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Infect Immun. 2010 June; 78(6): 2620–2630.
Published online 2010 April 5. doi:  10.1128/IAI.01169-09
PMCID: PMC2876546

Importance of CXC Chemokine Receptor 2 in Alveolar Neutrophil and Exudate Macrophage Recruitment in Response to Pneumococcal Lung Infection[down-pointing small open triangle]

Abstract

Sustained neutrophilic infiltration is known to contribute to organ damage, such as acute lung injury. CXC chemokine receptor 2 (CXCR2) is the major receptor regulating inflammatory neutrophil recruitment in acute and chronic inflamed tissues. Whether or not the abundant neutrophil recruitment observed in severe pneumonia is essential for protective immunity against Streptococcus pneumoniae infections is incompletely defined. Here we show that CXCR2 deficiency severely perturbs the recruitment of both neutrophils and exudate macrophages associated with a massive bacterial outgrowth in distal airspaces after infection with S. pneumoniae, resulting in 100% mortality in knockout (KO) mice within 3 days. Moreover, irradiated wild-type mice reconstituted with increasing amounts of CXCR2 KO bone marrow (10, 25, 50, and 75% KO) have correspondingly decreased numbers of both neutrophils and exudate macrophages, which is associated with a stepwise increase in bacterial burden and a reciprocal stepwise decrease in survival in S. pneumoniae-induced pulmonary infection. Finally, application of the CXCR2 antagonist SB-225002 resulted in decreased alveolar neutrophil and exudate macrophage recruitment in mice along with increased lung bacterial loads after infection with S. pneumoniae. Together, these data show that CXC chemokine receptor 2 serves a previously unrecognized nonredundant role in the regulation of both neutrophil and exudate macrophage recruitment to the lung in response to S. pneumoniae infection. In addition, we demonstrate that a threshold level of 10 to 25% of reduced neutrophil recruitment is sufficient to cause increased mortality in mice infected with S. pneumoniae.

Innate immune responses to acute bacterial infections in the lung are characterized by successive waves of early recruited polymorphonuclear neutrophils (PMN) followed by recruited mononuclear phagocytes (24). Recruitment of neutrophils by alveolar macrophages is essential for reducing the bacterial burden in the alveolar space. Newly recruited exudate macrophages, which mature into alveolar macrophages, may also contribute to bacterial containment and the resolution/repair phase of pulmonary infections (14, 22, 31). Despite their unquestionably important role in lung innate immunity, recruited neutrophils rapidly undergo apoptosis or necrosis and subsequently contribute to the formation of consolidated infiltrates, which disrupt alveolar gas exchange and may impair host defense functions of the lung (31). In addition, neutrophils, due to their enormous capacity to release reactive oxygen species and proteolytic enzymes, can directly contribute to acute lung injury as “collateral damage” (1, 13, 32). Even nonlethal challenge per se of the lung with bacteria and/or bacterial toxins results in the recruitment of huge numbers of neutrophils into both the lung interstitial and alveolar compartments, which is characteristic of acute respiratory distress syndrome (ARDS) (15, 17). However, whether such an uncontrolled neutrophilic burden developing in response to bacterial infections in the lungs of both humans and mice is helpful or harmful for elimination of pulmonary pathogens is incompletely defined.

Therapeutic strategies to accelerate the removal of consolidated infiltrates in infected organs may either target macrophage pool sizes or aim at inhibiting chemokine receptor signaling that results in the neutrophil recruitment and activation process. Our group has recently reported that constitutively increased, CC chemokine receptor 2 (CCR2)-dependent mononuclear phagocyte subset recruitment significantly improves the lung resolution/repair process and outcome in response to Streptococcus pneumoniae challenge (31). On the other hand, CXC chemokine receptor 2 (CXCR2) antagonists were found to reduce neutrophil recruitment and effector cell functions in lipopolysaccharide (LPS)-induced lung neutrophilia in rodent models and could have a therapeutic benefit in neutrophilic pulmonary diseases, including chronic obstructive pulmonary disease (COPD) (5, 6).

The murine CXCR2 (mCXCR2) and its ligands, CXCL1 (keratinocyte-derived chemokine [KC]) and CXCL2/3 (macrophage inflammatory protein 2 [MIP-2]), functional homologues of human growth-related oncogenes (GROα, -β, and -γ), have been identified as the primary chemokine receptor system mediating neutrophil recruitment in various models of acute and chronic inflammation, including Escherichia coli endotoxin-induced acute lung inflammation and injury (6, 19, 20, 23). Both CXCR2 knockout (KO) mice as well as small-molecule inhibitors that interfere with CXCR2 receptor function have shown a role for neutrophils in mediating hyperoxic or ventilator-induced lung injury and in collagen deposition in bleomycin-induced fibrosis (23). Unlike in influenza virus pneumonia, where CXCR2 is required for neutrophil recruitment but not viral clearance, CXCR2 is important for neutrophil recruitment as well as pathogen elimination in Pseudomonas aeruginosa or Nocardia asteroides pneumonia (18, 29). Also, a role for CXCR2 in mediating at least neutrophil recruitment and neutrophil-dependent killing in S. pneumoniae-induced lung infection was reported recently (26). However, it is currently not clear whether the abundant neutrophil recruitment observed in pneumococcal lung infection is really required for mounting protective immunity against this pathogen and what threshold level of CXCR2 inhibition/deletion causes decreased lung protective immune responses to challenge with S. pneumoniae. In addition, the role of CXCR2 in regulating S. pneumoniae-induced lung exudate macrophage recruitment is also unknown. Therefore, wild-type (WT) mice with or without pretreatment with CXCR2 antagonist SB-225002 or CXCR2 KO mice as well as mixed bone marrow chimeric mice reconstituted with an increasingly CXCR2-deficient hematopoietic system were infected with S. pneumoniae, and neutrophil versus monocyte-derived exudate macrophage recruitment profiles and the respective CC and CXC chemokine responses as well as bacterial loads and survival were determined over time.

