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Published ahead of print on September 25, 2002, doi:10.1164/rccm.200207-698OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 171-179, (2003)
© 2003 American Thoracic Society

Original Article

Alveolar Macrophages Have a Protective Antiinflammatory Role during Murine Pneumococcal Pneumonia

Sylvia Knapp, Jaklien C. Leemans, Sandrine Florquin, Judith Branger, Nico A. Maris, Jennie Pater, Nico van Rooijen and Tom van der Poll

Laboratory of Experimental Internal Medicine, Departments of Pathology and Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam; and Department of Cell Biology and Immunology, Free University Amsterdam, Amsterdam, The Netherlands

Correspondence and requests for reprints should be addressed to Sylvia Knapp, M.D., Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, G2-132, 1105 AZ Amsterdam, The Netherlands. E-mail: s.knapp{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar macrophages (AMs) are considered major effector cells in host defense against respiratory tract infections by virtue of their potent phagocytic properties. In addition, AMs may regulate the host inflammatory response to infection by production of cytokines and by their capacity to phagocytose apoptotic polymorphonuclear cells (PMNs). To elucidate the in vivo contribution of AM to host defense against pneumococcal pneumonia, we depleted mice of AMs via pulmonary application of liposomal dichloromethylene-bisphosphonate (AM- mice) before inoculation with Streptococcus pneumoniae; control mice received saline (AM+sal) or liposomal phosphate-buffered saline (AM+lip) before bacterial inoculation. AM- mice displayed a significantly higher mortality compared with AM+ control mice, whereas bacterial clearance did not differ. Poor outcome of AM- mice was accompanied by a pronounced increase of local proinflammatory cytokine production as well as strongly elevated and prolonged pulmonary PMN accumulation. Closer examination of infiltrating PMN in AM- mice disclosed high proportions of apoptotic and secondary necrotic cells, reflecting the lack of efficient clearance mechanisms in the absence of AMs. Furthermore, caspase-3 staining showed only slightly higher activity in AM- mice, arguing against accelerated apoptosis per se. These data suggest that AMs are indispensable in the host response to pneumococcal pneumonia by means of their capacity to modulate inflammation, possibly via elimination of apoptotic PMNs.

Key Words: bacterial • lung • macrophage • inflammation • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pneumoniae is a leading causative pathogen in community-acquired pneumonia (1–3). Despite adequate antimicrobial therapy, pneumococcal pneumonia remains a major cause of morbidity and mortality. Furthermore, with an increasing incidence of antibiotic resistance in this pathogen, there is an urgent need to expand our knowledge of the pathogenic and host defense mechanisms that influence outcome in S. pneumoniae pneumonia (2, 4).

Alveolar macrophages (AMs), located at the interphase between air and lung tissue, provide the first line of cellular defense against microbes (5, 6). Most in vivo data concerning the role of AMs in pulmonary host defense come from studies in which AMs were depleted by administration of liposome-encapsulated dichloromethylene bisphosphonate (Cl2MBP) to the pulmonary tract. In this way, the role of AMs as phagocytes has been demonstrated in mice challenged with Pseudomonas aeruginosa or Klebsiella pneumoniae. Mice lacking AMs showed a delayed and impaired bacterial clearance as compared with control mice (7, 8), although conflicting data exist about the phagocytic properties of AMs in a mouse model with unopsonized P. aeruginosa (9). In sharp contrast to uncapsulated bacteria, S. pneumoniae are known to bind poorly to macrophages without prior opsonization (10–12). Moreover, mouse AMs show minimal expression of receptors for C3b/iC3b (CR1/CR3/CR4) (13), indicating that AMs use other nonopsonic phagocytosis mechanisms. Alternatively, the contribution of AMs to pulmonary host defense in S. pneumoniae pneumonia may rely on different, as yet unidentified mechanisms.

