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 x 10(5) 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 x 10(7) 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.

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. 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.

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.

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.

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.