MATERIALS AND METHODS

Mice.

Wild-type BALB/c mice (weight, 20 to 22 g) were purchased from Charles River (Sulzfeld, Germany). CXCR2 KO mice (CXCR2−/−) on the BALB/c background were purchased from Jackson Laboratories (Bar Harbor, ME). Both female wild-type mice and female CXCR2 KO mice (20 to 22 g) were kept under specific-pathogen-free conditions with free access to autoclaved food and water and were routinely screened for major pathogens according to the Federation of European Laboratory Animal Science Associations recommendations to exclude any opportunistic infections that would possibly confound the experimental results. Mice were used in all experiments at 8 to 12 weeks of age in accordance with the guidelines of our Institutional Animal Care and Use Committee of Hannover School of Medicine, Germany. Animal experiments were approved by our local government authorities. Experiments were performed under blinded conditions.

Reagents.

A Roti-Quick kit for total RNA isolation was purchased from Carl Roth (Karlsruhe, Germany). All other reagents for reverse transcription (random hexamer primers, Moloney murine leukemia virus reverse transcriptase [MMLV-RT], recombinant RNase inhibitor, deoxynucleoside triphosphates, and dithiothreitol) were purchased from Promega (Madison, WI). A SYBR green 1 kit was purchased from Eurogentec (Seraing, Belgium). Selective nonpeptide CXCR2 antagonist SB-225002 was purchased from Biozol (Eching, Germany) and was dissolved at 10 mg/ml in ethanol, according to the manufacturer's instructions. For in vivo application, aliquots of the inhibitor were dissolved in vehicle (0.9% NaCl solution containing 0.33% Tween 80) just before use (3).

Total cellular RNA isolation, cDNA synthesis, and real-time RT-PCR.

Total cellular RNA was isolated from bone marrow cells using the Roti-Quick kit (Roth) following the instructions of the manufacturer. RNA quantification and purity were determined on an Agilent Bioanalyzer 2100 (Agilent Biosystems), and only those RNA preparations exceeding absorbance ratios (A260/A280) of >1.90 were further processed for reverse transcription and real-time RT-PCR experiments. Synthesis of cDNA and real-time RT-PCR experiments were performed as recently described (25). Primers for detection of mCXCR2 mRNA were purchased from Operon Biotechnologies (Cologne, Germany) and had the following sequences: forward primer, 5′-AGCAAACACCTCTACTACCCTCTA-3′; reverse primer, 5′-GGGCTGCATCAATTCAAATACCA-3′. Real-time RT-PCR experiments were run on an Applied Biosystems ABI-7300 analyzer.

Culture and quantification of Streptococcus pneumoniae.

We used a capsular type 19 S. pneumoniae strain (EF3030) for infection experiments. This strain has been demonstrated to primarily cause lobar pneumonia in mice without bacteremia (4, 30). S. pneumoniae was grown in Todd-Hewitt broth (THB; Difco) supplemented with 0.1% yeast extract to mid-log phase, and aliquots were snap-frozen in liquid nitrogen and stored at −80°C until use, essentially as described recently (14, 27). Quantification of S. pneumoniae stocks was done by plating serial dilutions on sheep blood agar plates (BD Biosciences, Heidelberg, Germany), followed by incubation of the plates at 37°C with 5% CO2 for 18 h and the determination of CFU.

Generation of bone marrow chimeric mice.

Bone marrow chimeric mice were generated as recently described (16, 27). Briefly, bone marrow cells were isolated under sterile conditions from tibias and femurs of sex-matched wild-type and CXCR2-deficient donor mice. Single-cell suspensions were carefully prepared from the bone marrow isolates and filtered through 70- and 40-μm nylon meshes (BD Biosciences, Heidelberg, Germany) to remove residual cell aggregates. The cells were washed in RPMI 1640 supplemented with 10% fetal calf serum (Gibco) before transplantation. Recipient wild-type mice received a total body irradiation of 8 Gy at a dose rate of 1.8 Gy/min delivered by a linear accelerator (Siemens MD 2) operating in a 6-MV high-energy photon delivery mode (27). Subsequently, sedated wild-type mice received donor bone marrow cell preparations collected from wild-type and CXCR2 KO mice mixed at ratios of 90:10; 75:25; 50:50, and 25:75 (WT:KO ratios; 1 × 107 cells/mouse) with Leibovitz L-15 medium (Gibco) via lateral tail vein injections. Resulting mixed chimeric wild-type mice were then housed under specific-pathogen-free (SPF) conditions with free access to autoclaved food and water for at least 6 weeks prior to infection experiments.

Experimental groups.