Until recently, most investigations dealing with pneumonia concentrated on the onset of inflammation. Within the past few years, more effort has been made to study mechanisms responsible for the resolution of inflammation and restitution of tissue homeostasis. Through this, it has become evident that persistent inflammation, leading to tissue injury and organ malfunction, may not only be due to prolonged proinflammatory events but may equally likely arise from inefficient resolution processes (14). AMs have been implicated as major effector cells in this resolution process, mainly by phagocytosing apoptotic polymorphonuclear cells (PMNs) (15). The rapid elimination of extravasated, apoptotic PMNs by AMs may provide an injury-limiting mechanism as the membrane of PMNs remains intact, preventing potential injurious granule contents from being released. AMs can therefore be regarded as major modulators of pulmonary host defense. They readily phagocytose and eliminate certain inhaled pathogens and act as effector cells in the equally important resolution process (14, 15).

To our knowledge, the proposed dual function of AMs, facilitating both the clearance of bacteria and the resolution of the ensuing inflammatory response in the pulmonary compartment, has never been investigated directly in a model of bacterial pneumonia. We therefore sought to obtain insight into the in vivo contribution of AMs to the host response to pneumococcal pneumonia in a well-established animal model. For this purpose, mice were depleted of AMs by administration of liposomal Cl2MBP before intranasal infection with S. pneumoniae. Intranasal Cl2MBP administration selectively depletes AMs without damaging other cells (16). We thereby investigated the traditional role of AMs as phagocytes as well as their contribution to resolution processes.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Pathogen-free 6- to 8-week-old female BALB/c mice were obtained from Harlan Sprague-Dawley (Horst, The Netherlands). The Animal Care and Use Committee of the University of Amsterdam (The Netherlands) approved all experiments.

In Vivo AM Depletion
Cl2MBP was a gift from Roche Diagnostics (Mannheim, Germany). Preparation of liposomes containing Cl2MBP was performed as described previously (16); 100 µl of Cl2MBP was administered intranasally (AM- mice) 48 hours before bacterial inoculation according to methods described by our laboratory previously (17). Control mice received liposomal phosphate-buffered saline or saline, respectively.

Induction of Pneumonia
Pneumonia was induced as described previously (18–20). S. pneumoniae serotype 3 was obtained from American Type Culture Collection (ATCC 6303; Rockville, MD), and 50 µl (approximately 5 x 104 CFU) was inoculated intranasally.

Preparation of Lung Homogenates
At 20 and 44 hours after infection, mice were anesthetized and killed; whole lungs were homogenized for CFU determination and for cytokine measurements exactly as described previously (17–20). Cytokines and chemokines were measured using specific enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The detection limits were 31 pg/ml for tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin (IL)-10, 12 pg/ml for the mouse chemokine KC, and 8 pg/ml for IL-1ß.

Fluorescence-activated Cell Sorter Analysis of Bronchoalveolar Lavage Fluid and Lung Suspension
Bronchoalveolar lavage and leukocyte differentiation were done as described previously (17, 18, 21). Pulmonary cell suspensions were obtained using an automated disaggregation device (Medimachine-System; Dako, Glostrup, Denmark) exactly as described previously (17, 20). For fluorescence-activated cell sorter (FACS) analysis of apoptotic PMNs, bronchoalveolar lavage fluid (BALF) and lung cells were gated for PMNs by forward and side scatter and were stained with Annexin-V PE and 7-amino-actinomycin D. In these experiments, BALF and lung suspension cells were obtained from the same mice to address simultaneously both alveolar and interstitial lung compartments. Annexin V–positive and 7-amino-actinomycin D–negative cells were considered apoptotic, and double-positive PMNs were considered necrotic (22). To correct for aspecific staining, an appropriate isotype-control antibody was used. Staining was performed in the presence of 20% normal mouse serum to block nonspecific binding to Fc{gamma}-receptor. All reagents were purchased from Pharmingen (San Diego, CA) and were used in concentrations recommended by the manufacturer. Samples were analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA).

