Prolastin, a pharmaceutical preparation of purified human alpha1-antitrypsin, blocks endotoxin-mediated cytokine release

Abstract

Background: Alpha1-antitrypsin (AAT) serves primarily as an inhibitor of the elastin degrading proteases, neutrophil elastase and proteinase 3. There is ample clinical evidence that inherited severe AAT deficiency predisposes to chronic obstructive pulmonary disease. Augmentation therapy for AAT deficiency has been available for many years, but to date no sufficient data exist to demonstrate its efficacy. There is increasing evidence that AAT is able to exert effects other than protease inhibition. We investigated whether Prolastin, a preparation of purified pooled human AAT used for augmentation therapy, exhibits anti-bacterial effects.

Methods: Human monocytes and neutrophils were isolated from buffy coats or whole peripheral blood by the Ficoll-Hypaque procedure. Cells were stimulated with lipopolysaccharide (LPS) or zymosan, either alone or in combination with Prolastin, native AAT or polymerised AAT for 18 h, and analysed to determine the release of TNFalpha, IL-1beta and IL-8. At 2-week intervals, seven subjects were submitted to a nasal challenge with sterile saline, LPS (25 microg) and LPS-Prolastin combination. The concentration of IL-8 was analysed in nasal lavages performed before, and 2, 6 and 24 h after the challenge.

Results: In vitro, Prolastin showed a concentration-dependent (0.5 to 16 mg/ml) inhibition of endotoxin-stimulated TNFalpha and IL-1beta release from monocytes and IL-8 release from neutrophils. At 8 and 16 mg/ml the inhibitory effects of Prolastin appeared to be maximal for neutrophil IL-8 release (5.3-fold, p < 0.001 compared to zymosan treated cells) and monocyte TNFalpha and IL-1beta release (10.7- and 7.3-fold, p < 0.001, respectively, compared to LPS treated cells). Furthermore, Prolastin (2.5 mg per nostril) significantly inhibited nasal IL-8 release in response to pure LPS challenge.

Conclusion: Our data demonstrate for the first time that Prolastin inhibits bacterial endotoxin-induced pro-inflammatory responses in vitro and in vivo, and provide scientific bases to explore new Prolastin-based therapies for individuals with inherited AAT deficiency, but also for other clinical conditions.

Figures

Figure 1

A concentration-response inhibition of lipopolysaccharide-stimulated TNFα (A) and IL-1β (B) release by Prolastin in human blood monocytes. Isolated blood monocytes were treated with LPS (10 ng/ml) alone or together with various concentrations of Prolastin (0–16 mg/ml) for 18 h. TNFα and IL-1β levels were measured by ELISA. Data are the means of quadruplicate culture supernatants ± S.E. and are representative of three separate experiments.

Figure 2

Comparisons of the effects of native (nAAT), polymeric (pAAT) and Prolastin on lipopolysaccharide – stimulated TNFα (A) and IL-β (B) production by human blood monocytes isolated from four healthy donors. Isolated blood monocytes were treated with LPS (10 ng/ml) alone or together with 0.5 mg/ml nAAT, pAAT or Prolastin for 18 h. TNFα and IL-1β levels were measured by ELISA. Each bar represent the mean ± S.E. *** p

Figure 3

Effects of AATs on neutrophils activated with zymosan. (A) Concentration-dependent effects of Prolastin on IL-8 release from neutrophils activated with opsonised zymosan. Freshly isolated blood neutrophils were treated with zymosan (0.3 mg/ml) alone or together with various concentrations of Prolastin (0–8 mg/ml) for 18 h. IL-8 levels were measured by ELISA. Data are the means of quadruplicate culture supernatants ± S.E. and are representative of three separate experiments. (B) Effects of opsonised zymosan alone or together with native (nAAT), polymeric (pAAT) AAT or Prolastin on IL-8 release from neutrophils. The release of neutrophil IL-8 was measured in cell free supernatants as described in Materials and methods. Neutrophils were treated for 18 h with a constant amount of zymosan (0.3 mg/ml) alone or together with nAAT, pAAT or Prolastin (0.5 mg/ml) for 18 h. IL-8 levels were measured by ELISA. Each bar represents the means ± S.E. of three separate experiments carried out in duplicate repeats. *** p

Figure 4

IL-8 analysis in nasal lavage of subjects challenged with LPS alone or LPS+Prolastin combination. Seven healthy volunteers were treated with LPS (25 μg/nostril) or with LPS followed 30 min later with Prolastin (2.5 mg/nostril), nasal lavage was collected at different time points (0, 2, 6 and 24 h) as described in Material and Methods. The concentration of IL-8 (pg/ml) was measured by ELISA. IL-8 values are expressed as a ratio of IL-8 concentration at selected time point and the basal level. Independent two sample t-test shows after 6 and 24 h significantly higher levels of IL-8 in subjects treated with LPS compared to LPS+Prolastin. * p