Fas ligand triggers pulmonary silicosis

Abstract

We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-alpha, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand-deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-alpha production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand-deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand-dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis.

Figure 1

FasL-deficient mice do not develop silicosis. (A) Weight loss. Mice were instilled with saline (white bars) or silica (black bars) intratracheally. BALB.wt, but not FasL-deficient BALB.gld mice lost weight (P < 0.05, n = 6; NS, n = 6, respectively). (B) TNF-α production. BALB.wt, but not BALB.gld mice increased BAL levels of TNF-α after silica instillation (P < 0.05, n = 4; NS, n = 4, respectively). (C) Respiratory mechanics. BALB.wt, but not BALB.gld mice increased lung total (ΔP_tot), resistive (ΔP₁), and viscoelastic/inhomogeneous (ΔP₂) pressures after silica instillation (P < 0.01, n = 6; NS, n = 6, respectively). SIL, silica. (D–F) Histopathology of lung sections from wt and gld mice. Top and bottom fields highlight areas of lung parenchyma and bronchial cross-sections, respectively. (D) Normal aspect of lung tissue from BALB.wt mice after saline instillation. (E) Intense alveolitis and heavy nodular infiltration of neutrophils and macrophages in lung from BALB.wt mice after silica instillation. (F) Normal aspect of lung tissue from BALB.gld mice after silica instillation. Hematoxylin and eosin staining. Original magnification: ×200.

Figure 2

Deficient neutrophil extravasation in FasL-deficient mice. (A) Macrophage and neutrophil numbers in BAL fluid from mice instilled with PBS or silica intratracheally. Silica exposed BALB.gld mice (black bars) showed a 5.8-fold reduction in neutrophil number (P < 0.01, n = 4), and a 4.0-fold increase in macrophage number (P < 0.01, n = 4), compared with BALB.wt mice (white bars). SIL, silica. (B) MPO activity. Mean MPO activity of both BAL cells and lung parenchyma of wt and gld mice instilled with PBS alone (n = 4 pools of 3 mice each) was taken as 100% of control value. Left: MPO activity of BAL cells from mice instilled with silica. Cells from BALB.gld (▪) mice had much less MPO activity than cells from BALB.wt (•) mice (P < 0.05). Right: after BAL removal, lung parenchyma from BALB.wt (•), but not from BALB.gld (▪) mice, had significant MPO activity, compared with PBS-instilled controls (P < 0.01, and NS, respectively). (C) Cytospin aspect of BAL cells. BAL cells were cytocentrifuged and stained with May-Grunwald. Top: BAL cells from BALB.wt mice after saline instillation were unstimulated alveolar macrophages. Middle: BAL cells from BALB.wt mice after silica instillation were mainly neutrophils, a minority were large macrophages. Bottom: BAL cells from BALB.gld mice after silica instillation were mainly large macrophages, a minority were neutrophils. Original magnification: ×630.

Figure 3

FasL-expressing macrophages initiate silicosis. (A) The cell type initiating silicosis is of bone marrow origin. MPO activity in the lungs of BALB.wt, BALB.gld mice, and the indicated chimeras, instilled with either saline (○,□) or silica (•,▪). Only mice with functional (wt) FasL on bone marrow cells accumulated neutrophils in the lung (P < 0.01). (B) FasL-expressing lung macrophages initiate silicosis. Left: MPO activity of BALB.wt mice treated with saline (□) or silica (▪) were taken as controls. Right: MPO activity in the lung parenchyma of wt (•) or FasL deficient gld mice (▴,▾), adoptively transferred intratracheally with wt (•,▴) or gld (▾) lung macrophages before silica instillation. Local transfer of wt (P < 0.01), but not of gld macrophages (NS), restored the ability of gld mice to develop silicosis.

Figure 4

FasL and Fas expression in vivo. (A–C) Immunohistochemistry of lung tissue from BALB.wt mice instilled with silica. Lung sections were stained with control hamster IgG (A), anti-FasL (B), or anti-Fas (C) mAbs, and revealed by immunoperoxidase. Silica deposition induces FasL (B) and increases Fas (C) expression, compared with control hamster IgG (A). FasL staining is restricted to granulomatous nodules containing silica; both unaltered areas of parenchyma and epithelia were negative. Original magnification: ×200. (D–G) Higher magnification aspects of FasL (D and E) and Fas (F and G) expression in lung tissue from BALB.wt mice instilled with either saline (D and F) or silica (E and G). FasL expression is absent, and Fas expression is low in saline-instilled lung sections. Original magnification: ×630.

Figure 5

Silica induces FasL expression in macrophages. (A) Macro-phages express FasL in vivo. Double staining immunofluorescence of lung macrophages from wt mice instilled with saline (left) or silica (right). Top: differential image contrast (DIC) microscopy of selected fields. Middle: staining of FITC-labeled F4/80⁺ macrophages in the same fields. Bottom: simultaneous staining of PE-labeled FasL⁺ macrophages in the same fields. Only silica-exposed F4/80⁺ macrophages stained for FasL expression. After Fc block, normal and silica-exposed macrophages did not stain for PE-labeled control hamster IgG (not shown). Original magnification: ×630. (B) Macrophages express FasL in vitro. FasL expression in normal wt BAL macrophages cultured with medium or silica in the absence (white bars) or in the presence (black bars) of deferoxamine (DFO). FasL expression was measured by cell ELISA. Silica induced FasL expression (P < 0.05), which was prevented by DFO (P < 0.05). (C) Silica induces FasL-dependent macrophage apoptosis. Apoptosis in BAL macrophages from normal gld and wt mice treated with silica in the presence of control hamster IgG or anti-FasL. Silica induced apoptosis in wt (P < 0.01), but not in gld (NS) macrophages; apoptosis was prevented by anti-FasL (P < 0.01). SIL, silica.

Figure 6

FasL blockade prevents silica-induced inflammation. (A) MPO activity in mice instilled with silica. Treatment with anti-FasL mAb MFL4 (NS compared with saline; n = 4), but not with control IgG (P < 0.01, n = 4), prevented increased MPO activity induced by silica in lung parenchyma. SIL, silica. (B) TNF-α production. Treatment with anti-FasL (NS compared with saline, n = 4), but not with control IgG (P < 0.01, n = 4), prevented increased TNF-α production in BAL fluid from silica exposed mice. (C–E) Histopathology of lung sections. (C) Normal aspect of lung tissue after saline instillation. (D) Intense pulmonary inflammation in mice instilled with silica and treated with control hamster IgG. (E) Lung tissue from mice instilled with silica and treated with anti-FasL mAb. Morphology comparable to C, except for a discrete macrophage infiltrate. Hematoxylin and eosin staining. Original magnification: ×200.