The following experimental groups were defined: Mock-infected mice received intratracheal instillations of vehicle (50 μl of THB/mouse) and were analyzed at 24 h posttreatment. Wild-type mice and CXCR2 KO mice were either mock infected or were infected with serotype 19 S. pneumoniae (~3 × 106 CFU/mouse) and were analyzed at the indicated time points postinfection. Moreover, groups of irradiated wild-type mice transplanted with mixtures of CXCR2 KO and wild-type bone marrow cells (at ratios of 10:90, 25:75, 50:50, 75:25 [KO:WT]; 1 × 107 cells/mouse) (Table (Table1)1) were infected with serotype 19 S. pneumoniae (~3 × 106 CFU/mouse) and subsequently analyzed for bacterial load, inflammatory leukocyte recruitment, and CC and CXC chemokine profiles, as indicated. In selected experiments, wild-type mice received intraperitoneal applications of vehicle or CXCR2 antagonist SB-225002 (1.5 or 15 μg/g of body weight) twice daily prior to infection of mice with S. pneumoniae. Twenty-four hours after infection, bronchoalveolar lavage (BAL) fluid and lung tissue CFU counts and numbers of alveolar recruited neutrophils and exudate macrophages were determined.

TABLE 1.
Treatment groups and hematopoietic CXCR2 expression profiles

Infection of mice with Streptococcus pneumoniae.

CXCR2 KO mice, wild-type mice, and chimeric wild-type mice were infected with serotype 19 S. pneumoniae, using freshly prepared dilutions of thawed aliquots, as recently described (14, 27). For infection, mice were fixed in a vertical position followed by visualization of the pharynx via transillumination of the neck with a cold light source (Leica, Wetzlar, Germany). Subsequently, the trachea was intubated with a 26-gauge catheter (Abbocath) followed by careful intratracheal instillation of S. pneumoniae into the lungs. After instillation, mice were kept in individually ventilated cages (IVC) with free access to autoclaved food and water and were monitored twice daily for disease symptoms and survival during the entire observation period (2).

Determination of bacterial loads in bronchoalveolar lavage fluid and lung tissue homogenate.

Bacterial loads in the lungs of S. pneumoniae-infected mice of the various experimental groups were determined in both BAL fluid and lung tissue homogenates. Briefly, mice were euthanized with an overdose of isoflurane, and tracheas of the mice were exposed and cannulated with a shortened 20-gauge needle that was firmly fixed to the trachea. Subsequently, 300-μl aliquots of ice-cold sterile phosphate-buffered saline (PBS) were instilled and carefully aspirated until a first BAL fluid volume of 1.5 ml was collected. Subsequently, the collection of BAL was continued until an additional BAL fluid volume of 4.5 ml was collected. The 1.5-ml and 4.5-ml BAL fluid samples (whole lung washes) collected from S. pneumoniae-infected mice of either treatment group were immediately processed for determination of bacterial loads by plating 100 μl of the respective BAL fluid aliquot in 10-fold serial dilutions on sheep blood agar plates followed by incubation of the plates at 37°C with 5% CO2 for 18 h. Subsequently, CFU were counted and bacterial loads in whole lung washes were calculated. Whole lung washes were further subjected to centrifugation at 1,400 rpm (4°C, 10 min), and cell pellets were pooled to determine total numbers of BAL fluid leukocytes. In addition, BAL fluid CC (CCL2 [JE, monocyte chemoattractant protein 1, or MCP-1], and CCL12 [MCP-5]) and CXC chemokines (CXCL1 [KC] and CXCL2 [MIP-2]) were measured in cell-free BAL fluid supernatants of the individual 1.5-ml BAL fluid aliquots. Subsequent to the collection of BAL fluid, lung tissue was homogenized in 2 ml of Hanks balanced salt solution without supplements using a tissue homogenizer (IKA, Staufen, Germany), and 10-fold serial dilutions of the lung tissue homogenates were plated on sheep blood agar plates followed by incubation of the plates at 37°C with 5% CO2 for 18 h for CFU determination. Since the chimeric wild-type mice transplanted with 75% CXCR2-deficient bone marrow demonstrated 100% mortality during day 3 after infection with S. pneumoniae, determinations of leukocyte counts and CFU in BAL fluids and lung tissue of the 75% chimeric mice were done immediately after reaching the 72-h time point of infection, in order to avoid unnecessary repetition of this experiment due to increased mortality.

Immunophenotypic analysis of mononuclear phagocyte subsets in bronchoalveolar lavage fluid.

