doses of 20
µg/mouse of neutralizing polyclonal Ab against MIP-1 or 5
µg/mouse of mAb against MCP-1 based on reported neutralizing efficacy
in animal models (4, 18), or 1 µg/mouse of polyclonal Ab
against RANTES based on the recommended dose of manufacturer. A last
group received a mixture of 20, 1, and 5 µg/mouse of
anti-MIP-1, anti-RANTES, and anti-MCP-1, respectively.
For each Ab, the first administration was performed 30 min before the
inhalation of S. pneumoniae, and subsequent doses were
repeated on days 1 and 2. Control mice ("preimmune") received
normal goat IgG for anti-MIP-1 and for anti-RANTES, and
normal hamster IgG for anti-MCP-1 (Jackson ImmunoResearch
Laboratories, West Grove, PA). Mice were then killed 6 h after
final administration of Abs (54-h postinfection) on day 2, and BALs
were collected to analyze leukocyte counts.
Inhibition of fMLP activity in vivo
The fMLP antagonist Boc-PLPLP (Sigma) (19) was injected i.v. at a concentration of 2 x 10-4 M, 0.1 ml/mouse, 48 h after infection with S. pneumoniae. Animals were sacrificed 6 h after injection of the drug. This product, when injected at this dose to uninfected mice, did not induce leukopenia.
Statistical analysis
All statistical analyses were performed using a computer package (Statview+SE Software; Abacus Concepts, Berkeley, CA). The significance between groups was statistically evaluated using a one-way ANOVA test, followed by the t test with Fisher’s corrections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our streptococcal pneumonia model, neutrophils started to
appear in alveoli (where the infection occurred) 4 h after
inoculation with S. pneumoniae, as we published before
(12). The number of neutrophils recruited in alveoli
sharply increased at 12 h, maintained high levels until they
peaked at 48 h, then were reduced at the point of death 72–96 h
after infection (Fig. 1
). A small number
(1.4 x 104 cells/ml) of resident alveolar
macrophages were present in alveoli before the infection, and newly
recruited macrophages became evident into alveoli at 48 h
postinfection. Lymphocytes and eosinophils remained essentially absent
in BAL over the duration of the experiment.
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Chemokine production
To investigate whether biosynthesis of C-X-C chemokines
(chemoattractants for neutrophils) and C-C chemokines (chemoattractants
for monocytes/macrophages) is up-regulated during murine pneumococcal
pneumonia, chemokine mRNA expression in whole lungs was first analyzed
at various times (0–72 h) by Northern blots and RPA. The RPA had the
capacity to simultaneously detect several chemokine mRNA species in a
single sample of RNA. The chemokine protein production in whole lungs
and their release in alveoli (BAL) were also estimated by specific
sandwich ELISAs. Northern and RPA analysis (Fig. 2) demonstrated that substantial amounts
of C-X-C chemokine MIP-2 (Fig. 2A) and KC (Fig. 2B) mRNA were present in lungs as early as 2 h after
infection. They peaked at 4 h, and appeared to plateau from 8 to
24 h after the infection. Thereafter, from 48 to 72 h, mRNA
levels gradually decreased for KC but persisted to a certain extent for
MIP-2. KC and MIP-2 proteins were also detected at 2 h and peaked
at 4 h in both lungs and BAL fluid (Fig. 3, A and B). The
same pattern as for mRNA detection was observed, as KC decreased
gradually over time to reach basic level at 72 h, whereas MIP-2 in
lungs persisted at a significant level until death of the animals.
Therefore, KC and MIP-2 protein levels were consistent with their
respective mRNA expression. Moreover, the peak concentrations of KC and
MIP-2 proteins coincided with the initiation of neutrophil infiltration
into lung interstitium (MPO activity) and alveoli (cell counts), but
only the persistence of MIP-2 coincided with the sustained recruitment
of neutrophils late after infection (Figs. 1
and 3A).