Histology
Lungs for histology were fixed in 4% formaline and were embedded in paraffin, and 4-µm sections were stained with hematoxylin and eosin. Granulocyte staining was done as described previously (19, 21). To detect apoptotic bodies, deparaffinized slides were boiled in citrate buffer (pH 6.0). After blocking of nonspecific binding and endogenous peroxidase activity, slides were incubated with rabbit anti-human active caspase-3 polyclonal antibody (Cell Signaling, Beverly, MA) followed by biotinylated swine anti-rabbit antibody (Dako) and further revealed as described for granulocyte staining. Mucosa-associated lymphatic tissue of mice treated with Staphylococcal enterotoxin B for 8 hours (known to induce apoptosis strongly) served as positive control in these stainings. All antibodies were used in concentrations that were recommended by the manufacturers.

Myeloperoxidase Assay
Myeloperoxidase activity was determined by measuring the H2O2-dependent oxidation of 3,3'5,5' tetramethylbensidine as described previously (23). Myeloperoxidase activity is expressed as activity/gram lung tissue/reaction time. All reagents were purchased from Sigma (St. Louis, MO).

Statistical Analysis
Differences between groups were calculated by Mann-Whitney U test. For survival analyses, Kaplan-Meier analysis followed by a log rank test was performed. Values are expressed as mean ± SEM. A p value of 0.05 or less was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Depletion of AMs by Cl2MBP
Others and we have previously demonstrated that the intrapulmonary delivery of liposomal Cl2MBP results in a selective depletion of AMs in BALF (7, 8, 16, 17, 24, 25). To confirm this finding for this series of experiments, we administered saline (AM+sal), liposomal phosphate-buffered saline (AM+lip), or liposomal Cl2MBP (AM-) intranasally (n = 6 per group) and determined the number of AMs in BALF 48 hours later (i.e., at the time point mice were designated to receive S. pneumoniae). Cl2MBP led to a depletion of 74 ± 1.4% of AM relative to AM+sal mice (p < 0.05). The number of AM in AM+lip mice did not differ from that in AM+sal mice (data not shown).

AM- Mice Are More Susceptible to S. pneumoniae Pneumonia
To determine the role of AMs in host defense against pneumonia in vivo, we first assessed the survival of mice depleted of AMs (AM-) and control mice (AM+sal or AM+lip) after intranasal inoculation with 5 x 104 S. pneumoniae CFU. All AM- mice died within 5 days after the bacterial challenge, whereas 33% (4 of 12) of AM+lip (p = 0.04 versus AM-) and 50% (6 of 12) of AM+sal mice (p = 0.001 versus AM-) survived (Figure 1) . All mice surviving 5 days appeared to no longer be clinically ill.



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  		Figure 1. Increased mortality of AM- mice after S. pneumoniae infection. BALB/c mice (n = 12 per group) were intranasally administered Cl2MBP (AM-), liposomes (AM+lip), or saline (AM+sal) 48 hours before bacterial challenge with 5 x 104 CFUs S. pneumoniae. +p <= 0.05 AM- mice versus AM+lip; *p <= 0.05 AM- versus AM+sal.

 
The Increased Mortality of AM- Mice Is Not Related to an Impaired Bacterial Clearance
Because AMs are considered an important first-line phagocytic defense against inhaled pathogens, we next sought to investigate whether the increased lethality in AM- mice was related to an impaired clearance of S. pneumoniae. At both 20 and 44 hours after inoculation with S. pneumoniae, the number of CFUs in lungs was similar in all three groups (Figure 2A) . Of interest, the lowest bacterial outgrowth at 44 hours was found in AM- mice, although the difference with the two control groups did not reach statistical significance. In addition, blood from all three groups contained the same amount of bacteria at 20 and 44 hours after inoculation (Figure 2B). Hence, bacterial clearance was not impaired in the absence of AMs and therefore cannot explain differences in survival.



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  		Figure 2. AM depletion does not influence the outgrowth of pneumococci. Similar bacterial outgrowth in lungs (A) and blood (B) of AM- and AM+ mice 20 and 44 hours after inoculation with S. pneumoniae. Data are mean ± SEM of eight mice per group at each time point.