Mononuclear phagocyte subset populations (alveolar macrophages and alveolar exudate macrophages) contained in the bronchoalveolar lavage fluid of mock-infected or S. pneumoniae-challenged mice of the various experimental groups were subjected to immunophenotypic analysis of their cell surface antigen expression profiles, using a BD FACSCanto flow cytometer equipped with an argon ion laser operating at a 488-nm excitation wavelength and a helium neon laser operating at a 633-nm wavelength (BD Biosciences, Heidelberg, Germany), according to recently published protocols (14, 27, 30). Briefly, cells preincubated with octagam (Octapharma, Langenfeld, Germany) were stained for 15 min at 4°C with various combinations of appropriately diluted fluorochrome-conjugated monoclonal antibodies with specificities for the following cell surface molecules: phycoerythrin (PE)-Cy7-conjugated anti-CD11b, PE-Cy5.5-conjugated anti-CD11c, PE-conjugated anti-CD86, PE-conjugated anti-major histocompatiblity complex (MHC) class II (all from BD Biosciences), and allophycocyanin (APC)-conjugated anti-F4/80 (Serotec, Düsseldorf, Germany). Subsequently, cells were washed in PBS-0.1% bovine serum albumin (BSA)-0.02% sodium azide, and cell acquisition was performed on a BD FACSCanto flow cytometer (BD Biosciences). Gating of the respective mononuclear phagocyte subsets in BAL fluid was done according to their forward scatter area (FSC-A) versus side scatter area (SSC-A) characteristics and FSC-A versus F4/80-APC fluorescence emission characteristics to exclude debris and neutrophils from further analysis. Resident alveolar macrophages, which were collected by bronchoalveolar lavage, were identified according to their CD11cpos, CD11bneg/low, CD86neg, and MHC-IIneg cell surface antigen expression profile. In contrast, alveolar exudate macrophages contained in BAL fluid were identified according to their cell surface expression of CD11cpos, CD11bhigh, CD86neg, and MHC-IIneg, essentially as described recently (14). Data analysis and careful postacquisition compensation of spectral overlaps between the various fluorescence channels were performed using BD FACSDiva software (BD Biosciences). For quantification of neutrophils in BAL fluids, cells were counted in Pappenheim-stained cytocentrifuge preparations according to their staining pattern and morphological criteria, including their nuclear shape, followed by multiplication of this value with total BAL fluid cell numbers. In selected experiments, we determined CXCR2 and CCR2 chemokine receptor expression profiles on peripheral blood neutrophils and inflammatory monocytes or alveolar exudate macrophages. Briefly, cells were gated according to their FSC area versus SSC area characteristics, followed by hierarchical subgating of the Gr-1high F4/80neg neutrophils or the Gr-1pos F4/80pos inflammatory monocytes (13) or alveolar exudate macrophages, as described above, and subsequent determination of their CXCR2 and CCR2 receptor expression profiles. Anti-CCR2 antibody MC21 was employed for these experiments as described recently (13, 15). Detection of CXCR2 expression on neutrophils and exudate macrophages was done using a previously described protocol (16). Briefly, after incubation with Fc block (10 μl; BD Biosciences), cells were incubated with anti-CXCR2 antibody or isotype control antibody (R&D Systems, Wiesbaden, Germany), washed three times, and incubated on ice with biotinylated F(ab′)2 for 30 min. Cells were then washed and stained with PE-conjugated streptavidin (BD Biosciences) for 15 min on ice in the dark.

ELISA.

CC chemokines CCL2 and CCL12 and CXC chemokines CXCL1 and CXCL2 were determined in BAL fluids of mock-infected or S. pneumoniae-infected wild-type mice or CXCR2 KO mice or the various groups of chimeric wild-type mice by using commercially available enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer's instructions (R&D Systems, Wiesbaden, Germany).

Statistics.

All data are given as means ± standard errors of the means (SEM). Differences between treatment groups over time were analyzed by analysis of variance followed by a post hoc Dunnett test. Significant differences between groups were analyzed by Levene's test for equality of variances followed by Student's t test using SPSS 15.0 for Windows software package. Survival curves were compared by the log-rank test. Statistically significant differences between groups were assumed when P values were <0.05.

RESULTS

CXCR2 KO mice are highly susceptible to infection with S. pneumoniae.

To determine the role of CXCR2 in lung protective immunity to pneumococcal infection, wild-type mice and CXCR2 KO mice were challenged with serotype 19 S. pneumoniae. As shown in Fig. Fig.1,1, low-dose pneumococcal infection of wild-type mice with 3 × 106 CFU/mouse did not cause any mortality during an observation period of 8 days. In sharp contrast, CXCR2 KO mice showed a progressive mortality starting at day 2 postinfection, with 100% mortality in this group by day 4 of infection (Fig. (Fig.1A).1A). Bacterial loads in BAL fluids and lung tissue homogenates of wild-type mice rapidly declined within 3 days of infection (Fig. 1B and C), while at the same time, CXCR2 KO mice demonstrated a drastic outgrowth of S. pneumoniae in BAL fluids and lung parenchymal tissue up until day 3 postinfection (Fig. 1B and C), which was accompanied by a sharp decline in survival just on days 3 and 4 postinfection (Fig. 1A to C). Such immediate early mortality observed in the CXCR2 KO mice suggests impaired innate immune responses to infection with S. pneumoniae, thereby strongly impacting survival.

FIG. 1.
Survival and bacterial pathogen elimination in wild-type mice and CXCR2 KO mice infected with S. pneumoniae. (A) Wild-type mice and CXCR2 KO mice were infected with serotype 19 S. pneumoniae (~3 × 106 CFU/mouse), and survival (12 mice ...

Deletion of CXCR2 attenuates alveolar neutrophil and exudate macrophage recruitment in response to S. pneumoniae infection.