Interestingly, KC was detected in plasma as early as 4 h after
infection (Fig. 4) and remained high
until 72 h, whereas MIP-2 appeared only after 48 h. As for
IP-10, the mRNA expression of this C-X-C chemokine occurred later than
that of KC or MIP-2, but high levels were seen from 8 to 24 h and
were maintained to some extent until 72 h (Fig. 2).
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, MIP-1,
MCP-1, TCA-3, and RANTES, which was undetectable in uninfected lungs,
increased as early as 4 and 8 h after infection (Fig. 2). (In
fact, in the multiple probes present in the RPA, only eotaxin was not
expressed at any time during the experiment). However, the kinetics
and/or magnitude of expression of these mRNA were distinct for each C-C
chemokine, suggesting their involvement at different time points during
pneumonia.
There was a marked difference in the time-dependent expression of mRNA
and protein between MCP-1 and MIP-1 during the course of infection.
At the protein level, MIP-1 was the first to be recovered in BAL,
with a significant increase from 2 to 8 h after infection (Fig. 3D) when there was no significant macrophage recruitment
(Fig. 1). Despite the fact that evolution of the infection accelerated
macrophage recruitment into alveolar spaces 48 h after infection,
levels of MIP-1 in BAL were reduced significantly after 24 h
(Fig. 3D). However, levels of MIP-1 in total lungs
remained detectable throughout infection (Fig. 3C). MCP-1
protein production in the lungs became evident at 4 h and reached
a plateau at 12 h that was sustained up to 72 h when animals
started to die (Fig. 3C), indicating that the kinetics of
MCP-1 expression in lungs preceded actual macrophage recruitment. In
BAL, MCP-1 protein secretion to alveoli was delayed in the first
12 h but strongly increased between 24 and 48 h when
macrophage recruitment into alveoli became evident (Figs. 1 and 3D). MCP-1 proteins in BAL decreased to background levels
thereafter despite continuous biosynthesis in lung tissues. These data
demonstrate that the kinetics of MCP-1 protein in alveolar spaces
correlates better than that of MIP-1 to the emigration of
macrophages to alveoli, which took place within 48 h after
infection (Figs. 1 and 3, C and D). Finally,
MCP-1 was recovered in plasma from 8 to 72 h, whereas MIP-1
remained essentially absent from bloodstream throughout the evolution
of pneumonia (Fig. 4).
ISH for MCP-1
We had detected an early and sustained MCP-1 release in lungs and
alveolar spaces, suggesting that MCP-1 might form a gradient to induce
monocyte/macrophage recruitment to infected alveoli. Thus, we
investigated the distribution of MCP-1 mRNA by ISH using
digoxigenin-conjugated antisense probes on paraffin sections of mouse
lung tissues infected with S. pneumoniae. In normal lung
tissue, no MCP-1 mRNA was detected (Fig. 5A). In the first 24 h
after infection, MCP-1 mRNA was mainly detected in peribronchovascular
areas (Fig. 5, B–D). Over that period, some bronchiolar
epithelial cells (Fig. 5G), capillary endothelial cells
(Fig. 5H), and alveolar macrophages (Fig. 5I)
localized in these areas expressed MCP-1 mRNA. Then, as infection
progressed, labeled mRNA expanded from large peribronchovascular sites
to surrounding tissues and alveolo-capillary spaces where endothelial
cells, alveolar macrophages, and numerous recruited leukocytes were the
main expressing cells (Fig. 5E). In fact, maximal labeling
in the lung was noted at 48 h postinfection (Fig. 5E),
when strong recruitment of macrophages occurred (Fig. 1). At 72 h,
when active emigration of macrophages to alveoli was observed, MCP-1
mRNA expression declined in peribronchovascular spaces but remained
present at various pulmonary sites (Fig. 5F). Control ISH
sections exposed to sense probe or without any probe were unreactive,
thereby confirming specificity of the detection signals (data not
shown).
