Role of Alveolar Macrophages in Initiation and Regulation of Inflammation in Pseudomonas aeruginosa Pneumonia

Abstract

To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 × 10⁵ CFU of P. aeruginosa. In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 × 10⁷ CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.

Alveolar macrophages (AMs), the resident mononuclear phagocytes of lung alveoli, are the first line of cell-mediated defenses against organisms inhaled into the lower respiratory tract (1). Besides their scavenger functions, such as phagocytosis and subsequent digestion of pathogens, AMs can release various chemical mediators upon contact with pathogens or pathogenic substances (13, 17). The mediators released from AMs promote the generation of subsequent inflammatory responses (15). Among those inflammatory responses, early recruitment of polymorphonuclear leukocytes (PMNs) to the site of infection is one of the critical responses for rapid clearance of organisms (5, 11). To examine the role of AMs in initial PMN recruitment after gram-negative bacterial infection, we previously depleted AMs in rats by aerosol inhalation of negatively charged large oligolamellar liposomes encapsulating clodronate disodium (Cl₂MDP-liposomes) (8). This method cleared over 95% of AMs from the lungs without causing lung damage or airspace neutrophilia. Depletion of AMs attenuated initial PMN recruitment and chemokine (macrophage inflammatory protein 2 [MIP-2] and CINC/GRO) production in Pseudomonas aeruginosa pneumonia. These results suggest that AM-derived mediators, such as MIP-2 and CINC/GRO, contribute to neutrophil recruitment.

Although an inflammatory response is essential to clear pathogens from the site of infection, a prolonged inflammatory response might worsen lung injury and might actually interfere with pathogen elimination (12, 16). Along with the fact that macrophages release various mediators (tumor necrosis factor alpha and nitric oxide, etc.) which activate PMNs, the role of AMs as regulators of inflammatory responses was recently reported; AMs engulf apoptonic PMNs and release anti-inflammatory mediators (6, 14). These findings imply that the role of AMs in the inflammatory response is not limited to generation of the initial inflammation but extends to regulation of the inflammatory responses, including effective elimination of pathogens phagocytosed by PMNs at a later phase (7).

In this study, we depleted >95% of AMs by aerosol inhalation of Cl₂MDP-liposomes in mice to evaluate the role of AMs in the initiation and regulation of inflammation induced by Pseudomonas infection. This was done by monitoring PMN recruitment, releases of chemoattractants, such as MIP-2 and KC, lung injury, and survival at an early time (8 h) and a later time (48 h).

MATERIALS AND METHODS

Bacterial strain and culture.

P. aeruginosa PAO1 was generously provided by K. Okuda (Yokohama City University, Kanagawa, Japan). Bacteria from frozen stock were streaked on tryptic soy agar (Difco Laboratories, Detroit, Mich.) for 24 h at 37°C and then grown in tryptic soy broth (TSB; Difco) for 12 h at 32°C in a shaking incubator. The bacteria in the broth culture were sedimented by centrifugation at 4,000 × g for 20 min and resuspended in Ringer’s lactate solution. The concentration of bacteria was measured by spectrophotometry. The number of bacteria was also determined by diluting all samples and plating them for quantitative culture. Bacteria were serially suspended to a density of 10⁷ or 10⁹ CFU/ml in phosphate-buffered saline (PBS) without calcium [PBS(−)].

Animal model of P. aeruginosa pneumonia.

Male specific-pathogen-free CD-1 mice (Japan S.L.C. Co., Ltd., Shizuoka, Japan) weighing 35 g to 37 g were lightly anesthetized with inhaled sevoflurane. Fifty microliters of bacterial solution was then slowly instilled into the left lung through a gavage needle (modified animal feeding needle, 24 gauge; Popper & Sons, Inc., New Hyde Park, N.Y.) inserted into the trachea via the mouth. A total of 5 × 10⁷ CFU (large-inoculum model) of bacteria were instilled for the survival study, and 5 × 10⁵ CFU (small-inoculum model) were instilled for other experiments. The mice recovered from anesthesia within 2 min. All animal procedures were approved by the Animal Care Committee of the Kyoto Prefectural University of Medicine.

Preparation of Cl₂MDP-liposomes.

