Figure 1. Purified IgA1 protease induces dose-dependent release of TNF-a from PBMCs. On the top axis, LPS concentrations were set ac- cording to the endotoxin content in the IgA1 protease protein on the bot- tom axis. TNF-a synthesis induced by LPS and IgA1 protease is shown by , and d, respectively. PBMCs were cultured for 18 h in the presence of IgA1 protease or LPS. TNF-a was measured in pooled cell-free culture supernatants by a specific ELISA. TNF-a levels in nonstimulated control cells (medium control) did not exceed 0.02 ng/ml. Data were confirmed by three experiments using PBMCs from different donors. l-glutamine, 25 mM Hepes (GIBCO BRL), 500 U/ml penicil- lin, 500 mg/ml streptomycin, and 5% heat-inactivated human AB serum (pooled male serum, ,,1 ng/ml endotoxin; Sigma Chem- ical Co.). This medium is further designated as culture medium. A total of 105 cells was cultured in 200 ml culture medium in round-bottomed 96-well plates at 378C, 5% CO2. MonoMac 6 cells (105 cells/well) were incubated for 24 h in 96-well flat-bot- tomed plates with culture medium. Cell-free supernatants were har- vested after 18 h (150 ml/well, three wells per independent experi- ment), pooled, and frozen in 50 ml aliquots at 2208C until assayed. To investigate the effect of CD31 T lymphocytes on the IgA1 protease–induced cytokine production of CD141 monocytes, 2 3 104 CD141 cells were cultured either with or without CD31 cells keeping the monocyte/lymphocyte ratio constantly at 1:4. A total of 105 PBMCs were cultured as a control with a monocyte content ranging from 10 to 20%. Cytokine ELISAs. The levels of the released cytokines TNF-a, IL-1b, IL-6, IL-8 (R&D Systems), and IFN-g, GM-CSF, and IL-10 (PharMingen) in the culture supernatants were quantified with commercially available sandwich ELISA kits. The assays were performed as recommended by the manufacturer. In brief, polysty- rene microtiter plates were coated with the catching mAb over- night, washed intensively (washing buffer: PBS, 0.02% Tween 20), and blocked with PBS, 5% AB serum. Different dilutions of all sam- ples and the standard cytokine dilutions were assayed in duplicate or triplicate, respectively, with a total volume of 100 ml for 2 h at room temperature. After removing the unbound detecting mAb by washing (10 times), streptavidin-peroxidase solution was added to each well and incubated for 20 min. After 10 washes, the chromo- genic substrate (TMB substrate with H2O2; Kirkegaard & Perry) was added to each well and color development was stopped after 20 min by adding 50 ml 1 M H3PO4. The absorbance of the samples was measured at 450 nm. The cytokine concentrations were deter- mined by extrapolating from the standard curve. The results are ex- pressed as means of triplicates in ng/ml. The detection limits of the ELISA kits were as follows: TNF-a, IL-1b, IL-10, GM-CSF, and IFN-g # 0.01 ng/ml; IL-6 and IL-8, # 0.05 ng/ml. Flow Cytometry. For immunofluorescence staining, we used the following: anti-CD14–PE as a monocytic marker (LeuM3, IgG2a; Becton Dickinson), and FITC-conjugated anti–HLA-DR (B.812.2, IgG2a; Coulter Immunotech) and anti-CD25 for detection of the IL-2 receptor (ACT-1, IgG1; Dako). Control mouse Ig (IgG1- FITC/IgG2a-PE) were obtained from Becton Dickinson. An FITC-conjugated goat anti–mouse F(ab) fragment (Coulter Im- munotech) was used as a secondary antibody. The cells were washed, adjusted to 2 3 105/200 ml in PBS with 0.2% BSA, and incubated with the antibodies for 30 min on ice. After washing, the samples were analyzed on a FACScalibur™ (Becton Dickin- son) using the CELLQuest™ program. The cells were gated due to their forward scatter/side scatter profile. Background levels of immunofluorescence were determined using isotype control Ig. For the detection of IL-2 receptor, cells were first labeled with unconjugated anti-CD25, washed, and then incubated with FITC-conjugated goat anti–mouse F(ab)2 fragment (5 mg/ml). Proliferation Assay. Proliferation assays with PBMCs (105 cells/well in culture medium) were performed at 378C in 96-well round-bottomed plates at a total of 150 ml/well. The cells were cul- tured for various time periods (1–4 d) and pulsed with 18.5 kBq of [3H]thymidine per well. Cells were then harvested on filters, and incorporation of [3H]thymidine was measured using a Micro- beta Scintillation Counter (Wallac Instruments). Unstimulated cells and PHA-stimulated (5 mg/ml) cells were used as controls. Table I. Dose-dependent Cytokine Induction by Purified IgA1 Protease and LPS from E. coli IgA1 protease (mg/ml) LPS (ng/ml) Cytokine* IL-1b IL-6 IL-8 Control‡ ,0.01 0.2 6 0.0 7 6 0.5 0.1 0.02 6 0.0 0.3 6 0.1 15 6 1.9 1.0 0.1 6 0.0 19.5 6 0.5 29.8 6 3.6 3.0 0.31 6 0.02 14.3 6 2.3 33.1 6 1.8 10 0.75 6 0.05 24.5 6 1.7 64.24 6 6.1 0.03 ,0.01 ND 21 6 1.1 30 0.99 6 0.09 26.1 6 2.5 45.3 6 2.6 *Cytokine levels in culture supernatants from PBMCs incubated for 18 h in the presence of different concentrations of IgA1 protease and E. coli LPS. Values are the means 6 SD from triplicate wells. Amounts are given in ng/ml; IL-1b (detection limit 10 pg/ml), and IL-6 and IL-8 (detection limit 50 pg/ml). ‡Culture medium was used as a control. 1051 Lorenzen et al.