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D) while
most of the recruited leukocytes expressed MCP-1 mRNA at later time
points (Fig. 5E) in the whole lung surface, we performed an
additional assay to determine which type of infiltrating cells
expressed MCP-1 in our model. ISH was made on cells obtained from BAL
fluid 48 h postinfection, when neutrophil and macrophage
recruitment was active. Interestingly, among the leukocytes present in
BAL, expression of MCP-1 mRNA was observed in macrophages but not in
neutrophils (Fig. 5J). Influence of neutralizing Abs specific to C-C chemokines
The above results suggested that sustained MCP-1 expression and
late monocyte/macrophage recruitment characterized our lethal pneumonia
model. To further evaluate the specific contribution of C-C chemokines
(MCP-1, MIP-1, and RANTES) to monocyte/macrophage recruitment,
chemokine-inhibition experiments were performed using specific
neutralizing Abs that have been characterized extensively in vitro and
in vivo. Infected mice were injected with three doses of
anti-MCP-1, anti-MIP-1, or anti-RANTES alone, or a
combination of the three neutralizing Abs 30 min before infection, as
well as 24 and 48 h after infection. The extent of
monocyte/macrophage emigration to alveoli (BAL fluid) was studied
6 h after final administration of Abs (active macrophage
emigration to alveoli is observed at 48 h postinfection). Control
mice received control preimmune IgG.
As shown in Table I, none of the
neutralizing Abs given alone could reduce monocyte/macrophage
recruitment in BAL fluid at 54 h postinfection. Neutrophil counts
were not reduced as well (data not shown). In contrast to
administration of Ab against individual chemokine, passive
immunization with the combination of Abs against three C-C
chemokines (MCP-1, MIP-1, and RANTES) reduced the number of
monocytes/macrophages emigrated to alveoli by 33%
(p < 0.05) compared with the amount recovered
in mice administered the control IgG (Fig. 6). Administration of the combined Abs
slightly reduced neutrophil counts (6.4 ± 1.1 x
104 cells/ml for controls vs 4.9 ± 0.6
x 104 cells/ml for treated mice), which was not
statistically significant. Those data suggest that three major C-C
chemokines participate in monocyte/macrophage recruitment to infected
alveoli, but neutralization of a single chemokine among those three
does not affect monocyte extravasation to alveoli.
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We further evaluated the contribution of chemotactic formyl
peptides derived from bacteria (such as fMLP) in our model of
pneumonia. Blocking experiments were performed using an antagonist of
formyl peptide receptor, Boc-PLPLP. Mice were injected with Boc-PLPLP
48 h after infection, and the amount of leukocytes emigrated to
infected alveoli was analyzed 6 h later. Fig. 7 shows a significant
(p < 0.05) 42% reduction in
monocyte/macrophage counts recovered in BAL fluid of mice treated with
the antagonist as compared with the untreated infected group.
Emigration of neutrophils in infected alveoli of mice treated with
Boc-PLPLP also significantly (p < 0.05)
decreased (5.9 ± 1.4 x 104
neutrophils/ml) compared with infected controls receiving placebo
(9.6 ± 1.4 x 104 neutrophils/ml).
Together, these data suggest that formyl peptides contribute to
extravasation of both macrophages/monocytes and neutrophils to infected
alveoli during pneumococcal pneumonia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this experiment, pneumococcal pneumonia induced a rapid,
time-dependent accumulation of neutrophils and monocytes/macrophages
into the lungs. In our murine model of pneumonia, sustained neutrophil
recruitment was observed from 4 h after infection until death, and
it was followed by macrophage exudation that was significant from
48 h postinfection. Therefore, we consider this pneumonia model as
an interesting in vivo approach to investigate how macrophages emigrate
in the presence of multiple conflicting chemoattractant stimuli. In
this study, our data demonstrate that biosynthesis and release of C-C
chemokines, MCP-1, MIP-1, MIP-1, TCA-3, and RANTES, was
up-regulated. Although MCP-1 expression in alveolar spaces relatively
well paralleled the accumulation of macrophages in alveoli among the
C-C chemokines, passive immunization with anti-MCP-1 alone did not
significantly affect macrophage and neutrophil extravasation at a time
when active monocyte/macrophage emigration and MCP-1 release were
evident (48 h postinfection). Although passive immunization with Abs
against MCP-1, MIP-1, or RANTES alone could not affect
monocyte/macrophage emigration to alveoli, administration of a
combination of the three Abs against MCP-1, MIP-1, and RANTES did
reduce monocyte/macrophage recruitment significantly. Therefore, those
three chemokines apparently participate in extravasation of
monocytes/macrophages in a complementary manner, whereas each chemokine
alone can replace the others to some degree. Administration of the
formyl peptide antagonist Boc-PLPLP significantly reduced the
emigration of both macrophages and neutrophils, suggesting that
bacterial-derived formyl peptides also participate in the exudation
process of macrophages in the late stage of pneumococcal pneumonia.
