press-5.qxd 11/28/07 3:01 PM Page 400 400 Table 1. Human plasmatic α-1-antitrypsin preparation modifications1 B. Mosheimer et al.: Recombinant A1PI and granulocyte migration Company Plasma half-lives (days) Specific activity (mg functional A1PI/mg protein)* Glycoisoforms Deamidation (qualitative) Deamidation (quantitative, %) C-terminal Lys truncation (%) Cys232 cysteinylation Methionin oxidation 1 Data in part taken from product specifications or from Kolarich et al. [12]. * Plasma A1PI, 0.02. Aralast Prolastin Baxter Healthcare Talecris Biotherapeutics 5.9±1.2 5.1±0.5 ≥0.55 ≥0.35 No major difference Detectable 6 10 67 2 + + Not observed (A1PI fully functional active) Zemaira CSL Behring 5.1±2.4 ≥0.07 7 6 – levels and inflammation had already been observed in another study using recombinant A1PI [16]. Recombinant technology has also been used to pro- duce human A1PI [4, 11, 14, 17], as regulatory authori- ties have recommended the development of recombi- nant counterparts to blood-derived therapeutic human proteins [18]. Recombinant A1PI is not yet commercial- ly available, but it is under investigation as a therapy for A1PI deficiency for both intravenous application and inhalation. In the development of drug therapy for A1PI deficiency, aerosol administration of recombinant pro- teins to the airways has been the preferred method of delivery because of their shorter half-lives after intra- venous infusion compared with that of A1PI purified and concentrated from human plasma [5]. Recent studies suggest a role for A1PI as an anti- -inflammatory modulator [19] independent of its antiproteinase effect, which may explain some of the observations in recent clinical studies. A1PI inhibited lipopolysaccharide (LPS)-induced tumor necrosis fac- tor-α and interleukin (IL)-1β release from monocytes dose dependently and IL-8 release from neutrophils in vitro. Furthermore, A1PI instillation after LPS chal- lenge prevented LPS-induced IL-8 production in vivo [19]. It is currently unknown to what extend recombi- nant A1PI affects leukocyte chemotaxis. Alteration of leukocyte migration could have relevance to the cells’ pulmonary accumulation, which is of major pathophysi- ological importance [10]. We therefore investigated the direct effects of human recombinant (rh)A1PI on human peripheral blood granulocyte chemotaxis in vitro. We report that rhA1PI from yeast does not induce in vitro chemotaxis of leukocytes in micropore filter assays. MATERIALS AND METHODS rhA1PI preparation and other material The rhA1PI used in this study, supplied by Baxter (Vienna, Austria), was produced by yeast transformed with an expressing plasmid containing a human cDNA encoding the mature normal Ml (Val213) human A1PI protein [21]. This rhA1PI preparation has a molecular mass of about 45 kDa, similar to human plasma A1PI, and functions in an identical manner as human plasma rhA1PI as an inhibitor of neutrophil elastase [1]. Formyl-Met-Leu-Phe, dextran, and penicillin-strepto- mycin were from Sigma Chemical Corp. (St. Louis, MO, USA). RANTES was from Pepro Tech EC Ltd. (London, UK). RPMI 1640 with phenol red purchased from Biochrom AG (Berlin, Germany). Bovine serum albumin (BSA) was from Dade Behring (Marburg, Germany). Lymphoprep was from Nycomed Pharma AS (Oslo, Norway). MACS separation columns and microbeads were from Miltenyi Biotech (Auburn, CA, USA). Fetal calf serum and phosphate-buffered saline (PBS) were from PAA Laboratories GmbH (Pasching, Austria). Trypsin-EDTA in HBSS from Gibco (Paisley, UK). The microchemotaxis chambers were from Neuroprobe (Bethesda, MD, USA) and the cellulose nitrate filters were from Sartorius (Goettingen, Germany). Preparation of granulocytes Neutrophils and eosinophils were obtained from peripheral EDTA-anticoagulated blood of healthy volun- teers by Lymphoprep density gradient centrifugation followed by dextran sedimentation and hypotonic lysis of contaminating erythrocytes as described previously [6]. The neutrophil preparations yielded above 95% neu- trophils (by morphology in GIEMSA stains) of more than 99% viability (by trypan dye exclusion). For the eosinophil preparation, the cells were resuspended in 50 ml of ice-cold MACS buffer (PBS with 5 mmol/l EDTA and 0.5% BSA) per 5×107 cells and an equal volume of MACS colloidal superparamagnetic microbeads conju- gated with monoclonal anti-human CD16 monoclonal antibody was added and incubated (30 min, 4°C). Recommended volumes of ice-cold MACS buffer were added to the cell-microbead mixture and the cell suspen- sion was loaded onto the separation column. The eluate containing CD16-negative eosinophils was collected, washed, resuspended in RPMI 1640/0.5% BSA, and the separation procedure was repeated to increase purity. The purity of the sorted eosinophils was 98% as deter-