press-5.qxd 11/28/07 3:01 PM Page 401 B. Mosheimer et al.: Recombinant A1PI and granulocyte migration mined by morphology and FACS analysis. Contaminating cells were 1% lymphocytes, 1% neutrophils and baso- phils, and negligible numbers of monocytes/macrophages. Migration assay The migration assays were performed using a modi- fied 48-well Boyden microchemotaxis chamber (Neuroprobe, Bethesda, MD, USA) in which a 5-µ m- -pore-size cellulose nitrate filter separated the upper and lower chambers [6]. Neutrophils or eosinophils were resuspended in RPMI 1640/0.5% BSA (1×106 cells/ml). Fifty µ l of the cell suspension were placed into the upper chamber and allowed to migrate toward 10 nmol/l of formyl-Met-Leu-Phe or toward 10 ng/l of RANTES chemokines, which were used as positive-control chemoattractants at optimal concentrations for neu- trophils and eosinophils, respectively; various dilutions of rhA1PI probes 1–4; or respiratory cell culture super- natants placed in the lower chamber for 30 min (neu- trophils) or 60 min (eosinophils) at 37°C in a humidified atmosphere (5% CO2). After the migration period, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. The migration depth of the cells into the filters was quantified by means of microscopy, mea- suring the distance (µ mm) from the surface of the filter to the leading front of three cells. Data are expressed as the chemotaxis index, which is the ratio of the distances of directed and random migration of neutrophils or eosinophils into the nitrocellulose filters. Statistical methods Data are expressed as mean ± standard error of the mean (SEM). Means were compared by the Mann- 401 -Whitney U-test and Kruskal-Wallis ANOVA. A differ- ence with p<0.05 was considered to be significant. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkeley, CA, USA). RESULTS AND DISCUSSION To investigate whether granulocyte recruitment to the lung could be a direct effect of rhA1PI, in vitro neu- trophil or eosinophil chemotaxis was assessed. The human plasmatic A1PI protein in a range of concentra- tions from 0.1–3 mg/ml had no effect on granulocyte chemotaxis (data not shown). Data from different preparations of yeast rhA1PI and a vehicle (MOCK) preparation which contained the preparation’s solution but devoid of the protein are shown for neutrophils (Fig. 1) and for eosinophils (Fig. 2). The positive-control attractants 10 nmol/l of formyl-Met-Leu-Phe and 10 ng/l of RANTES for neutrophils and eosinophils, respectively, significantly stimulated directed migration. Neither of the rhA1PI preparations affected migration in a significant manner at doses ranging from attomo- lar to micromolar concentrations. The MOCK prepara- tion had no effect on eosinophil migration. It stimulated neutrophil directed migration to some extent, reach- ing statistical significance at the highest dose tested, cor- responding to the rhA1PI vehicle concentration of >1 µ mol/l. Inhaled rhA1PI may directly contribute to leukocyte accumulation in the lung as human plasmatic A1PI may function as a neutrophil chemoattractant when poly- merized [15, 20, 22]. However, no direct effect was seen for rhA1PI on neutrophil or eosinophil chemotaxis. Fig. 1. Human peripheral blood neutrophil migration toward the chemoattractant formyl-Met-Leu-Phe or α(1)-antitrypsin (rAAT). Mean distances of random migra- tion were 66, 77, 74, and 85 µ m for rAAT-1, rAAT-2, rAAT-3, and MOC, respectively. *p<0.05, Mann-Whitney U-test versus me- dium after multiple group comparison by using the Kruskal-Wallis test.