Polymers of Z alpha1-antitrypsin co-localize with neutrophils in emphysematous alveoli and are chemotactic in vivo

Abstract

The molecular mechanisms that cause emphysema are complex but most theories suggest that an excess of proteinases is a crucial requirement. This paradigm is exemplified by severe deficiency of the key anti-elastase within the lung: alpha(1)-antitrypsin. The Z mutant of alpha(1)-antitrypsin has a point mutation Glu342Lys in the hinge region of the molecule that renders it prone to intermolecular linkage and loop-sheet polymerization. Polymers of Z alpha(1)-antitrypsin aggregate within the liver leading to juvenile liver cirrhosis and the resultant plasma deficiency predisposes to premature emphysema. We show here that polymeric alpha(1)-anti-trypsin co-localizes with neutrophils in the alveoli of individuals with Z alpha(1)-antitrypsin-related emphysema. The significance of this finding is underscored by the excess of neutrophils in these individuals and the demonstration that polymers cause an influx of neutrophils when instilled into murine lungs. Polymers exert their effect directly on neutrophils rather than via inflammatory cytokines. These data provide an explanation for the accelerated tissue destruction that is characteristic of Z alpha(1)-antitrypsin-related emphysema. The transition of native Z alpha(1)-antitrypsin to polymers inactivates its anti-proteinase function, and also converts it to a proinflammatory stimulus. These findings may also explain the progression of emphysema in some individuals despite alpha(1)-antitrypsin replacement therapy.

Figure 1-4410

Demonstration of the partially loop-inserted Z α₁-AT (red) that opens up β-sheet A (green) to favor insertion of the reactive loop of another molecule to form an AT dimer (center) and polymers (right) (adapted from R. Mahadeva et al15).

Figure 2-4410

Immunohistochemistry of the anti-polymer antibody on emphysematous lung tissue from Z-AT individuals. A: Z-AT lungs stained with the anti-polymer antibody—note the staining within the alveolar walls, particularly around capillaries. B: Z-AT lungs stained with isotype control antibody—note the absence of staining within the alveolar walls and the lung vasculature. C: The anti-polymer antibody was preincubated with a 10-fold molar excess of native α₁-AT before immunostaining Z-AT lungs. Staining was not inhibited, indicating that the anti-polymer antibody recognizes polymeric AT and not native AT. D: The anti-polymer antibody was preincubated with a 10-fold molar excess of polymers of α₁-AT before immunostaining. Note the absence of staining within the alveolar walls. However, staining is not inhibited within the endothelium of the larger pulmonary vessels (arrow).

Figure 3-4410

Immunohistochemistry of the anti-polymer antibody on emphysematous lung tissue from M-AT individuals. A: M-AT lungs stained with the anti-polymer antibody. There was an absence of staining within the alveolar walls, but there was however (B, arrow) positive staining within the endothelium of a pulmonary artery, a pattern also seen within Z-AT endothelium. C: IgG isotype control of M-AT patient. There was an absence of staining within the pulmonary artery and surrounding alveolar walls.

Figure 4-4410

Staining of M-AT and Z-AT emphysematous lungs with an antibody to neutrophil elastase and double staining with the anti-polymer antibody. A–C: Z α₁-AT emphysematous lungs. A: Neutrophils demonstrated with anti-neutrophil elastase antibody. There was an abundance of neutrophils within the alveolar walls at both low power (A) and high power (B). C: The polymers co-localized with neutrophils in Z-AT alveoli. Oil immersion image of neutrophils (red, arrows) and polymers (brown) stained with neutrophil elastase and the anti-polymer antibody, respectively. D–F: M α₁-AT emphysematous lungs. Neutrophils demonstrated with neutrophil elastase staining (low power, D; high power, E). Neutrophils were present within the alveolar walls. F: M-AT lungs: co-localization of neutrophil elastase (red, arrows) and the anti-polymer antibody (brown). There was no polymer staining within the alveolar walls. G: Graph of PMN counts in alveoli from 10 M-AT and 10 Z-AT emphysematous lungs. Six random fields were counted in each individual and the number of neutrophils documented by two independent observers blinded to the patient phenotype. *, P < 0.01 for neutrophil counts in the alveolar wall in Z-AT versus M-AT emphysematous lung tissue. Original magnifications: ×10 (A, D); ×40 (B, E); ×100 (C).

Figure 5-4410

a: Graph demonstrating the effect of intratracheal instillation of native and polymeric α₁-AT on polymorphonuclear leukocyte (PMN) numbers in BALF from C57BL/6J mice. Each bar graph represents the data from six mice. *, P < 0.01 for polymeric AT compared with native AT. b: C57BL/6J mice were anesthetized and intubated. Native or polymeric α₁-AT in 40 μl of PBS was instilled via the intratracheal route. At 4, 24, and 72 hours after instillation, BAL was performed and aliquots were assessed on a 7.5% (w/v) nondenaturing PAGE followed by Western blot analysis for α₁-AT using a polyclonal antibody that recognizes all forms of α₁-AT. Lane 1: Native AT, starting material 0.1 μg; lane 2: polymeric AT, starting material 0.1 μg; lane 3: BAL fluid 4 hours after native AT instillation; lane 4: BAL fluid 4 hours after instillation of polymeric AT; lane 5: BAL fluid 24 hours after native AT instillation; lane 6: BAL fluid 24 hours after instillation of polymeric AT; lane 7: BAL fluid 72 hours after native AT instillation; lane 8: BAL fluid 72 hours after instillation of polymeric AT.

Figure 6-4410

Graph demonstrating the effect of native and polymeric AT on murine neutrophil chemotaxis compared with positive controls 10⁻⁴ formylated Met-Leu-Phe (fmlp) and 4% (w/v) zymosan-activated serum. *, P < 0.01 for polymeric AT compared with native AT. The data are the mean and SD of five wells for each potential chemoattractant repeated between three and six times.