 
AM- Mice Display an Increased Inflammatory Response within the Pulmonary Compartment
Neutrophilic influx to the lungs as well as the production of proinflammatory cytokines and CXC chemokines is regarded as a major host defense mechanism in bacterial pneumonia (26, 27). AMs are thought to play a key role in the initial recruitment of PMN through their capacity to secrete many of these proinflammatory mediators. We therefore investigated these early host responses to obtain insight into the mechanism by which AM- mice are more susceptible to pneumococcal pneumonia. The highest level of pulmonary cell recruitment was found in AM- mice (Table 1) . Differential counts of whole-lung cell suspensions displayed an impressive predominance of PMNs. This observation was confirmed by elevated myeloperoxidase activity in lungs of AM- mice (p = 0.0002 versus AM+sal and p = 0.04 versus AM+lip) and a profoundly elevated PMN number in BALF of AM- mice (p = 0.002 versus AM+sal and p = 0.002 versus AM+lip) (Figure 3) .


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  		TABLE 1. Cellular composition of lungs

 


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  		Figure 3. Increased pulmonary PMN influx in AM- mice. (A) Increased myeloperoxidase activity in lungs of AM- mice 44 hours after bacterial challenge. (B) PMN counts obtained in BALF 44 hours after inoculation with S. pneumoniae. Data are from eight mice per group (mean ± SEM); +p <= 0.05 AM- mice versus AM+lip; *p <= 0.05 AM- versus AM+sal (A and B). MPO = myeloperoxidase.

 
Forty-four hours after S. pneumoniae inoculation, both groups of AM+ control mice had a predominantly interstitial pulmonary infiltrate (Figures 4A and 4C) , composed of monocytes, lymphocytes, and only few granulocytes (Figures 4B and 4D) compatible with the clearance phase of the infection. In sharp contrast, the lungs of AM- mice displayed dense and diffuse inflammatory infiltrates with focal destruction of the lung parenchyma (Figure 4E). These inflammatory infiltrates were mostly composed of PMNs, as demonstrated by immunostaining with the mouse granulocyte marker Ly-6G (Figure 4F). This increased PMN influx in AM- mice was accompanied by an early and marked increase in pulmonary TNF-{alpha}, IL-1ß, and KC levels, whereas in both AM+ control groups, a less impressive and less sustained elevation in these cytokines was found (Figure 5) . Interestingly, lung concentrations of the antiinflammatory cytokine IL-10 were significantly lower in AM- mice than in AM+ mice. Theoretically, the administration of Cl2MBP could have been responsible for the increased inflammatory response in AM- mice, rather than the infection with S. pneumoniae. Therefore, we treated mice (n = 6 per group) with liposomal Cl2MBP, liposomal phosphate-buffered saline, or saline at 48 hours before intranasal administration of sterile saline (instead of S. pneumoniae). At 44 hours after saline administration, lung myeloperoxidase and TNF-{alpha} levels and histopathology were similar in the three groups and were not different from normal control mice (data not shown). Together, these data suggest that S. pneumoniae induces an exaggerated inflammatory response in AM- mice, which likely accounts for the increased mortality in these animals.



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  		Figure 4. Histopathology at 44 hours after infection. Both lungs of AM+ mice showed a histologic picture compatible with the clearance phase of the pneumonia characterized by a slight interstitial influx, mostly composed of monocytes (A, AM+sal; C, AM+lip, hematoxylin and eosin staining, x50) with sparse granulocytes as shown in the higher magnified inserts (A and C, x400) and by immunostaining with Ly-6G (B, AM+sal; D, AM+lip, anti-Ly-6G immunostaining, x50). In contrast, lungs of AM- mice displayed diffuse and dense inflammatory infiltrates with effacement of the lung parenchyma (E, AM-, hematoxylin and eosin staining, x50). As shown in the insert (E, x400) and on F, granulocytes were the predominant cell type in this process (anti-Ly-6G immunostaining, x50).