Cl₂MDP-liposomes were prepared as described previously (8). Briefly, 80 mg of phosphatidylcholine and 30 mg of cholesterol were dissolved in 4 ml of chloroform in a 200-ml round-bottom flask. After low-vacuum rotary evaporation of the chloroform, a thin film formed on the inner surface of the flask. Then, 3 ml of distilled water containing 0.4 g of disodium clodronate (dichloromethylene-biphosphonate; Boehringer Mannheim GmbH, Mannheim, Germany) and 2 ml of ether were added to the flask. The fluid was sonicated for 2 min and then reduced by low-vacuum evaporation until the ether was slowly removed at 30°C. The liposome suspension was then extruded sequentially through both 0.8- and 0.2-μm-pore-size cellulose acetate syringe-type filters (Corning Glass Works, Corning, N.Y.). The unencapsulated drug was separated from drug-containing liposomes by high-speed centrifugation (10,000 × g for 60 min), and the pellet was resuspended in 10 ml of PBS(−).

Depletion of AMs by aerosol inhalation of Cl₂MDP-liposomes.

Cl₂MDP-liposomes were aerosolized to deplete AMs 72 h before bacterial instillation. An aerosol device was employed to deliver the liposomes. Mice were placed in a nose-only aerosol chamber (Intox Products, Albuquerque, N. Mex.). An Aerotech II (Bedford, Mass.) nebulizer (CIS-II) supplied with compressed air at a flow rate of 12 liters/min was used to generate the liposome aerosol. To assess the effectiveness of Cl₂MDP-liposomes in depleting AMs, the mice were subjected to bronchoalveolar lavage (BAL) 24 h, 48 h, 72 h, 96 h, 5 days, 14 days, and 28 days after liposome aerosol inhalation. Control mice were exposed to saline aerosol in the same manner.

BAL.

At predetermined intervals after the instillation of Pseudomonas into the air space, the mice were anesthetized with pentobarbital (200 mg/kg of body weight) and then exsanguinated by aortic transection. The trachea was exposed by a midline incision and cannulated with a sterile polypropylene 18-gauge catheter. The lungs were lavaged with 5 ml of PBS supplemented with 0.6 mM EDTA in 0.2-ml increments. BAL fluid was centrifuged at 1,000 × g for 8 min at 4°C. The total returns after lavage averaged 4 to 4.5 ml/mouse. Occasionally, lesions in lung tissue caused intrathoracic leakage of the lavage fluid. This resulted in smaller recovery volumes, and quantitative data from these animals were discarded. The cell pellet obtained from centrifugation of BAL fluid was diluted in 1.0 ml of PBS(−). The total cell counts were performed with a hemacytometer after staining the cells with gentian violet. Cell viability was determined by trypan blue exclusion. Differential cell counts were done on cytocentrifuge preparations (Cytospin 3; Shandon Southern Instruments, Sewickley, Pa.) stained with modified Giemsa stain (Diff-Quick; Baxter, McGaw Park, Ill.).

Measurement of the permeability of alveolar epithelium by albumin.

A radioactive tracer was used to measure the flux of albumin across the alveolar epithelial barrier, as described previously (10). An instillation that included an alveolar protein tracer (0.05 μCi of ¹²⁵I-labelled albumin) was prepared. Four or 44 h after the instillation of P. aeruginosa, 50 μl of the alveolar protein tracer was instilled into the left lung. Four hours after the instillation of the tracer, the mice were anesthetized with 2.0 mg of pentobarbital intraperitoneally and blood was collected by carotid arterial puncture. After the mice received additional pentobarbital, sternotomy was done. The lungs, trachea, and oropharynx were harvested, and the radioactivity of these samples was measured. The percentage of radioactive albumin that left the lung and entered the circulation was calculated by multiplying the counts measured in the terminal blood sample (per milliliter) times the blood volume [body weight × 0.07 (1 − haematocrit)].

Measurement of excess lung water.

The wet-weight-to-dry-weight (W/D) ratio of the whole lungs was estimated. The lungs were dissected, weighed, and then dried at 60°C for 5 days. The W/D ratio was then calculated by dividing the wet weight by the final dry weight.

Bacterial culture.

The lungs were removed and homogenized in 3 ml of sterile water. Lung homogenates were sequentially diluted and plated on sheep blood agar plates for quantitative culture. The number of bacteria in each lung was calculated by multiplying the weight of the lung by the number of CFU. The detection limit was 100 CFU/lung.