Infection with S. pneumoniae led to the rapid, coordinated,
time-dependent expression of MIP-2 and KC chemokines in lung tissue and
in BAL fluid. Although MIP-2 was secreted in larger amounts than KC,
both pulmonary profiles for these C-X-C chemokines were quite similar
and peaked closely before a strong neutrophil recruitment occurred in
tissue and alveoli. This C-X-C chemokine kinetics suggests that MIP-2
and KC might be involved in neutrophil recruitment. Although this
correlation is consistent with other lung infection models where
overproduction or inhibition of KC or MIP-2 were respectively
associated with increased (25) or attenuated (23, 24, 26, 27) neutrophil accumulation in alveoli, the exact roles
for KC and MIP-2 in neutrophil recruitment in pneumococcal pneumonia
remain to be clarified. As those C-X-C chemokine protein levels
increase in blood in the late stage of infection, it is possible that
the appropriate chemotactic gradient of C-X-C chemokines to attract
neutrophils may not be formed in the late stage. Lack of appropriate
chemotactic gradient by a particular chemokine could lead to the
failure of leukocyte extravasation toward that chemokine. For example,
it has been demonstrated that MCP-1-transgenic mice that express high
levels of MCP-1 in blood and in a variety of organs do not display
monocyte infiltration (28). Series of works by Doerschuk
et al. (29, 30, 31, 32) clearly demonstrate that in the case of
pneumococcal pneumonia, a significant proportion of neutrophils
emigrates in the alveolar spaces independently of two classical
adhesion molecules expressed in neutrophils,
2-integrins, and selectins. As those adhesion
molecules participate dominantly in Gram-negative bacteria-induced
acute lung infection and injury, pulmonary infection with S.
pneumoniae seems to induce an alternative or "nonclassical"
neutrophil diapedesis pathway. Therefore, it is possible that not only
general C-X-C chemokines but also other classical chemoattractants are
involved in the diapedesis of neutrophils in pneumococcal pneumonia,
and this should be further investigated.
In our model, maximal monocyte/macrophage recruitment occurred between
days 2 and 4 after infection. The mechanisms that govern specific
monocyte/macrophage recruitment to alveoli are poorly understood.
Previous investigations in other models of inflammation have suggested
that monocyte recruitment depends upon both the activation of
endothelium and the generation of monocyte chemoattractants
(33). Yet this study demonstrated that a large array of
C-C chemokines are expressed during S. pneumoniae infection,
including MCP-1, MIP-1, MIP-1, RANTES, and TCA-3, whose mRNA
expressions were detected in the lung as soon as 4 h or 8 h
after infection. Although eotaxin has already been suggested to be a
colony-stimulating factor for both granulocytes and macrophages in lung
allergic inflammation (34), its main target cells
are the eosinophils, which were not recruited in our model, and
which also coincided with the absence of eotaxin mRNA
expression in lung tissues. Interestingly, despite the fact that
various C-C chemokines known to display overlapping activity for
lymphocyte and monocyte recruitment were expressed in our model, very
few, if any, lymphocytes emigrated into lung tissues. It is possible
that recruitment and/or retention of lymphocytes in S.
pneumoniae-infected alveoli need additional factors besides C-C
chemokines. Alternatively, a chemoattractant gradient by formyl
peptides that might not be chemotactic toward lymphocytes could play
roles in leukocyte diapedesis in pneumococcal pneumonia.