 


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  		Figure 5. Proinflammatory cytokines predominate in AM- mice. Elevated lung concentrations of the proinflammatory mediators TNF-{alpha}, IL-1ß, and KC, and reduced pulmonary IL-10 levels in AM- mice at 20 and 44 hours after infection. Data represent mean ± SEM of eight mice per time point and group; +p <= 0.05 AM- mice versus AM+lip; *p <= 0.05 AM- compared with AM+sal.

 
AM Deficiency Impedes the Clearance of Apoptotic Neutrophils
The deleterious effects of AM depletion in mice with pneumonia seemed to result from prolonged and exaggerated inflammation rather than from impaired bacterial clearance. Apart from participating in the first line of defense, AMs have been implicated in the resolution of inflammation because of their ability to phagocytose and degrade PMNs that are undergoing apoptosis (14, 15). Indeed, in BALF cytospins of AM+ mice with moderate persistent pulmonary inflammation 44 hours after inoculation, we not only found apoptotic PMNs but also AMs with phagocytosed apoptotic bodies (Figure 6) . These data indicate that the depletion of AM may impair physiologic repair mechanisms, including elimination of apoptotic PMNs, thereby promoting prolonged inflammation. If this is the case, an increase of apoptotic and/or secondary necrotic PMNs should be detectable in lungs of these mice. We therefore investigated the proportion of apoptotic and necrotic PMNs in BALF and whole-lung suspensions. Among the elevated number of PMNs, we indeed found higher proportions of apoptotic (Annexin V positive, 7-amino-actinomycin D negative) PMNs in BALF and lung suspensions of AM- mice (Figure 7) . Moreover, the percentage of dead (Annexin V and 7-amino-actinomycin D double positive) PMNs in BALF was significantly higher in AM- mice as compared with AM+lip and AM+sal control mice. This was confirmed by histologic analysis of AM- lungs, showing at higher magnification large numbers of shrunken cells, cellular debris, and pyknotic nuclei characteristic of apoptotic bodies (Figure 8A) .



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  		Figure 6. AM phagocytose apoptotic PMNs. Cytospin preparation (Giemsa staining) of a BALF specimen of AM+ mouse showing PMN undergoing apoptosis (arrowhead) and phagocytosed apoptotic bodies within the cytoplasm of AM (arrows).

 


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  		Figure 7. Accumulation of apoptotic and dead PMNs in AM- mice. (A) BALF and lung suspension obtained 44 hours after bacterial inoculation were analyzed for apoptotic and dead PMNs by FACS as described in METHODS. Annexin V/7-amino-actinomycin D double positive PMNs were considered "dead" and single Annexin V positive PMN "apoptotic." (B) Representative FACS plots of lung suspension samples are shown. Quadrants were set according to respective isotype controls. Data are mean ± SEM of eight mice per group. +p <= 0.05 AM- mice versus AM+lip; *p <= 0.05 AM- compared with AM+sal.

 


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  		Figure 8. Many apoptotic bodies but low caspase-3 activity. Close up on the inflammation observed in AM- lungs 44 hours after infection showing large amounts of pyknotic nuclei, cellular debris, and shrunken cells, compatible with apoptotic bodies (A, hematoxylin and eosin staining, x160). Immunostaining for active caspase-3 showed a discrepancy between the amount of apoptotic bodies and the active caspase-3 activity in the lungs of AM- mice (B, x60). Moreover, given the 10-fold higher number of PMN in lungs of AM- mice, active caspase-3 activity seemed to be only marginally higher than in both control AM+ groups (C, AM+sal; D, AM+lip, x60). Arrows indicate caspase-3–positive cells.