Alveolar macrophages and neutrophils are centrally involved in uptake and elimination of inhaled bacteria (7, 24). Therefore, we next determined the impact of CXCR2 deletion on profiles of resident and inflammatory leukocyte subsets in BAL fluids of wild-type and CXCR2 KO mice challenged with S. pneumoniae. Both mock-infected wild-type mice and CXCR2 KO mice had similar numbers of resident alveolar macrophages and few recruited exudate macrophages in their BAL fluids, as shown in Fig. 2A and B (0-day time points). In response to low-dose challenge with S. pneumoniae, both wild-type and CXCR2 KO mice developed significantly reduced numbers of resident alveolar macrophages by days 1 and 2 postinfection compared to mock-infected mice on day 0, most probably due to S. pneumoniae-induced apoptosis induction in these cells, according to recent reports (7, 27) (Fig. 2A and B). Unexpectedly, however, we observed that unlike wild-type mice, which developed a coordinated recruitment of exudate macrophages into their lungs in response to S. pneumoniae, CXCR2 KO mice had significantly reduced numbers of exudate macrophages in the bronchoalveolar compartment, particularly on days 2 and 3 postinfection (Fig. (Fig.2B).2B). Furthermore, repopulation of the alveolar air space with alveolar macrophages, as observed in S. pneumoniae-infected wild-type mice, was significantly impaired in the CXCR2 KO mice on day 3 postinfection (Fig. (Fig.2A).2A). In addition, CXCR2 KO mice had a markedly reduced neutrophil recruitment in response to S. pneumoniae challenge on days 1 and 2 postinfection compared to the wild-type control mice (Fig. (Fig.2C).2C). Together, these data show that deletion of CXCR2 causes severe perturbations of both neutrophil and exudate macrophage trafficking in response to pneumococcal infections, which results in a marked diminution of the entire pulmonary macrophage population in infected lungs of CXCR2 KO mice.

FIG. 2.
Analysis of professional phagocyte subsets in the lungs of wild-type mice and CXCR2 KO mice infected with S. pneumoniae. Wild-type mice and CXCR2 KO mice were infected with serotype 19 S. pneumoniae (~3 × 106 CFU/mouse). At the indicated ...

Increasing levels of deletion of hematopoietic CXCR2 result in an increasing loss of lung protective immunity to pneumococcal challenge.

Although acute bacterial infections of the lung are characterized by a dramatic recruitment of neutrophils into both the lung parenchymal tissue and bronchoalveolar space (24), the pathophysiological value of an abundant neutrophilic response is currently unclear. Since global deletion of CXCR2 in mice led to 100% mortality within 3 to 4 days of infection, we next examined (i) at what threshold level of hematopoietic CXCR2 ablation a reduced lung neutrophil and/or exudate macrophage burden would be observed and (ii) to what degree partial CXCR2 ablation affected lung protective immune responses to challenge with S. pneumoniae. We therefore generated chimeric wild-type mice reconstituted with a successively CXCR2-deleted hematopoietic system (Table (Table11).

Figure Figure3A3A shows a representative real-time RT-PCR amplification curve of CXCR2 mRNA levels in wild-type mice compared to CXCR2 KO mice and chimeric wild-type mice with a 50% CXCR2-negative hematopoietic system. Moreover, we also determined baseline expression profiles of chemokine receptors CXCR2 and CCR2 on the cell surface of peripheral blood neutrophils and monocytes, as well as on the cell surfaces of recruited neutrophils and exudate macrophages in BAL fluids of S. pneumoniae-infected mice (13). As shown in Fig. Fig.3,3, Gr-1high peripheral blood neutrophils from wild-type mice (Fig. (Fig.3B),3B), but not CXCR2 KO mice (Fig. (Fig.3G),3G), demonstrated strong CXCR2 expression, while neutrophils collected from chimeric wild-type mice with a 50% CXCR2-negative hematopoietic system demonstrated an ~1:1 distribution of CXCR2-positive to CXCR2-negative Gr-1high neutrophils (Fig. (Fig.3I),3I), thus confirming the validity of the chosen experimental approach. Moreover, we found that inflammatory Gr-1pos blood monocytes from wild-type mice (Fig. (Fig.3D)3D) or CXCR2 KO mice (Fig. (Fig.3H)3H) demonstrated similar cell surface expression of CCR2 but not of CXCR2 (Fig. (Fig.3C).3C). However, in response to pneumococcal challenge, alveolar recruited neutrophils maintained their cell surface CXCR2 expression (Fig. (Fig.3E),3E), whereas alveolar recruited exudate macrophages (Fig. (Fig.3F),3F), in contrast to their circulating precursors, demonstrated increased CXCR2 expression, suggesting that pneumococcal infection triggered increased CXCR2 expression on the cell surface of recruited exudate macrophages but not circulating inflammatory monocytes.

FIG. 3.
CXCR2 gene and protein expression patterns in wild-type mice, chimeric wild-type mice, and CXCR2 KO mice. (A) Bone marrow cells were isolated from wild-type mice or chimeric wild-type mice (50:50 KO:WT) or CXCR2 KO mice and subjected to analysis of CXCR2 ...

We next determined the effect of graded CXCR2 ablations in peripheral blood on the lung host defense against pneumococcal infection. As shown in Fig. Fig.4A,4A, we found that more than 90% of irradiated wild-type mice reconstituted with 100% wild-type bone marrow (serving as transplantation controls) survived a low-dose infection with serotype 19 S. pneumoniae, which was nonsignificantly reduced compared to mock-infected wild-type mice (Fig. (Fig.4A4A versus 1A). Unexpectedly, however, we found that just a 10% CXCR2 ablation in the hematopoietic system (90:10 WT:KO) led to a 20% reduced survival of mice, relative to transplantation controls (P < 0.05) (Fig. (Fig.4A).4A). Moreover, mice reconstituted with a 75:25 WT:KO bone marrow mixture demonstrated a sharp decline in survival, resulting in ~50% mortality by day 4 of infection relative to transplantation controls and further progressing toward ~80% mortality at day 7 postinfection. In addition, mice transplanted with a 50:50 or 25:75 WT:KO bone marrow mixture demonstrated an overall mortality of 100% on days 5 and 3, respectively, in response to low-dose infection with S. pneumoniae (Fig. (Fig.4A4A).