MPO assay.

The enzyme myeloperoxidase (MPO) was used as a tracer to quantitate PMN sequestration in tissues, because the MPO assay has been found to be more sensitive than quantitative histology for detecting sequestered neutrophils. A sample of blotted lung tissue (0.5 g) was homogenized in 5 ml of 0.01 M potassium phosphate buffer (PPB; pH 7.4) containing 1 mM EDTA. Two milliliters of the homogenate and 5 ml of 0.01 M PPB containing 1 mM EDTA were mixed gently and then centrifuged at 10,000 × g for 20 min at 4°C. The pellet was rehomogenized in 5 ml of 0.05 M PPB (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. This suspension was frozen, thawed, and sonicated with a Branson cell disrupter (Heat Systems; Ultronics, Plainview, N.Y.) at 65 W for 1 min. Then, 0.1-ml aliquots were mixed with 0.79 ml of 0.08 M PPB (pH 5.4) and 0.1 ml of 1 mM tetramethylbenzidine dissolved in N,N-dimethylformamide at 37°C. After 2 min, 0.01 ml of 30 mM H₂O₂ was added. This solution was incubated for 3 min at 37°C, and then a 300-μg/ml concentration of a 0.05-ml catalase solution was added. This mixture was then diluted with 4 ml of 0.2 M sodium acetate (pH 3.0) and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was read in a spectrophotometer (U-2000; Hitachi, Tokyo, Japan). One unit of enzyme activity was defined as the amount of MPO that produced an absorbance change of 1.0 optical density unit per minute per milligram of tissue at 37°C.

Determination of MIP-2 and KC by ELISA.

MIP-2 and KC are known as major chemoattractants for neutrophil or C-X-C chemokines in rodents (3). Thus, MIP-2 and KC concentrations in BAL fluid were measured at 8 and 48 h after bacterial inoculation by using enzyme-linked immunosorbent assay kits (IBL, Gunnma, Japan). The detection limits for MIP-2 and KC were 2 and 25 pg/ml, respectively.

Histopathological observation.

Six mice (of 24 examined) were presented for their representative light microscopic findings. Three mice were exposed to aerosolized Cl₂MDP-liposomes (AM-depleted group), and the other mice were exposed to PBS(−) liposomes (control group). One mouse from each group was processed 72 h after the aerosolization. Two mice of each group received 5 × 10⁵ CFU of bacteria in the trachea 72 h after the aerosolization and were processed 8 or 48 h after the bacterial instillation. All mice were exsanguinated by transection of the abdominal aorta under anesthesia. The lungs were then removed en bloc and processed for hematoxylin-eosin staining.

Statistical analysis.

All data are reported as the mean ± standard deviation (SD). Multiple comparisons were performed by using one-way analysis of variance, followed by Scheffe’s F test. Survival data were compared by Fisher’s exact test. All other data were compared by Student’s unpaired t test. Significance was assigned at a P of <0.05.

RESULTS

More than 95% of AMs were depleted by aerosolization of Cl₂MDP-liposomes.

The effect of Cl₂MDP-liposomes was evaluated by counting the remaining macrophages in the BAL fluid (Fig. 1; see Fig. 4b). After aerosol inhalation of Cl₂MDP-liposomes, the number of AMs recovered from BAL fluid was reduced to less than 5% of normal within 3 days. After day 5, the number of AMs recovered gradually and finally reached the normal level on day 28.

FIG. 1.

Changes of neutrophil and macrophage numbers in BAL fluid from mice given an aerosol of Cl₂MDP-liposomes. Mouse lungs were lavaged 1, 2, 3, 4, 5, 8, 14, and 28 days after aerosol administration (n ≥ 5 per group). At 3 days, 95% ± 2% of AMs were depleted. There was a significant reduction in the number of macrophages up to 14 days compared to that of saline-treated controls. The Cl₂MDP-liposome aerosolization hardly induced neutrophil infiltration into alveolar spaces. Data are expressed as means + SD. ∗, significant at P of <0.05 (Student’s t test) compared to value before aerosol delivery.

FIG. 4.