Interestingly, the MCP-1 and MIP-1 protein kinetics were quite
different during the evolution of pneumonia. MIP-1 secretion into
infected alveoli peaked early (within 12 h after infection) and
sharply decreased thereafter. As diapedesis of macrophages started
48 h after infection, it is unlikely that MIP-1 participated
directly in the recruitment of macrophages to alveoli. This is also
suggested by our experiment with anti-MIP-1 Ab, as
neutralization of MIP-1 did not result in the reduction
of macrophage recruitment in alveoli. In contrast to alveolar sites, a
significant amount of MIP-1 protein was expressed in total lung
tissue throughout infection, with no level of MIP-1 detected in
plasma at any time. Thus, it is possible that MIP-1 might be
involved in macrophage recruitment (or activation) into pulmonary
interstitium.
The different patterns of MIP-1 and MCP-1 secretion that we observed
in alveolar air spaces and blood suggest that expression of these two
C-C chemokines might be mediated by disparate regulatory pathways, that
these chemokines might be elaborated by mutually exclusive cells and/or
tissues, and that they may serve nonoverlapping functions in modulating
pulmonary host defenses. In fact, MCP-1 protein production in lungs and
its accumulation in alveoli preceded and correlated more closely to the
monocyte/macrophage infiltration in lungs than MIP-1 did. This
sustained release of MCP-1 with live pneumococci is consistent with the
same observation by Huffnagle et al. (5) in a mouse model
of pulmonary C. neoformans infection. Although (among the
C-C chemokines examined in this study) MCP-1 expression showed the
closest correlation to the kinetics of pulmonary macrophage
recruitment, the production of MCP-1 in lung and alveoli preceded
macrophage recruitment in alveoli infected with S.
pneumoniae (the time lag was between 24 and 36 h). This delay
is in contrast to the C. neoformans infection where exact
correlation between macrophage infiltration and MCP-1 accumulation in
alveoli was observed (5). It is not entirely clear why
such early C-C chemokine production in our model fails to induce
immediate infiltration of macrophages. It is possible that
monocytes/macrophages have been desensitized to C-C chemokines in the
early stage of infection or that additional signals are necessary for
emigration and differentiation/activation of these cells.
To better identify cells that synthesize MCP-1 and to follow the chemotactic gradient that is formed after pneumococcal infection, ISH was performed on lung tissue sections. Our results demonstrate that a variety of immune and nonimmune cells do synthesize MCP-1 mRNA in murine pneumococcal pneumonia. Endothelial cells in the peribronchovascular regions were among the main MCP-1 mRNA-expressing cells early after infection while labeling expanded to epithelial cells and monocytes/macrophages disseminated throughout the inflamed lungs at later stages of infection, suggesting that biosynthesis of MCP-1 is not exclusively limited to a particular cell type but rather is up-regulated in a synchronized manner that might be important for appropriate presentation of chemokines toward leukocytes. The difference in the profile of MCP-1 protein levels in lung tissue and BAL fluid determined by ELISA also suggests the existence of a gradient of chemokines to a certain degree. As macrophages that emigrate toward infected alveoli synthesize MCP-1, macrophages themselves are likely to participate in the establishment of such gradient. Although the complex events that initiated cell recruitment cannot fully be delineated from the present experiment, it is possible that bacterial chemotactic components or signals may have diffused freely from alveoli to surrounding tissues to blood vessels, as will be discussed later.