FIG. 4.
Effects of successive CXCR2 ablation or inhibition on survival, bacterial pathogen elimination, and professional phagocyte recruitment profiles in chimeric wild-type mice infected with S. pneumoniae. (A to F) Irradiated wild-type mice were reconstituted ...

Analysis of pneumococcal loads in BAL fluids and lung parenchymal tissue of chimeric wild-type mice on days 1 and 3 after infection with S. pneumoniae showed increased bacterial counts starting on day 1 and particularly by day 3 postinfection (Fig. 4B and C) relative to transplantation controls. More specifically, a less pronounced, but detectable, bacterial outgrowth was observed in the 10% and 25% chimeric wild-type mice, whereas 50% and 75% chimeric wild-type mice demonstrated a significantly increased bacterial outgrowth both in BAL fluids and lung parenchymal tissue relative to transplantation controls (Fig. 4B and C).

We next examined the effect of partial CXCR2 ablation on numbers of alveolar macrophages, exudate macrophages, and neutrophils in the various experimental groups on days 1 and 3 of infection with S. pneumoniae. As shown in Fig. Fig.4D,4D, all treatment groups demonstrated reduced numbers of alveolar macrophages at day 1 postinfection, consistent with recent reports (27), which were found to again increase toward baseline levels in the transplantation controls and the 10% and 25% CXCR2-ablated chimeric wild-type mice. In contrast, the 50% and 75% CXCR2-ablated chimeric wild-type mice demonstrated highly reduced numbers of alveolar macrophages in their BAL fluids by day 3 of infection (Fig. (Fig.4D),4D), demonstrating that a 50% CXCR2 ablation is sufficient to perturb alveolar macrophage repopulation in response to S. pneumoniae.

We observed that exudate macrophage recruitment, which typically develops after 3 to 4 days of bacterial infection (31), increased both in transplantation controls as well as the 10% and 25% CXCR2-ablated chimeric mice from day 1 until day 3 of infection, whereas strongly and significantly reduced numbers of exudate macrophages were noted in the 50% and 75% CXCR2-ablated mice by days 1 and 3 postinfection (Fig. (Fig.4E).4E). Analysis of neutrophil recruitment patterns in mice of the various treatment groups revealed a nearly linear inverse relationship between the degree of CXCR ablation and the numbers of BAL fluid neutrophils in S. pneumoniae-infected chimeric mice (Fig. (Fig.4F).4F). Transplantation controls and 10% CXCR2-ablated chimeric wild-type mice demonstrated nearly similar degrees of alveolar neutrophil recruitment in response to pneumococcal lung infection, whereas the 50% and 75% CXCR2-ablated chimeric wild-type mice, in particular, showed a nearly 70% decreased alveolar neutrophil recruitment at days 1 and 3 of infection with S. pneumoniae (Fig. (Fig.4F4F).

Finally, we determined the effect of a selective nonpeptide CXCR2 antagonist on bacterial loads and lung leukocyte recruitment profiles in mice infected with S. pneumoniae. As shown in Fig. 4G to J, application of the CXCR2 inhibitor SB-225002 in mice dose dependently reduced numbers of both alveolar recruited neutrophils and exudate macrophages and led to significantly increased bacterial loads as early as 24 h post-pneumococcal challenge (Fig. 4I and J). Collectively, these data add to the findings in chimeric mice and support the concept that interfering with CXCR2 signaling may severely perturb the lung host defense against inhaled bacterial pathogens.

Effect of a gradual CXCR2 ablation on inflammatory cytokine release in S. pneumoniae-infected mice.

We finally determined inflammatory CXC (KC [CXCL1] and MIP-2 [CXCL2]) and CC chemokine (MCP-1 [CCL2] and MCP-5 [CCL12]) responses in BAL fluids of wild-type and CXCR2 KO mice as well as the various groups of chimeric wild-type mice. Figure 5A and B demonstrates that relative to wild-type mice, CXCR2 KO mice had drastically increased neutrophil chemokine release in their BAL fluids (up to 10,000 pg/ml) on days 1 to 3 of infection with S. pneumoniae (Fig. 5A and B). In addition, we also found highly increased monocyte chemokines (CCL2 and CCL12) (Fig. 5C and D) in BAL fluids of CXCR2 KO mice but not wild-type mice on days 1 to 3 of infection, reflective of uncontrolled CXC and CC chemokine storms in CXCR2-deficient mice in response to S. pneumoniae. As observed in wild-type mice, transplant controls also demonstrated low levels of neutrophil and monocyte chemoattractants in their BAL fluids upon infection with S. pneumoniae (Fig. 5E to H).

FIG. 5.
Effects of partial or total CXCR2 deletion on the release of neutrophils compared to monocyte chemoattractants in bronchoalveolar lavage fluids of wild-type mice, CXCR2 KO mice, and chimeric wild-type mice challenged with S. pneumoniae. Wild-type mice, ...