Histologic assessment (hematoxylin-eosin staining) of mouse lung tissue. Mice were exposed to PBS(−)-containing liposomes (control group) (a, c, and e) or Cl₂MDP-liposomes (AM-depleted group) (b, d, and f). (a) Tissue from control mouse without P. aeruginosa instillation. Arrows indicate AMs. (b) Tissue from AM-depleted mouse without P. aeruginosa instillation. (c) Tissue from control mouse 8 h after P. aeruginosa instillation. (d) Tissue from AM-depleted mouse 8 h after P. aeruginosa instillation. (e) Tissue from control mouse 48 h after P. aeruginosa instillation. (f) Tissue from AM-depleted mouse 48 h after P. aeruginosa instillation. Original magnification, ×125.

Depletion of AMs delayed early neutrophil recruitment but prolonged subsequent inflammatory cell recruitment.

PMN and monocyte/macrophage recruitment after instillation of P. aeruginosa in AM-depleted mice and control mice were evaluated by whole-lung BAL (Fig. 2). After instillation of P. aeruginosa (5 × 10⁵ CFU), in control mice, the number of PMNs recovered from BAL fluid increased rapidly and reached a peak at 16 h (Fig. 2a). In AM-depleted mice, fewer PMNs were recovered from BAL fluid at an early phase (0 to 16 h) but larger quantities of PMNs were obtained in the late phase (24 to 96 h). The number of monocytes/macrophages was significantly lower in AM-depleted mice than that in control mice at 0 to 16 h (Fig. 2b). However, a rapid increase of cells was observed after 24 h in both groups and there was a significant increase of monocytes/macrophages in AM-depleted mice after 72 h. MPO activity in the supernatant of lung homogenates was measured to estimate the total number of PMNs trapped in the lungs. Eight hours after bacterial challenge, MPO activity was significantly increased in the control group compared to that in the AM-depleted group. In contrast, the MPO activity in the AM-depleted group significantly exceeded that in the control group after 48 h (Table 1).

FIG. 2.

Number of PMNs and macrophages in BAL fluid and number of bacteria (CFU) in lung tissue from mice inoculated with P. aeruginosa. AM-depleted mice (solid bars) and control mice (open bars) received 5 × 10⁵ CFU of P. aeruginosa intratracheally and were sacrificed and analyzed at the specified time intervals. Each data point represents the mean + SD of values for five or more mice. ∗, P < 0.05 versus value for control mice. (a) PMNs in BAL fluid; (b) AMs in BAL fluid; (c) bacterial counts in lung tissue.

TABLE 1.

MPO activity in the lungs of control and AM-depleted mice

Mouse group	MPO activitya (U/g [mean ± SD])
	Pre	8 h	48 h
Control	8 ± 2	102 ± 35	24 ± 14
AM depleted	10 ± 3	40 ± 26b	94 ± 41b

^aMPO activity in the lung was measured before (Pre; n = 3) or 8 or 48 h after (n = 6 for each group) P. aeruginosa instillation. 

^bP < 0.05, compared to control value by Student’s t test. 

Delayed bacterial clearance in AM-depleted mice.

The number of bacteria in the lungs of the mice is shown in Fig. 2c. There were significantly more bacteria in the lungs of AM-depleted mice than in those of control mice between 4 and 48 h.

Depletion of AMs significantly reduced early chemokine release but sustained later release.

Two chemokines, MIP-2 and KC, were investigated in BAL fluids after bacterial instillation. Eight hours after bacterial instillation, the MIP-2 concentration in the BAL fluid from control mice was significantly higher than the concentration in AM-depleted mice. The KC concentration was also higher in control mice. In contrast, 48 h after bacterial instillation, the levels of both chemokines were significantly higher in AM-depleted mice than in control mice (Table 2).

TABLE 2.

Concentrations of MIP-2 and KC in the BAL fluid of control and AM-depleted mice

Mouse group	Concna (pg/ml [mean ± SD])
	MIP-2			KC
	Pre	8 h	48 h	Pre	8 h	48 h
Control	NDb	706 ± 357	4 ± 5	ND	1,568 ± 1,604	56 ± 48
AM depleted	ND	290 ± 171c	62 ± 48c	ND	597 ± 310	288 ± 180c

^aThe concentrations of MIP-2 and KC in the BAL fluid were measured before (Pre; n = 3) or 8 or 48 h after (n = 5 for each group) P. aeruginosa instillation. 

^bND, not detected. 