 
Caspase-3 gets activated during the early stages of apoptosis and is involved in the subsequent disassembly of the cell. Active caspase-3 is therefore regarded as an early marker of cells undergoing apoptosis (28). In sharp contrast to the tremendous amount of characteristic apoptotic bodies seen by conventional hematoxylin and eosin staining in AM- mice, antiactive caspase-3 immunostaining of lung specimens showed only few positive cells (Figure 8B). Moreover, these mice displayed only slightly elevated active caspase-3 activity compared with both AM+ control mice (Figures 8C and 8D). Therefore, the increase in apoptotic cells in AM- mice is not due to an increase in apoptosis, further supporting the theory that it is due to an imbalance between apoptosis and rapid elimination.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AMs are considered major effector cells of innate immunity, capable of participating in both the initiation and resolution process of pulmonary inflammation (14). Previous publications have reported the importance of AM in the rapid elimination of Gram-negative pathogens such as P. aeruginosa and K. pneumoniae from the respiratory tract (7, 8), whereas others have emphasized the role of AMs in early PMN recruitment to the alveolar space (24). Together these reports concentrated on the role of AMs in initiating and orchestrating the immediate pulmonary host defense against invading pathogens. Thus far, however, evidence for the equally important regulatory role of AMs in the resolution process comes from in vitro experiments or in vivo studies with intact AMs using oleic acid, ozone, or lipopolysaccharides (15, 29, 30). Therefore, to examine the contribution of AM to resolution of pneumonia, we used a direct approach and depleted mice of AMs before infection with S. pneumoniae serotype 3.

In this study, we demonstrate the importance of AM as major effector cells in the resolution process of pneumococcal pneumonia in vivo. The lack of AM led to overwhelming inflammation and an insufficient clearance of apoptotic PMNs, which was associated with an increased lethality. The classic role of AM as phagocytes of invading S. pneumoniae seems to be of less importance, as illustrated by unaltered bacterial outgrowth in AM- mice.

AM- mice exhibited a pronounced and prolonged influx of PMNs within the alveoli and interstitial space. This finding is consistent with previous reports on Gram-negative pneumonia. However, in contrast to our observations of an unaltered bacterial clearance, these earlier investigations found increased bacterial loads in AM- animals, and the authors apparently contributed the prolonged influx of PMNs to the more extensive proinflammatory stimulus provided by the increased bacterial burden (7, 8). We further examined these pulmonary infiltrates and revealed a high proportion of apoptotic or dead PMNs. Apoptosis, the process of programmed cell death, is believed to play a major regulatory role in the inflammatory response as invading PMNs undergo apoptosis and are readily phagocytosed by surrounding AMs. This process prevents the release of potentially toxic or immunogenic intracellular contents and thereby allows an injury-limiting elimination of potentially harmful PMNs (14). Thus, the higher number of apoptotic and secondary necrotic PMNs that we found in AM- mice is very likely the result of inefficiency of the normal resolution process in the absence of AM, thereby tipping the balance toward persistent inflammation and tissue injury.

It is interesting to note that apoptosis of PMN is known to be delayed in both systemic and local inflammation, including pneumonia and acute respiratory distress disorder (31–33). This increased PMN longevity has been attributed to factors like granulocyte-macrophage colony-stimulating factor, granulocyte-colony-stimulating factor, interferon-{gamma}, and to some degree, TNF-{alpha} (33–36). In accordance with this, despite the high number of apoptotic/necrotic PMNs in lungs of AM- mice, we could not find signs of accelerated PMN apoptosis itself. In apoptosis, caspase-3 is known as a key effector protease that once activated by initiator proteases like caspase-8 or caspase-9 irreversibly leads to cell disassembly (28, 37). Immunostaining for active caspase-3 revealed only slightly increased numbers of positive cells in AM- lungs as compared with AM+ mice. This slight elevation of caspase-3 seems almost negligible compared with the enormous number of infiltrating PMNs in these mice (Figure 4).

