enzymatic activity is involved in that process, PBMCs were incubated in the presence of 100 mM of a specific IgA1 protease inhibitor (peptide prolyl boronic acid [37]). Al- though at this concentration the inhibitor completely blocked the cleavage of IgA1, it altered neither the LPS- nor the IgA1 protease–induced production of proinflammatory cy- tokines (data not shown). This demonstrates that the native form but not the enzymatic activity of IgA1 protease is re- quired for the induction of proinflammatory cytokines. Cytokine Release Induced by IgA1 Protease Is Augmented in the Presence of T Cells. IgA1 protease triggers a strong re- lease of proinflammatory cytokines from PBMCs after only a short incubation period, indicating that monocytes might be the main producer of these mediators. To determine the source of cytokine release, we cultivated lymphocyte-depleted peripheral blood monocytes (purity varied between 85 and 95%). As demonstrated in Fig. 3, IgA1 protease induced significantly lower amounts of TNF-a, IL-1b, IL-6, and IL-8 compared with LPS, which readily stimulated TNF-a production in purified monocytes. Direct comparison with PBMCs revealed that IgA1 protease–stimulated PBMCs can elicit up to 20-fold higher amounts of these cytokines compared with the respective lymphocyte-depleted mono- cyte cultures, which was particularly true for IL-6 and IL-8 (data not shown; see also Fig. 2). We also tested the mono- cytic cell line, MonoMac 6. This cell line shows phenotypic and functional characteristics of mature blood monocytes and produces IL-1, IL-6, and TNF-a upon stimulation with LPS or PMA (39, 41). Compared with LPS, we found a significantly lower production of these cytokines and IL-8 after stimulation with IgA1 protease (Table II). Direct contact with T cells can augment monocyte cyto- kine production (42). To examine the necessity of cell contact for TNF-a release in the different types of stimula- tion, monocytes stimulated with IgA1 protease, LPS, or PHA were incubated with autologous active and fixed (15 min, paraformaldehyde at 08C) lymphocytes at a monocyte/ lymphocyte ratio of 1:2. In parallel, the lymphocytes and monocytes were incubated alone. Such lymphocytes and purified monocytes produced only low levels of TNF-a upon stimulation with 10 mg/ml IgA1 protease (Fig. 4). The same results were obtained after stimulation with PHA. The addition of viable lymphocytes increased the TNF-a secre- tion in nonstimulated, IgA1 protease–, and PHA-treated monocytes, whereas the addition of prefixed lymphocytes did not. Monocytes stimulated with IgA1 protease or PHA also showed a significantly stronger TNF-a induction in the presence of lymphocytes compared with the control. Only LPS was able to induce TNF-a in purified monocytes alone, and this was not further enhanced by lymphocytes. To examine the particular role of T cells for IgA1 pro- tease–induced TNF-a release, monocytes were incubated Table II. Release of Proinflammatory Cytokines in the Monocytic Cell Line, MonoMac 6 Proinflammatory cytokines* induced by: Cytokine‡ Control§ TNF-a IL-1b IL-6 IL-8 ,dl ,dl ,dl 2.02 IgA1 protease (10 mg/ml) 0.03 0.02 0.59 12.21 LPS (30 ng/ml) 0.12 0.06 23.37 84.96 PHA (5 mg/ml) ,dl ,dl ,dl 11.28 PMA (100 ng/ml) 0.29 ND 4.73 67.96 Figure 3. Production of proinflammatory cytokines by purified mono- cytes in medium alone (white bars) and in response to 30 ng/ml LPS (hatched bars) or 10 mg/ml IgA1 protease (black bars). The level of each cytokine in the supernatant of stimulated monocyte cultures (105) was de- termined after 18 h. The results shown here are mean values from three different experiments 6 SEM. *Cytokine concentrations in culture supernatants are given in ng/ml. ,dl, below detection limit. ‡MonoMac 6 cells were incubated at 106 cells/ml for 24 h, and super- natants were analyzed for cytokine production. Values are means from duplicate assays. §Corresponding culture medium was used as a control. 1053 Lorenzen et al.