^cSignificantly different from control value (P < 0.05) by Student’s t test. 

Lung injury was improved in the early phase but was subsequently aggravated in AM-depleted mice.

Lung injury after bacterial instillation (5 × 10⁵ CFU) was evaluated by measuring efflux of the alveolar protein tracer from the lungs into the circulation and by measurement of lung edema (Fig. 3). Eight hours after bacterial instillation, there was no difference in the lung epithelial injury between control and AM-depleted mice (Fig. 3a). However, 48 h after the bacterial instillation, the lung epithelial injury was significantly increased in the AM-depleted mice compared to that in control mice. Although the lung edema in AM-depleted mice was significantly less than that in the control mice at 8 h, edema increased significantly by 48 h in AM-depleted mice (Fig. 3b).

FIG. 3.

Lung injury in five groups (untreated mice, n = 4; control mice instilled with saline, n = 4; AM-depleted mice instilled with saline, n = 4; control mice instilled with P. aeruginosa, n = 6; and AM-depleted mice instilled with P. aeruginosa, n = 6). (a) Movement of alveolar protein tracer, ¹²⁵I-albumin, from the lungs into the blood circulation. At 48 h, AM-depleted mice instilled with P. aeruginosa showed increased efflux of the tracer compared to that of control mice instilled with P. aeruginosa. (b) Pulmonary edema was assessed from the lung tissue W/D ratios. At 8 h after inoculation, the W/D ratio increased in control mice compared to that in AM-depleted mice. Conversely, the AM-depleted mice instilled with P. aeruginosa showed high W/D ratios compared to that in control mice instilled with P. aeruginosa at 48 h. ∗, P < 0.05 versus value for control mice instilled with saline; †, P < 0.05 for control mice instilled with P. aeruginosa versus AM-depleted mice instilled with P. aeruginosa. Statistical analysis was done by one-way analysis of variance and Scheffe’s F test. Values are means + SDs.

Histopathological aspects of lung injury.

Histopathological findings are shown in Fig. 4. In the control mouse without P. aeruginosa instillation, there was a normal distribution of AMs (Fig. 4a); these were absent in the AM-depleted mouse without P. aeruginosa instillation (Fig. 4b). Eight hours after the bacterial instillation, few PMNs were infiltrated into airspace in the AM-depleted mouse (Fig. 4d), in contrast to the control mouse which showed significant predominance of PMNs in the airspaces (Fig. 4c). On the other hand, 48 h after the bacterial instillation, destruction of alveolar structure and thickening of interstitial spaces were marked in AM-depleted mice (Fig. 4f).

Depletion of AMs improved early-phase infection but not later survival in mice with P. aeruginosa pneumonia in the large-inoculum study.

Survival after the instillation of P. aeruginosa (5 × 10⁷ CFU) in AM-depleted mice and control mice is shown in Fig. 5. After the instillation of P. aeruginosa, 22.8% of mice died within 12 h in the control group (non-AM depleted), whereas none of the mice in the AM-depleted group died by this time point. However, more than 90% of AM-depleted mice died by 96 h after bacterial instillation, while 50% of control mice survived. With an inoculum of 5 × 10⁵ CFU, none of the mice in either group were dead after bacterial instillation.

FIG. 5.

Effect of Cl₂MDP-liposome inhalation on survival of mice after P. aeruginosa instillation (5 × 10⁷ CFU) into the lungs. Mice were given an aerosol of Cl₂MDP-liposomes 72 h before bacterial instillation (AM-depleted group, n = 34). In the control group, mice received an inhalation of saline (control group, n = 36). There was a significant difference (∗ and †) between the values of the two groups at 12, 96, and 168 h by Fisher’s exact test.

DISCUSSION

Depletion of AMs with Cl₂MDP-liposomes.

Liposome-mediated elimination of macrophages has become a standard approach to evaluate the roles of macrophages in host defenses (21). Selective in vivo depletion of AMs by intratracheal instillation of liposome-encapsulated Cl₂MDP (clodronate) has been reported for rodent models (1, 4, 18, 19). Because only about 75% of AMs can be depleted by intratracheal instillation of Cl₂MDP-liposomes, this method may blunt the total effect of their depletion. We have recently shown that aerosol inhalation of negatively charged large oligolamellar liposomes complexed with clodronate disodium resulted in the depletion of over 95% of rat AMs without increasing neutrophils nor damaging the epithelium of the lung (8). In the present study, we depleted >95% of mouse AMs by this technique and investigated the roles of AMs in airspace inflammation. As shown in Fig. 3, AM depletion or aerosolization itself did not cause any detrimental effect on the airspace of the lung.