We found a nearly linear relationship between the degree of hematopoietic CXCR2 ablation and the amount of both neutrophil and monocyte chemoattractants released into the bronchoalveolar compartment: 10% CXCR2-deficient chimeric wild-type mice demonstrated low levels of both CXC (KC and MIP-2) and CC (CCL2 and CCL12) chemokines in their BAL fluids, whereas 50% and 75% CXCR2-ablated chimeric wild-type mice responded with a strongly increased release of CXC and CC chemokines upon infection with S. pneumoniae (Fig. 5E to H). Importantly, the strongest chemotactic responses in 50% and 75% chimeric wild-type mice were observed just on day 3 postinfection and coincided with the highest bacterial loads, lowest neutrophil and exudate macrophage responses, and a rapid decline in survival rates in these groups. Together, these data demonstrate that successive deletion of hematopoietic CXCR2 leads to a stepwise increased release of not only neutrophil but also monocyte chemotactic activities in the BAL fluids of mice infected with S. pneumoniae, with the highest chemokine responses noted in the mutant mice upon infection with S. pneumoniae. These data illustrate for the first time important effects of CXCR2 on both neutrophil and monocyte chemotactic and cellular responses to infection with S. pneumoniae.

DISCUSSION

The purpose of the current study was to determine whether partial CXCR2 ablation would reduce neutrophil burdens in the lungs of mice without immediately impairing lung protective immunity to challenge with inhaled S. pneumoniae. We infected wild-type mice and CXCR2 KO mice and a series of increasingly CXCR2-ablated chimeric wild-type mice with a low dose of S. pneumoniae. Our results indicated that CXCR2 is absolutely required for protective immunity against S. pneumoniae. Deletion of CXCR2 resulted in an excessive early mortality by 3 to 4 days after infection, accompanied by a drastic overgrowth of pneumococci in lung distal airspaces and a strongly decreased alveolar neutrophil and alveolar exudate macrophage recruitment that resulted in a markedly reduced resident pulmonary macrophage pool size. Ablation of CXCR2 in the hematopoietic system was sufficient to trigger substantial mortality, with levels of just 10 to 25% hematopoietic CXCR2 ablation causing strongly increased bacterial loads and early mortality in the chimeric WT/CXCR2 KO mice. Together, these results for the first time demonstrate the critical and nonredundant role of CXCR2 in lung innate immunity against a prototypic, Gram-positive bacterial pathogen and demonstrate that even partial deletion of CXCR2 is sufficient to interrupt recruitment profiles of all major professional phagocyte subsets, including neutrophils, exudate macrophages, and resident alveolar macrophages, with fatal consequences for the infected host.

There are several as-yet-unanswered questions regarding the role of CXCR2 in lung leukocyte subset recruitment in S. pneumoniae-induced lung infection. First, is CXCR2 essential for lung exudate macrophage recruitment in response to S. pneumoniae or is this simply secondary to lung neutrophil recruitment? Second, what degree of abundance of lung neutrophil recruitment is required to contain and eradicate bacterial burden in S. pneumoniae pneumonia, and is there a threshold level of reduced neutrophil recruitment that does not impair lung protective immunity against inhaled S. pneumoniae? Our results suggest that both alveolar neutrophil and alveolar exudate macrophage recruitment levels are attenuated in S. pneumoniae-infected CXCR2 KO mice, thus demonstrating the critical role of CXCR2 in regulating the recruitment of both neutrophils and exudate macrophages.

Recent reports have demonstrated a role for neutrophil- and endothelial/epithelial CXCR2 expression in contributing to alveolar neutrophil recruitment in a model of E. coli-induced acute lung inflammation (20), but monocyte recruitment was not a prominent feature. Notably, Gram-negative as opposed to Gram-positive bacteria and their bacterial toxins (E. coli versus S. pneumoniae) are known to trigger different molecular signaling and recruitment pathways that mediate lung neutrophil recruitment in response to bacterial infection (10, 11, 15), thus making a direct comparison between the previous study and our report difficult. However, a weak neutrophilic response was still detectable in the mutant mice, thus indicating possible involvement of chemokine receptor systems other than CXCR2 in such residual neutrophilic responses.

The current study addressed whether partial ablation of CXCR2 reduces the neutrophil burden of infected lungs without affecting lung protective immune responses against inhaled bacteria. We found that even a 10% ablation of CXCR2 expression resulted in impairment of the host defense as manifested by increased bacterial loads and reduced survival in chimeric wild-type mice without significantly affecting neutrophil or exudate macrophage counts in the lungs of these mice. Classically, G-protein-coupled chemokine receptors, including CXCR2 or CCR2, on macrophages and neutrophils are well known to mediate chemotactic recruitment of leukocyte subsets toward the site of inflammation. Against this background, the phagocytic capacity of professional phagocytes such as neutrophils per se is not expected to be altered by lack of the receptor. However, binding of a chemokine to its receptor is known to trigger a series of cellular events in leukocytes and other chemokine receptor-expressing cells like endothelial and epithelial barrier cells, such as intracellular calcium release, induction of a respiratory burst, and burst-dependent activation of antibacterial proteolytic activities, thereby affecting the killing potential of chemokine-activated leukocytes (20, 21). Also, changes in affinity profiles of cell surface adhesion molecules on circulating leukocytes and sessile barrier cells, which are needed for coordinated cellular transmigration, have been described as dependent on chemokine receptor signaling (12). All of these steps are integral components of the overall host inflammatory response to infection. In this view, even partial deletion of selected chemokine receptors in hematopoietic cells, as reported in the current study, will inevitably affect many different facets of the lung host response to bacterial challenge beyond the recruitment process itself.

