Statistical Analyses. The nonparametric Wilcoxon rank test was performed for comparison of cytokine data of PHA-, LPS-, and IgA1 protease–stimulated PBMCs. Statistical differences were considered significant at P , 0.01. Data from separate experiments were expressed as means 6 SEM and were evaluated for statistical significance by Student’s t test. Data from a single representative experiment are presented as means 6 SD obtained by triplicate determination. Results Induction of Proinflammatory Cytokine Release from Human PBMCs by IgA1 Protease. To assess the production of the proinflammatory cytokines TNF-a, IL-1b, IL-6, and IL-8, PBMCs from healthy human donors were incubated with purified IgA1 protease. LPS, PHA, and medium alone were used as control. Initial experiments showed that release of proinflammatory cytokines from PBMCs induced by IgA1 protease reached maximal levels between 12 and 24 h (data not shown). Furthermore, a rapid decrease in IgA1 protease– induced cytokine production could be observed after 24 h, and only small amounts of TNF-a were detectable after 72 h. Therefore, in subsequent experiments a single end-point of 18 h culture was chosen that allowed detection of TNF-a and other proinflammatory cytokines. IgA1 protease stimu- lated the release of proinflammatory cytokines by PBMCs in a dose-dependent manner (Fig. 1, and Table I). Significant amounts of TNF-a in the supernatants were already detect- able in the presence of 1 mg/ml IgA1 protease, and highest levels were reached using 10 mg/ml. Similar dose–response patterns were obtained for other proinflammatory cytokines such as IL-1b, IL-6, and the chemokine IL-8. Nonstimu- lated PBMCs produced negligible amounts of TNF-a, IL-1b, and IL-6, but some IL-8, which was probably induced by adherence as previously postulated by Kasahara et al. (40). To determine whether the cytokine induction was due to LPS present in the preparation of the recombinant IgA1 protease from E. coli, we tested the potential of E. coli LPS to induce TNF-a. The endotoxin content present in the protease preparation of 10 mg/ml did not exceed 20 pg/ml. As shown in Fig. 1, no significant cytokine production compared with the control was detected at 10–30 pg/ml LPS, indicating that endotoxin contamination was not the cause of cytokine induction by purified IgA1 protease. TNF-a synthesis in response to 10 mg/ml IgA1 protease reached the level synthesized by cells stimulated with a much higher dose of E. coli LPS (30 ng/ml). In subsequent experiments, low dose (30 pg/ml) and high dose (30 ng/ml) controls of LPS were always included. The cytokine release of PBMCs showed similar varia- tions between different donors upon stimulation with either IgA1 protease, LPS, or PHA. As shown in Fig. 2 (A–D), the median levels of the proinflammatory cytokines detected in the presence of 10 mg/ml IgA1 protease were approximately similar to those found after LPS stimulation and sometimes even higher than those incubated with the lectin PHA. Heat denaturation abolished the cytokine-inducing ca- pacity of IgA1 protease (data not shown). To test whether the Figure 2. Comparison of cytokine production by PBMCs from differ- ent donors. PBMCs were cultured under low endotoxin conditions (,10 pg/ml as determined by the Limulus amebocyte lysate [LAL] assay). The indicated stimuli were added to the medium at the following concentra- tions: LPS, 30 ng/ml; PHA, 5 mg/ml; and IgA1 protease, 10 mg/ml. The cell-free supernatants were harvested after 18 h, pooled, and analyzed for the indicated cytokines by ELISA. Plots include data from n 5 15 (for TNF-a [A] and IL-6 [C]) or n 5 12 (for IL-1b [B] and IL-8 [D]) inde- pendent experiments. Boxes represent second and third quartiles; the lines within each box are the median values; the error bars represent 5 and 95% confidence. To compare cytokine levels in the different stimulation groups, the nonparametric Wilcoxon rank test was used. Results are con- sidered as significantly different from values obtained with medium alone (*P , 0.05) and as significantly different from PHA (**P , 0.01). 1052 IgA1 Protease Stimulates Cytokine Production