Although recruited monocytes/macrophages were able to express chemokines once at the site of infection, MCP-1 mRNA was not expressed by neutrophils in our pneumococcal pneumonia model. Papers regarding the ability of neutrophils to synthesize MCP-1 in vivo are controversial (35, 36, 37). Certain rat models of chronic bleomycin-induced lung fibrosis and chronic collagen-induced arthritis show MCP-1 production by infiltrated neutrophils (35, 36), whereas MCP-1 synthesis is not readily detectable in exudated neutrophils in acute skin or peritoneal inflammation (37). Therefore, the production of MCP-1 may be strictly regulated in neutrophils, depending on the type of inflammation and whether it is acute or chronic.
In our pneumococcal pneumonia model, neutralization of single C-C
chemokine MCP-1, MIP-1, or RANTES didn’t enable us to
confirm strict dependence of monocytes/macrophages on any of these
three chemokines for emigration to infected alveoli. Significant
reduction occurred only with neutralization of the three chemokines
using a combination of appropriate Abs. In contrast, our data with
Boc-PLPLP as an antagonist to formyl peptides demonstrated that
N-formylated peptides released by pneumococci
strongly contribute to chemotaxis of both neutrophils and macrophages
in S. pneumoniae-infected alveoli. These small by-products
of bacterial protein catabolism can diffuse freely within intercellular
spaces (38) and are classical chemoattractants for
neutrophils and macrophages. It has been shown that formyl peptides can
be recovered in pneumococcal culture filtrates (39). Thus,
our data suggest that chemoattractant bacteria-derived formyl
peptides and host-derived chemokines were involved in the emigration of
macrophages into alveoli in the late stage of pulmonary infection with
S. pneumoniae. It is also possible that formyl peptides
abrogated the host response to C-C chemokines to a certain extent in
our model, as it has been demonstrated that preincubation of
neutrophils or macrophages with formyl peptide inhibits their ability
to respond to IL-8, or down-regulates CCR5, the receptor for MIP-1
and RANTES (8, 40). Interestingly, Campbell et al. (8)
suggest that this preincubation with formyl peptide does not inhibit
responsiveness to formyl peptide itself and proposed that
chemoattractants emanating directly from a target of phagocytosis such
as formyl peptide, would override signals generated from the
surrounding host tissues such as chemokines. It has been suggested that
fMLP and C5a receptors are the only receptors known to mediate
heterologous desensitization. None of the tested chemokine or lipid
attractant receptors examined to date appear to support uncoupling of
heterologous responses (10). In our model, high pulmonary
C-C chemokine (MCP-1, MIP-1, and RANTES) levels were seen very early
after infection, and macrophage recruitment was initiated relatively
late, at a time when active bacterial growth prevails in inflamed
lungs. Therefore, it is possible that, by the time peripheral monocytes
started to migrate, their C-C chemokine receptors were partly
insensitive to the chemoattractant gradient formed by C-C
chemokines.
In conclusion, our study suggests that pneumococcus-derived formyl peptides participate as chemoattractants in neutrophil and macrophage infiltration to the alveoli observed in late stage of pneumococcal pneumonia, and that formyl peptide activity together with preexisting chemoattractant signals formed by chemokines actually guide monocytes/macrophages to the appropriate site of infection. Additional in vivo and in vitro studies are necessary to further delineate the respective roles of fMLP and of C-X-C and C-C chemokines in community-acquired and nosocomial pneumonia.
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Acknowledgments |
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Footnotes |
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2 S.S. was supported by a fellowship from the Canadian Institutes of Health Research and received a salary support for a new investigator from the Fonds de la Recherche en Santé du Québec.
3 Address correspondence and reprint requests to Prof. Michel G. Bergeron, Infectious Diseases Research Center, CHUQ, CHUL Building, 2705 Laurier Boulevard, Sainte-Foy, Québec, Canada G1V 4G2. E-mail address: Michel.G.Bergeron{at}crchul.ulaval.ca
4 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; MCP, monocyte chemoattractant protein; BAL, bronchoalveolar lavage; RPA, RNase protection assay; Boc-PLPLP, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe; TCA, T cell activation; MPO, myeloperoxidase; DIG, digoxigenin; ISH, in situ hybridization.
Received for publication July 19, 2000. Accepted for publication April 5, 2001.
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