Acute-phase response to P. aeruginosa in AM-depleted mice.

In this study, a significant improvement in lung edema (<12 h) after P. aeruginosa instillation in AM-depleted mice was observed in comparison to that in nondepleted control mice, although there was a higher bacterial concentration in the lung of AM-depleted mice at 4 to 16 h. Significantly lower MPO activity at 8 h and fewer PMNs recovered from the BAL fluid at 4 to 16 h in AM-depleted mice suggest that fewer PMNs were recruited to the lungs after P. aeruginosa instillation in the early phase of infection. The significantly lower levels of the chemokines MIP-2 and KC at 8 h are consistent with low PMN recruitment. These findings suggest that AMs and/or AM-derived substances play an important role in the pathogenesis of acute lung injury and that the host inflammatory response initiated by AMs is responsible for acute host death in the early phase of infection. AMs contribute to rapid clearance of pathogens from the site of infection accompanied by increased tissue injury caused by the battle to eliminate bacteria, which can sometimes be life-threatening (16). As we previously found in an AM-depleted rat model, chemokines released from AMs appear to have a major role in early recruitment of PMNs to the site of infection (8). In addition, acute lung edema during the early phase of bacterial infection in our model seems to be mediated by macrophages or neutrophils. In the large-inoculum model (5 × 10⁷ CFU) of our study, the survival of AM-depleted mice was improved in the early phase. These data also show that AMs might make a major contribution to life-threatening condition in the acute phase of P. aeruginosa pneumonia.

Late-phase response to P. aeruginosa in AM-depleted mice.

In contrast to the early-phase results with the small inoculum of bacteria described above, lung injury (epithelial injury and edema) was significantly worse during the later phase of infection (48 h) in AM-depleted mice. Interestingly, the numbers of PMNs and monocytes/macrophages in the later phase were higher in AM-depleted mice than in control mice. More PMNs and monocytes/macrophages were recovered from the BAL fluid, and MPO activity and chemokine levels were higher in AM-depleted mice than in control mice. These findings suggest that there was prolonged inflammatory cell recruitment during the late phase in AM-depleted mice. These results correlated well with those of a recent study using Klebsiella pneumonia in an AM-depleted model (4). The higher levels of chemokines, as well as bacterial chemotactic factors and nonchemokine endogenous responses to unresolved infection, probably contributed to the greater PMN recruitment at this time point. Because the number of bacteria remaining in the lungs was greater in AM-depleted mice than in control mice between 4 and 48 h after bacterial instillation, depletion of AMs evidently decreased bacterial clearance and caused prolonged inflammatory responses, including greater PMN aggregation at the site of infection. Decreased bacterial clearance and prolonged inflammation appeared to aggravate lung injury in the later phase. The lung injury observed in the late-phase response of AM-depleted mice itself might have caused prolonged inflammatory responses, because PMN levels remained high at 72 to 144 h although bacteria were almost completely eliminated by 72 h. In addition, in our large-inoculum model (5 × 10⁷ CFU), AM-depleted mice hardly survived to day 7 after infection, although half of control mice could survive for the same period. This result also indicates that the AM-depleted condition could cause deleterious effects in the late phase of P. aeruginosa pneumonia.

Summary with clinical implications.

Recently, the use of various anti-inflammatory therapies has been proposed for acute inflammatory diseases (2, 20). However, we should not forget that an inflammatory response is essential to eliminate pathogens from the site of infection (16). The results obtained in AM-depleted mice illustrate both the beneficial and deleterious effects of AMs. Depletion of AMs in the early phase of Pseudomonas pneumonia may initially improve lung injury but may worsen the eventual outcome. However, temporary blockade of selective macrophage functions may contribute to decreasing neutrophil-mediated tissue injury and improving acute survival without compromising the subsequent beneficial effects of the inflammatory reaction on bacterial pneumonia. The findings obtained in this study using AM-depleted mice may provide same suggestions for clinical application of anti-inflammatory therapies.