In addition to the unquestionable importance of rapid elimination of aged PMNs, it has been shown that the uptake of apoptotic PMN induces an antiinflammatory phenotype in macrophages, as it actively inhibits the production of IL-1ß, IL-8, and TNF-{alpha} by human monocyte-derived macrophages (38–40). Local cytokine production in mice with intact AMs and, therefore, effective elimination of senescent PMNs were relatively diminished in parallel with a partial resolution of the inflammatory response. The substantial and prolonged elevation of proinflammatory cytokines such as TNF-{alpha} and IL-1ß in AM- mice is likely to result from ongoing inflammation fuelled by intracellular contents released from necrotic PMN. The potential cellular source of these mediators after depletion of AM can only be speculated on, but bronchial epithelial cells, alveolar epithelial cells type II, as well as interstitial macrophages are well known for their secretory capacity upon stimulation with proinflammatory cytokines, LPS, or reactive oxygen intermediates (41–44). One might argue that the prolonged and exaggerated inflammation in AM- mice results from the lack of antiinflammatory cytokines such as IL-10 after AM depletion. We certainly cannot exclude this possibility, but the low IL-10 levels found in AM- mice cannot be attributed to the lack of AM itself, as murine AMs (including BALB/c) do not produce IL-10 (45). Potential sources of IL-10 in the lungs are interstitial macrophages and T cells as well as infiltrating alveolar monocytes. We hypothesize that the impaired clearance of apoptotic cells in AM- mice and the concomitant prolonged inflammation precludes the "switch" of monocytic cells to an antiinflammatory cytokine production profile, as is known to occur in the resolution phase of inflammation (14, 38). This in turn could explain lower IL-10 levels in AM- mice.

The exposure of AM to liposome encapsulated Cl2MBP leads to selective apoptosis of AM. Both in vivo and in vitro experiments have demonstrated that PMNs are morphologically and functionally unaffected by this compound (16). Thus, an effect of Cl2MBP on PMNs cannot explain our findings. In addition, the intra-alveolar inflammatory reaction in response to Cl2MBP itself is minimal, as documented here and in earlier reports from other laboratories (7, 17, 24, 25, 46). Liposomes were used to encapsulate Cl2MBP to facilitate uptake by macrophages. As controls, we used mice pretreated with saline (AM+sal) or liposomes (AM+lip) only. Interestingly, both outcome and inflammatory responses in AM+lip mice ranged between those found in AM+sal and AM- mice, indicating that liposomes themselves somewhat impair or influence the functional properties of AM. Indeed, liposomes can reduce the phagocytic and migratory behavior of AMs (47). Similar observations have been made in earlier studies (16, 17).

Whole-lung suspensions revealed an increased proportion of monocytic cells in all three groups 48 hours after bacterial inoculation (Table 1). This observation can be explained by an influx of alveolar and interstitial monocytes, known to reach peak levels 48 hours after induction of inflammation (48). However, in sharp contrast to AM+ control mice with intact AM, the number of PMNs consistently outnumbered monocytic cells in AM- mice. This persistent imbalance of the PMN–monocyte/macrophage ratio in AM- mice most likely accounts for the observed impaired PMN clearance.

Pneumonia remains a leading cause of morbidity and mortality, and S. pneumoniae is the most frequently isolated pathogen in community-acquired pneumonia. Using a well-established model of murine pneumococcal pneumonia, we demonstrate that the selective depletion of AM results in an exaggerated inflammatory response and enhanced lethality together with an accumulation of apoptotic and necrotic PMNs. The clearance of pneumococci from the lungs was not influenced by AM depletion. These data indicate that AMs play an essential role in the regulation of the lung inflammatory response during pneumococcal pneumonia.


   Acknowledgments
 
The authors thank Joost Daalhuisen and Ingvild Kop for expert technical assistance and Nike Claessen for the immunohistochemical stainings.


   FOOTNOTES
 
Supported by grants from the Austrian Fonds zur Foerderung der wissenschaftlichen Forschung in Oesterreich (S.K.), the Netherlands Organization of Scientific Research (J.C.L. and S.F.), and the Netherlands Asthma Foundation (N.A.M.)

Received in original form July 15, 2002; accepted in final form September 18, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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