Elastin fragments drive disease progression in a murine model of emphysema

Abstract

Mice lacking macrophage elastase (matrix metalloproteinase-12, or MMP-12) were previously shown to be protected from the development of cigarette smoke-induced emphysema and from the accumulation of lung macrophages normally induced by chronic exposure to cigarette smoke. To determine the basis for macrophage accumulation in experimental emphysema, we now show that bronchoalveolar lavage fluid from WT smoke-exposed animals contained chemotactic activity for monocytes in vitro that was absent in lavage fluid from macrophage elastase-deficient mice. Fractionation of the bronchoalveolar lavage fluid demonstrated the presence of elastin fragments only in the fractions containing chemotactic activity. An mAb against elastin fragments eliminated both the in vitro chemotactic activity and cigarette smoke-induced monocyte recruitment to the lung in vivo. Porcine pancreatic elastase was used to recruit monocytes to the lung and to generate emphysema. Elastin fragment antagonism in this model abrogated both macrophage accumulation and airspace enlargement.

Figure 1

EFs display monocyte chemotactic activity. Human monocytes were isolated by elutriation, and chemotaxis assays were performed in a modified Boyden chamber. (A) BALF from smoke-exposed mice (2 months) was used as the chemoattractant. Data are from a representative experiment performed in triplicate. Net monocyte movement = total monocytes – media control (= 40). Bars represent SEM. *P < 0.01 vs. control. (B) The BAL samples from MMP-12^+/+ and MMP-12^–/– mice were subjected to gel filtration. The fractions were tested for monocyte chemotactic activity (control activity with PBS alone = 0). (C) Western blot was performed against gel filtration fractions (nos. 11 and 37 shown here) using a polyclonal anti-elastin antibody. A 45-kDa fragment in MMP-12^+/+ active fractions was detected. (D) The assays of monocyte chemotaxis were repeated using lung homogenates from mice exposed to smoke for 2 months as the chemoattractant. BA4 and IgG1 preimmune antibodies were used at a concentration of 50 nM. Data are from a representative experiment performed in triplicate. Net monocyte movement per high-powered field = total monocytes – media control (= 46). Bars represent SEM. *P < 0.01 vs. control. hpf, high-powered field; NS, non–smoke exposed; Sm, smoke exposed.

Figure 2

EF antagonism decreases cigarette smoke–induced lung macrophage accumulation in vivo. BAL macrophage counts are shown for mice (n = 5) exposed to 4 cigarettes a day for 7 days while receiving daily treatment with BA4, an antibody directed against EFs, or IgG1 control antibody. Negative-control treatments with PBS, IgG1, and BA4 without concomitant cigarette smoke exposure are also shown. Bars represent SEM. *P < 0.001 vs. control; **P < 0.001 vs. Sm + IgG1.

Figure 3

Effects of PPE and EF antagonism on lung macrophage content in vivo. PPE (1 U = 7.4 μg) or PBS control was administered into the lungs of WT mice. BAL was performed before the lungs were inflated and fixed with 10% buffered formalin at days 1, 3, 7, 14, and 21. (A and B) BAL macrophage (A) and BAL neutrophil (B) counts were tabulated using a hemocytometer and HEMA 3–stained cytospins. (C) Lung macrophage counts are expressed as macrophages per high-powered field adjusted for tissue density on mac-3–stained sections. n = 4 mice per group. Bars represent SEM. ^#P < 0.05 vs. control. (D) To determine the effect of EF antagonism on monocyte recruitment, PPE-recipient mice received 10 μg BA4 (n = 9), 10 μg anti–collagen type I (col) antibody (n = 4), 10 μg anti–laminin-5 (lam) antibody (n = 4), or 10 μg IgG1 isotype control antibody (n = 8) over the course of the 3-day experiment; and either BA4 or IgG1 isotype control over the course of 21 days. The macrophage counts are expressed as above. Bars represent SEM. *P < 0.01 vs. control; **P < 0.01 vs. PPE/IgG1. (E–G) Representative mac-3–stained sections are shown for PBS (E), PPE/IgG1 (F), and PPE/BA4 (G) at the 3-day time point. Magnification, ×40.

Figure 4

EF antagonism abrogates PPE-induced emphysema. WT mice received i.t. PPE at time 0. In addition, the mice received 10 μg i.t. doses of BA4 or IgG1 isotype control on days 1, 2, 3, 5, 7, 10, 13, 16, and 19 (n = 6 each group). A separate group of mice received i.t. PBS only at each time point. (A–C) The CLs (A), alveolar airspace areas (B), and alveolar tissue areas (C) are reported for PBS, PPE/IgG1, and PPE/BA4 at the 21-day time point. PPE alone at 24 hours is provided as a reference. Bars represent SEM. *P < 0.0001 vs. control; **P < 0.0001 vs. PPE/IgG1. (D–F) Representative H&E-stained sections are provided for PBS (D), PPE/IgG1 (E), and PPE/BA4 (F), all at the 21-day time point. Magnification, ×10.

Figure 5

Depiction of an elastin fiber. The schematic depicts an elastin fiber that consists of multiple tropoelastin monomers cross-linked together by lysyl oxidase. Potential degradation products and their cross-linked nature are shown.