and activation of Fc-a receptor–dependent phagocytosis, the cleavage of IgA1 has been postulated to impair IgA1- mediated effector functions (11, 12). However, most indi- viduals have neutralizing antibodies against IgA1 protease that may interfere with the function of the enzyme (21–23). Thus, the role of IgA1 protease in neisserial pathogenesis remains obscure. Inflammatory cytokines such as TNF-a, IL-1b, IL-6, and IL-8 are produced by activated monocytes/macrophages. They promote local inflammatory responses at the sites of microbial infections, and mediate adherence of leukocytes to the endothelial tissues and their transmigration by up- regulating the expression of adhesion molecules (24–27). Elevated levels of TNF-a, IL-1, IL-6, and IL-8 have been found in the cerebrospinal fluid (CSF)1 and plasma of patients with meningitis (28–31) and were detected in the urine after the intraurethral challenge of patients with N. gonorrhoeae (32). Besides LPS, several bacterial products that can stimulate cytokine synthesis in different cell types have been charac- terized (33). These cytokine-inducing agents include heat labile proteins, glycoproteins, lipoproteins, or peptides, which trigger cytokine synthesis by mechanisms distinct from LPS and therefore form an additional class of virulence factors termed modulins (34, 35). The purpose of this study was to assess the immunogenic and immunomodulatory properties of the neisserial IgA1 protease. We demonstrated that purified IgA1 protease acts like a modulin by stimulating the release of TNF-a, IL-1b, IL-6, and IL-8 from PBMCs. These effects were indepen- dent of the proteolytic activity but required the native form of the enzyme. Materials and Methods Purification and Characterization of Recombinant IgA1 Protease. In this study, we used the recombinant form of IgA1 protease ex- pressed in Escherichia coli strain H2053, which harbors plasmid pIP11 containing the iga gene of N. gonorrhoeae MS11 with the temperature-inducible bacteriophage l promoter PL, as described previously (18). In brief, the supernatant of a 4-liter stationary phase culture was recovered by centrifugation, supplemented with 0.1 M EDTA, and diluted 1:1 (vol/vol) with potassium phosphate buffer (20 mM, pH 7.0). The bacterial supernatant was applied to a cation exchange column. After washing, bound protein was eluted with 500 mM potassium phosphate, 8.6% glycerol. Frac- tions of 10 ml were collected, precipitated with ammonium sul- phate, and then redissolved in gel filtration buffer. Gel filtration was performed with a preequilibrated Superdex column (Amer- sham Pharmacia Biotech). Protein-containing eluates were rec- ognized by UV absorption, pooled, and precipitated with ammo- nium sulphate. Finally, the pellet was suspended in PBS and dialyzed against PBS, pH 7.4, and stored in aliquots at 2708C. Purity of the protein was assessed by SDS-PAGE with subsequent Coomassie staining showing the characteristic protein profile with the typical 109- and 106-kD protein bands as described pre- viously (18). The protein concentration was determined using a 1Abbreviation used in this paper: CSF, cerebrospinal fluid. commercial protein determination assay (Bio-Rad). Specificity for IgA1 protease was confirmed by immunoblotting using an IgA1 protease–specific rabbit antiserum (13). The cleavage activity of the recombinant IgA1 protease was determined with human IgA1 (20 ng/ml; Dakopatts) in Tris buffer (0.05 M Tris-HCl, 10 mM CaCl2, and 10 mM MgCl2, pH 7.5) at 378C according to the method described previously (36). Heat denaturation (boiling for 10 min) completely destroyed the enzymatic activity. The enzy- matic activity could also be blocked by addition of the specific peptide prolyl boronic acid inhibitor (37). 100 mM of the inhibi- tor (provided by A.G. Plaut, New England Medical Center, Bos- ton, MA) abrogated cleavage of IgA at concentrations up to 10 ng per ml of IgA1 protease (data not shown). Endotoxin Assays. The endotoxin contents of our IgA1 pro- tease preparations were determined by Limulus amebocyte lysate assay (LAL; Haemochrom Diagnostica). All preparations con- tained ,2 pg of endotoxin per mg protein. It is described that some proteins could interfere with the Limulus assay (38). To en- sure that IgA1 protease is free of LPS, we have tested in addition the priming potency of IgA1 protease for neutrophils, which are as sensitive as the Limulus assay in detecting LPS. Different types of LPS can prime neutrophils to increase their oxidative response after stimulation with the agonist fMLP (Sigma Chemical Co.) (38). PMNs preincubated with 10 mg/ml IgA1 protease for 30 min stimulated with 1 mM fMLP showed no alteration of the oxida- tive response compared with the control when measured as luminol- dependent chemiluminescence in a six-channel Biolumat LB 9505 (Berthold; data not shown). Cells. PBMCs were isolated from buffy coats or freshly citrated blood of adult, healthy volunteers by density gradient centrifuga- tion over Ficoll/Isopaque (Amersham Pharmacia Biotech) and washed twice in Ca2, Mg21–free PBS. For some experiments, lymphocytes and monocytes were separated (6 3 106 cells/ml in PBS plus 0.5% BSA) by counterflow centrifugation (elutriation) using the Beckman J6ML/JE-5.0 centrifuge system. Elutriation was performed at constant rotor speed with increasing flow rates, and lymphocytes were collected. To obtain purified T cells, the lymphocyte suspensions were positively sorted by anti-CD3 magnetic beads (MACS; Miltenyi Biotec). Purity of CD31 T cells was always .95% as determined by flow cytometry. Mono- cytes were prepared from PBMCs either by adherence to plastic dishes (1 h, 378C) or by negative selection using the MACS monocyte isolation kit (Miltenyi Biotec). The purity of CD141 cells was always .85% as measured by flow cytometry using a PE-conjugated anti-CD14 antibody (Leu M3; Becton Dickinson). The remaining 15% consisted of CD32, CD192, and CD562 flow cytometric counting events. The monocytic cell line MonoMac 6 (no. 124; American Type Culture Collection) was obtained from the German Col- lection of Microorganism and Cell Cultures (Braunschweig, Ger- many) and maintained in 24-well plates under low endotoxin conditions (,10 pg/ml) in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, and 9 mg/ml bovine insulin as de- scribed (39). The cell line normally expresses CD14 as controlled by flow cytometry. Cell Viability. Cytotoxic effects of IgA1 protease preparations were excluded by staining of PBMCs with propidium iodide (10 mg/ml) and subsequent evaluation by flow cytometry. Cell via- bility of freshly isolated or cultured cells (24 h with 10 mg/ml IgA1 protease) was .95% in all experiments. Cell Culture and Stimulation of Cytokine Release. All cell cul- tures were performed in RPMI 1640 supplemented with 20 nM 1050 IgA1 Protease Stimulates Cytokine Production