Therapy with plasma purified alpha1-antitrypsin (Prolastin®) induces time-dependent changes in plasma levels of MMP-9 and MPO

Abstract

The common Z mutation (Glu342Lys) of α1-antitrypsin (A1AT) results in the polymerization and intracellular retention of A1AT protein. The concomitant deficiency of functional A1AT predisposes PiZZ subjects to early onset emphysema. Clinical studies have implied that, among the biomarkers associated with emphysema, matrix metalloproteinase 9 (MMP-9) is of particular importance. Increased plasma MMP-9 levels are proposed to predict the decline of lung function as well as greater COPD exacerbations in A1AT deficiency-associated emphysema. The aim of the present study was to investigate the effect of A1AT therapy (Prolastin) on plasma MMP-9 and myeloperoxidase (MPO) levels. In total 34 PiZZ emphysema patients were recruited: 12 patients without and 22 with weekly intravenous (60 mg/kg body weight) A1AT therapy. The quantitative analysis of A1AT, MMP-9 and MPO was performed in serum and in supernatants of blood neutrophils isolated from patients before and after therapy. Patients with Prolastin therapy showed significantly lower serum MMP-9 and MPO levels than those without therapy. However, parallel analysis revealed that a rapid infusion of Prolastin is accompanied by a transient elevation of plasma MMP-9 and MPO levels. Experiments with freshly isolated blood neutrophils confirmed that therapy with Prolastin causes transient MMP-9 and MPO release. Prolastin induced the rapid release of MMP-9 and MPO when added directly to neutrophil cultures and this reaction was associated with the presence of IgA in A1AT preparation. Our data support the conclusion that changes in plasma levels of MMP-9 and MPO mirror the effect of Prolastin on blood neutrophils.

Fig 1. Quantitative analysis of serum A1AT, MMP-9 and MPO.

Measurement of (A) A1AT, (C) matrix metalloproteinase 9 (MMP-9) and (E) myeloperoxidase (MPO) levels in serum from augmented and non-augmented PiZZ A1ATpatients. From augmented patients serum samples were obtained at three different time points: before (pre, n = 22), two hours after (post, n = 22) and on day three after augmentation therapy (day 3, n = 12). From the non-augmented PiZZ A1AT deficient-patients serum samples were obtained only once (no augmentation, n = 12). To follow up the level of (B) A1AT as well as the biomarkers (D) MMP-9 and (F) MPO in a week course analysis, we obtained serum samples every day from one augmented PiZZ A1AT patient (GOLD stage III) and one non-augmented PiZZ A1AT-patient (GOLD stage II). Statistical significance between indicated groups at *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05.

Fig 2. The correlation between serum levels of A1AT, MMP-9 and MPO in PiZZ patients receiving Prolastin therapy.

(A) Spearman correlation analysis shows a significant positive correlation between the serum levels of MMP-9 and MPO (r = 0.7284, p<0.0001, n = 56); A1AT and MMP-9 (B) as well as A1AT and MPO (C) (r = 0.34, p = 0.009, n = 56; and r = 0.32, p = 0.016, n = 56, respectively). Line represents linear regression of data (MMP-9 with MPO: slope 0.1471±0.0175, r2 = 0.567; A1AT with MMP-9: slope 245.9±62.5, r2 = 0.223; A1AT with MPO: slope 35.54±12.98, r2 = 0.122).

Fig 3. Analysis of MMP-9 and MPO in supernatants of neutrophil isolated from a single PiZZ patient.

Neutrophils were isolated every day from one augmented PiZZ A1AT deficient-patient (GOLD stage III), and one non-augmented PiZZ A1AT deficient-patient (GOLD stage II). (A) matrix metalloproteinase 9 (MMP-9) and (B) myeloperoxidase (MPO) were analysed in the supernatants of 1x10⁶ cells/ ml (4 hours, 5% CO₂, 37°C). Neutrophil-associated A1AT was analysed in augmented (C) and non-augmented (D) PiZZ A1AT deficient-patient by Western blot using polyclonal antibody against A1AT.

Fig 4. Analysis of MMP-9 and MPO in supernatants of neutrophils treated with Prolastin.

Neutrophils were isolated from PiZZ A1AT deficient-patients before (pre, n = 8) and two hours after (post, n = 8) Prolastin therapy and from 8 PiZZ A1AT deficient-patients without Prolastin therapy. Quantification of the matrix metalloproteinase 9 (MMP-9) (A) myeloperoxidase (MPO) levels (B) was performed with or without pre-treatment of neutrophils (1x10⁶cells/ml) with Prolastin (1 mg/ml, 4 hours, 5% CO₂, 37°C). Statistical significance between the groups is indicated as: *** p ≤ 0.001 and ** p ≤ 0.01.

Fig 5. Analysis of MMP-9 and MPO in supernatants of neutrophils treated with Prolastin and Prolasin preparation without IgA.

(A) Prolastin (A1AT) and Prolastin preparation without IgA (A1AT*) were separated by non-reducing polyacrylamid gel gelelectrophoresis followed by Western blot analysis with anti-A1AT and Anti-IgA antibodies. (B) Neutrophils were isolated from healthy controls (Pi MM, n = 3) and treated with different concentrations of A1AT, or A1AT* or human serum albumin (protein control) for two hours, at 37°C, 5% CO₂. The concentration of 2.7 mg/ml mimics the average serum concentration post infusion of the augmented A1AT deficient patients. Quantification of the matrix metalloproteinase 9 (MMP-9, B) and myeloperoxidase (MPO, C) levels from cell culture supernatants were performed. Statistical significance between the groups is indicated as: ** p ≤ 0.009 and * p ≤ 0.02.