Figure 4. PBLs enhance TNF-a release of monocytes. PBMCs were separated by counterflow elutriation and adherence into monocytes (Mn) and lymphocytes (Ly) as described in Materials and Methods. After 1 h incubation, the autologous lymphocytes were added to the monocytes at a monocyte/lymphocyte ratio of 1:2. Fixed lymphocytes were treated with 2% paraformaldehyde (15 min at 08C). TNF-a levels in the superna- tants were measured 18 h after stimulation with 30 ng/ml LPS, 5 mg/ml PHA, or 10 mg/ml IgA1 protease. with autologous CD31 T lymphocytes at a monocyte/ lymphocyte ratio of 1:4. While T lymphocytes produced no or very low amounts of TNF-a, the monocytes released reduced levels of TNF-a in response to IgA1 protease compared with crude PBMCs. Such reduced TNF-a secre- tion of monocytes was enhanced in the presence of autolo- gous purified T cells (Fig. 5). However, when the two cell populations were separated by a permeable membrane dur- ing the cell culture, the TNF-a release by monocytes was not increased in response to IgA1 protease (data not shown). These results suggest that direct contact between T cells and monocytes is necessary for a maximum of cyto- kine production by monocytes in response to IgA1 protease. It is well known that lymphokines such as GM-CSF or IFN-g can augment cytokine release in monocytes (43, 44). We found increased levels of GM-CSF in PBMC cultures after incubation with IgA1 protease for 18 h, whereas IgA1 protease failed to induce release of IFN-g after that time (Fig. 6, A and B). Very low levels of IFN-g were finally de- tectable after 4 d (data not shown). However, the IFN-g levels in these cultures were always more than 100-fold lower than peak levels of IFN-g in PHA-stimulated PBMC cultures. Furthermore, in the first 4 d of culture, neither cell proliferation nor increase of IL-2 receptor–positive lympho- cytes could be measured after incubation with IgA1 protease (data not shown). These results suggest that viable T cells are required for the cytokine release by monocytes stimulated with IgA1 protease, but unlike PHA, IgA1 protease does not seem to provide a strong activation signal for T lymphocytes. Effect of IgA1 Protease on IL-10 Production by PBMCs. IL-10 is a cytokine with immunosuppressive and antiinflam- matory effects on monocytes. Reportedly, IL-10 synthesis is delayed relative to the synthesis of inflammatory cyto- kines TNF-a, IL-1b, and IL-6 (45). In contrast to the in- duction of proinflammatory cytokines, IgA1 protease failed to induce IL-10, whereas LPS as well as PHA could stimu- late IL-10 production in PBMCs (Fig. 7). Kinetic experiments revealed that IgA1 protease does not induce IL-10 produc- tion in PBMCs within a period up to 96 h. In contrast, cul- ture supernatants of PHA- or LPS-stimulated PBMCs con- tained high IL-10 levels at different time points (data not shown). Figure 5. Purified CD31 T lymphocytes enhance TNF-a production of CD141 monocytes. The autologous CD31 T lymphocytes (T) were added to the monocytes (Mn) at a monocyte/lymphocyte ratio of 1:4. TNF-a levels in the supernatants were measured after 18 h of culture with 10 mg/ml IgA1 protease. The results shown here are mean values from three experiments from different donors 6 SEM (., s, and d). Figure 6. GM-CSF (A) and IFN-g (B) release of PBMCs in response to IgA1 protease (10 mg/ml), PHA (5 mg/ml), and LPS (30 ng/ml). The levels of these cytokines were determined in supernatants after 18 h. The results (means 6 SD of three determinations) were confirmed by three independent experiments. 1054 IgA1 Protease Stimulates Cytokine Production