Only a 25% ablation of hematopoietic CXCR2 expression reduced neutrophil and exudate macrophage recruitment and resulted in a fatal outcome in mice, which was also confirmed in experiments employing selective CXCR2 antagonists. These data, in whole, indicate that partial CXCR2 interference is not beneficial in limiting lung injury due to uncontrolled neutrophilic responses in mice with severe Gram-positive bacterial infections. At the same time, these observations may also be important and relevant in view of currently emerging clinical trials to evaluate CXCR2 antagonists for the treatment of chronic pulmonary disorders (6). Our data are biologically important and relevant to these emerging clinical trials that are evaluating small-molecule inhibitors against CXCR2 for the treatment of noninfectious chronic neutrophilic pulmonary disorders, such as COPD, ARDS, and IPF. It is also possible that this approach could be applied to postviral neutrophilic airway disease and neutrophilic asthma. Based on our study in a mouse model, caution is needed with regard to interruption of CXCR2 signaling in patients with either acute or chronic pulmonary infections like pneumonia and bronchiectasis, and whether this treatment would be safe in patients with latent pulmonary infections, such as tuberculosis and cytomegalovirus (CMV) infection, will require careful monitoring in our view.

Another unexpected finding of the current study was the observation that progressive 50% and 75% CXCR2 ablation, as well as complete CXCR2 deletion, severely affected the alveolar exudate macrophage recruitment into the lungs of S. pneumoniae-infected mice. Of note, the main chemokine receptor/ligand system mediating alveolar exudate macrophage recruitment is binding of the major monocyte chemoattractant CCL2 (JE, MCP-1) to its CC chemokine receptor CCR2 (13, 21, 30). Importantly, we observed here that CCR2 was abundantly expressed on Gr-1-positive inflammatory monocytes acting as the critical precursors of alveolar recruited exudate macrophages (13), and we found greatly increased monocyte chemotactic activities in BAL fluids of CXCR2 KO mice and of 50% and 75% chimeric wild-type mice on day 3 postinfection. Although these inflammatory mediators usually drive inflammatory monocyte recruitment, we observed in mutant mice that there is a lack of a coordinated exudate macrophage mobilization. These data illustrate the critical importance of CXCR2 in regulating monocyte/macrophage recruitment pathways in pneumococcal lung infection.

Several possibilities may explain the heavily increased neutrophil and monocyte chemokine responses observed in chimeric mice and also the mutant mice. First, increased chemokine responses may reflect the severe disease progression in CXCR2 KO mice and chimeric mice, leading to exaggerated chemokine storms. Of note, baseline levels of monocyte and neutrophil chemoattractants in BAL fluids of mutant mice were similar to those in wild-type mice. Second, we previously observed that CCR2 KO mice challenged with E. coli LPS responded with greatly reduced exudate macrophage recruitment despite increased alveolar CCL2 protein levels, thus supporting the view that chemokine receptors on the cell surface of recruited leukocytes may internalize their ligands and thus downregulate local chemokine levels (17). This interpretation may explain increased neutrophil and monocyte chemokine levels in BAL fluids of mutant and chimeric wild-type mice, in which both numbers of BAL fluid neutrophils and exudate macrophages were significantly reduced. However, another issue is how CXCR2 deletion may cause reduced alveolar exudate macrophage recruitment in response to pneumococcal challenge. Another possible explanation was provided by a recent in vitro study which demonstrated that the amount of monocyte chemotaxis to suboptimal concentrations of monocyte chemokines CCL2 and CCL7 was synergistically increased by the binding of CXC chemokines interleukin-8 (CXCL8) and stromal cell-derived factor 1 (SDF-1; CXCL12) to their respective chemokine receptors, CXCR2 and CXCR4, on human monocytes and monocytic THP-1 cells (8).

In the current study, we also observed CXCR2 chemokine receptor expression on the cell surface of alveolar exudate macrophages but not circulating blood monocytes of wild-type mice. Such CXCR2 expression by recruited exudate macrophages in S. pneumoniae-challenged wild-type but not mutant mice provides a molecular basis for inflammatory monocyte chemotaxis toward CXCL1 to -3 chemokines. Along this line, Hartl and colleagues also demonstrated that recruited but not circulating neutrophils from patients with chronic inflammatory lung diseases demonstrated increased chemokine receptor expression profiles in vivo and chemokine responsiveness in vitro (9). In addition, other reports have demonstrated a role for enhanced CXCR2-dependent monocyte migration in the pathogenesis of COPD (28). Thus, our data support the concept that CXCR2 and its respective ligands, together with CC chemokine receptors such as CCR2, synergistically regulate the overall inflammatory recruitment and/or local activation of a given leukocyte subset, such as exudate macrophages, in both humans and mice (8).

In summary, these data demonstrate a critical and nonredundant role of CXCR2 in lung protective immunity to infection with the prototype Gram-positive bacterial pathogen S. pneumoniae. Just 10 to 25% ablation of hematopoietic CXCR2 expression was sufficient to reduce survival in mice. Moreover, successive CXCR2 ablation (stepwise) decreased alveolar neutrophil recruitment but also severely impaired the alveolar exudate macrophage recruitment in response to S. pneumoniae. These data have identified a previously unrecognized role of CXCR2 in the regulation of inflammatory monocyte/macrophage recruitment in acute infections of the lungs induced by Gram-positive bacterial pathogens and may be of significant clinical impact in view of emerging clinical trials to evaluate CXCR2 antagonists for the treatment of chronic neutrophilic pulmonary disorders.

Acknowledgments

This study was supported by the German Research Foundation, grant SFB 587 to U.A.M. and T.W.

Notes

Editor: J. N. Weiser

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 April 2010.

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