Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Abstract

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.

Fig. 1.

Total alveolar macrophages (A) and PMN (B) counts in the bronchoalveolar lavage fluid (BALF) of mice 24, 48, or 72 h after intratracheal instillation of liposomes containing PBS (white) or clodronate (gray) (n = 3/group for each time). C and D show representative lung tissue sections from mice studied 24 h after intratracheal instillation of liposomes containing PBS or clodronate, respectively [hematoxylin and eosin (H&E) staining, magnification ×400]. Note the absence of macrophages and neutrophils in the lungs from the mouse treated with clodronate-liposomes (D).

Fig. 2.

Total BALF macrophage (A), PMN (B), and lymphocyte (C) counts and lung homogenate MPO activity (D) in mice treated with intratracheal liposomes containing PBS or clodronate followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later; n = 6/group except for the PBS + IgG group, n = 5.

Fig. 3.

Cytokine concentrations in lung homogenates from mice treated with intratracheal liposomes containing PBS or clodronate, followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later. The cytokines were measured simultaneously using a multiplex assay and include TNFα (A), IL-1β (B), MIP-2 (C), KC (D), GM-CSF (E), VEGF (F), IFNγ (G) and IL-6 (H); n = 6/group except for the PBS + IgG group, n = 5.

Fig. 4.

Lung tissue sections from mice treated with intratracheal liposomes containing PBS or clodronate (Clod), followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later. Left: representative lung tissue sections stained with H&E (×400, insets ×200). Right: immunohistochemistry for alveolar macrophages (AM; arrows) using anti-Mac-2 antibody.

Fig. 5.

Number of cells staining for F4/80 in lung tissue sections from mice treated with intratracheal liposomes containing PBS or clodronate, followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later (A). B: percentage of F4/80 cells costaining for CX3CR1 or CCR2 in the same sections. Data are shown as means ± SE. *P < 0.05 compared with percentages of CX3CR1 or CCR2 positive cells.

Fig. 6.

Lung homogenate caspase-3 activity (arbitrary fluorescence units) (A) or TUNEL-positive cells per 12 high-power fields (hpf; B) in mice treated with intratracheal liposomes containing PBS or clodronate, followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later; n = 6/group except for PBS + IgG, n = 5.

Fig. 7.

Total BALF protein (A) and IgM (B) concentrations in mice treated with intratracheal liposomes containing PBS or clodronate, followed 24 h later by intratracheal instillations of Jo2 MAb or control IgG MAb, 2.5 μg/g, and studied 18 h later; n = 6/group except for PBS + IgG, n = 5.

Fig. 8.

Concentrations of KC in the supernatant of MLE-12 cells incubated with serial concentrations of a control IgG (black), Jo2 MAb (light gray), or Jo2 MAb + the broad caspase inhibitor zVAD (100 μM) (dark gray) (A). Cells incubated with VP16 (100 μM) serve as positive control. B: cell survival in the same cells as determined by the alamar Blue assay. Data are from at least 3 independent experiments and are shown as means ± SE; *P < 0.05 compared with cells incubated with control IgG at each time point.