Relationship of acute lung inflammatory injury to Fas/FasL system

Abstract

There is mounting evidence that apoptosis plays a significant role in tissue damage during acute lung injury. To evaluate the role of the apoptosis mediators Fas and FasL in acute lung injury, Fas (lpr)- or FasL (gld)-deficient and wild-type mice were challenged with intrapulmonary deposition of IgG immune complexes. Lung injury parameters ((125)I-albumin leak, accumulation of myeloperoxidase, and wet lung weights) were measured and found to be consistently reduced in both lpr and gld mice. In wild-type mice, lung injury was associated with a marked increase in Fas protein in lung. Inflamed lungs of wild-type mice showed striking evidence of activated caspase-3, which was much diminished in inflamed lungs from lpr mice. Intratracheal administration of a monoclonal Fas-activating antibody (Jo2) in wild-type mice induced MIP-2 and KC production in bronchoalveolar lavage fluids, and a murine alveolar macrophage cell line (MH-S) showed significantly increased MIP-2 production after incubation with this antibody. Bronchoalveolar lavage fluid content of MIP-2 and KC was substantially reduced in lpr mice after lung injury when compared to levels in wild-type mice. These data suggest that the Fas/FasL system regulates the acute lung inflammatory response by positively affecting CXC-chemokine production, ultimately leading to enhanced neutrophil influx and tissue damage.

Figure 1

Lung injury parameters permeability index (A), MPO content (B), and wet lung weights (C) 4 hours after IgG immune complex deposition in wild-type (WT IC) and lpr mice (lpr IC). For each vertical bar, n ≥ 5 animals. Two-way analysis of variance: factor 1 (condition), factor 2 (strain), and the interaction between the factors showed significant effects on the permeability index and MPO content. For wet lung weights the effect of factor 1 was significant, factor 2 not quite significant (P = 0.0541), and the interaction was not significant. Bonferroni’s post tests: *, P < 0.001 when compared to the corresponding noninjured animals (WT Ctrl and lpr Ctrl); **, P < 0.001 (A); **, P < 0.01 (B); and **, P < 0.05 (C) when compared to immediately preceding vertical bar.

Figure 2

Total cell counts (A) in BAL fluids 4 hours after onset of lung injury. Each vertical bar represents n ≥ 5 animals of noninjured wild-type mice (Ctrl), injured wild-type (WT IC), and injured lpr (lpr IC) mice. *, P < 0.001 when compared to noninjured mice (Ctrl); no statistical significant differences (n.s.) between injured wild-type and lpr animals (WT IC versus lpr IC). Differential white blood cell counts (B) in the same BAL fluids. No significant differences (n.s.) in neutrophil and mononuclear cell counts are found between the injured animal groups (WT IC and lpr IC); n.d., not detectable.

Figure 3

H&E-stained paraffin-embedded lung sections (6 μm) from wild-type (A, B) and lpr mice (C, D). A and C: Noninjured lungs. B and D: Lungs 4 hours after intrapulmonary deposition of IgG immune complexes. Shown are representative sections of at least three animals for each condition. Original magnifications, ×400.

Figure 4

BAL fluid content of KC (A), MIP-2 (B), MCP-1 (C), and TNF-α (D) in wild-type (WT) and Fas-deficient (lpr) animals from noninjured (Ctrl) and injured (IC) lungs 4 hours after onset of injury. For each vertical bar, n ≥ 5. Two-way analysis of variance: factor 1 (condition), factor 2 (strain), and the interaction between the factors showed significant effects on content of KC and MIP-2. For MCP-1 and TNF-α only the effect of factor 1 was significant, whereas factor 2 and the interaction were not significant. Bonferroni’s post tests: *, P < 0.001 when compared to the corresponding noninjured lungs; **, P < 0.001 (A and B) relative to inflamed WT lungs.

Figure 5

Western blot analysis for Fas protein in lung homogenates from normal (WT Ctrl) lungs and 4 hours after onset of injury (WT IC). Equivalent amounts of protein (25 μg) were loaded in each lane. For each group, n = 2. Lower frame is relative density.

Figure 6

Western blot analysis of lung homogenates for activated (cleaved) caspase-3 (17 kd) in noninjured (WT Ctrl) lungs and injured lungs from wild-type (WT IC) and lpr mice (lpr IC), 4 hours after IgG immune complex deposition. Equal amounts of protein (25 μg) were loaded in each lane. For each group, n = 2. Lower frame is the corresponding relative density. Data are representative of three separate and independent experiments.

Figure 7

Lung permeability (A) in wild-type and lpr noninjured mice (Ctrl), injured (IC) mice, injured mice treated with the pan-caspase inhibitor (IC+Z-VAD), or its inactive derivative (IC+Z-FA) 4 hours after induction of lung injury. *, P < 0.01 when compared to the noninjured control (Ctrl) lungs; **, P < 0.05 when compared to immediately preceding and following vertical bar; n.s., no significant difference. MPO (B) and wet lung weights (C) in wild-type mice under the same conditions as in A (Ctrl, IC, IC+Z-VAD, and IC+Z-FA). *, P < 0.001 when compared to the noninjured control (Ctrl) lungs (B and C); **, P < 0.05 when compared to immediately preceding vertical bar (C); n.s., no significant difference; for each vertical bar, n ≥ 5.

Figure 8

Lung injury parameters permeability index (A), MPO content (B), and wet lung weights (C) 4 hours after IgG immune complex deposition in wild-type (WT IC) and gld mice (gld IC). For each vertical bar, n ≥ 5 animals. Two-way analysis of variance: factor 1 (condition), factor 2 (strain), and the interaction between the factors showed significant effects on permeability index and MPO content. For wet lung weights the effect of factor 1 and factor 2 were significant, but not the interaction between the factors. Bonferroni’s post tests: *, P < 0.001 (A and B) and *, P < 0.01 (C) when compared to the corresponding noninjured animals (WT Ctrl and gld Ctrl); **, P < 0.05 (A and C) or **, P < 0.001 (B) when compared to the immediately preceding vertical bar.

Figure 9

A: MIP-2 and KC levels in BAL fluids of wild-type mice 4 hours after intratracheal administration of Fas-activating monoclonal antibody (mAb) Jo2 or isotype control IgG. *, P < 0.05 when compared to isotype control IgG; for each condition, n = 6; n.d., not detectable. B: MIP-2 content in cell culture supernatants of murine alveolar cell line macrophages (MH-S) treated with different doses of either anti-Fas mAb (Jo2) or isotype control IgG for 6 hours. Shown are representative data from two separate and independent experiments; for each condition, n = 3. Two-way analysis of variance: factor 1 (dose) and factor 2 (antibody) showed significant effects on MIP-2 content whereas the interaction between the two factors was not significant. Bonferroni’s post tests: *, P < 0.01 when compared to the corresponding isotype IgG control.