Figure 7. IL-10 release by PBMCs stimulated with IgA1 protease (10 mg/ml), LPS (30 ng/ml), or PHA (5 mg/ml) after 18 h incubation. Cul- ture medium alone was used as a control. The means 6 SEM of five in- dependent experiments with PBMCs from five different donors are shown. Discussion IgA1 protease, a putative virulence factor of human pathogenic Neisseriae, was initially discovered as an enzyme that cleaves human IgA1 molecules, thus interfering with the biological functions of IgA1 (10). Here we report on a novel property of neisserial IgA1 protease, the induction of the proinflammatory cytokines TNF-a, IL-1b, and IL-6, and the chemokine IL-8 in PBMCs. The stimulation of cytokine synthesis by IgA1 protease occurred in the nanomolar range and increased in a dose- dependent manner. The level of cytokine release was sub- stantial but varied to some extent depending on the donor. The rapid cytokine induction within 24 h indicates a role of monocytes/macrophages in this process. However, com- pared with PBMCs, significantly lower amounts of TNF-a and IL-8 and practically no IL-6 and IL-1b were elicited by purified monocytes in response to IgA1 protease, whereas LPS induced a strong cytokine secretion that included IL-6 and IL-1b. The monocytic cell line MonoMac 6 also showed a low production of TNF-a, IL-1b, IL-6, and IL-8 in response to IgA1 protease. These results suggest that the presence of lymphocytes is required for full cytokine induc- tion by IgA1 protease. It is known that the activation of monocytes/macrophages and the release of monocyte-derived cytokines can be pro- voked by either direct cell–cell contact with activated anti- gen-specific T cells or secreted lymphokines (42–44, 46). This is consistent with our finding that IgA1 protease– induced TNF-a secretion by monocytes was enhanced by the addition of lymphocytes or CD31 T cells to the cultures. To investigate the role of activated T cells in this process, we measured IFN-g secretion, CD69, and IL-2 receptor ex- pression, representing typical markers of activated T cells. Although CD69 was slightly upregulated, the low number of IL-2 receptor–positive cells suggests that no large T cell population was activated or that only partial activation oc- curred (46; and data not shown). The T cell cytokine IFN-g 1055 Lorenzen et al. can enhance the cytokine expression in monocytes syner- gistically in combination with GM-CSF (43, 44). Although GM-CSF was detected after IgA1 stimulation, the release of IFN-g was low and the addition of anti–IFN-g antibod- ies had no effect on the monokine production (data not shown). However, the fact that cocultivation with T cells separated from the monocytes by a permeable membrane could not enhance the production of proinflammatory cy- tokines indicates that T cells may also participate by a yet unknown contact-dependent mechanism or by nonclassical presentation of antigen (47–49). We also did not find bind- ing of IgA1 protease to T cells, whereas CD141 monocytes were clearly stained with FITC-labeled IgA1 protease (data not shown). The profile of the cytokine response of PBMCs to IgA1 protease shows some similarities to LPS stimulation. Al- though the IgA1 protease was produced as a recombinant protein in E. coli, our preparations contained usually ,2 pg LPS/mg protein. This LPS content could not sufficiently induce the observed cytokine production. Furthermore, ef- fects which are typically triggered by LPS, such as priming of phagocytes for an enhancement of the oxidative burst, could not be confirmed in cells incubated with IgA1 pro- tease (38; and data not shown). On the other hand, LPS does not require T cell engagement, suggesting that IgA1 protease functions by a different mechanism. Both mole- cules can have synergistic effects in vitro (data not shown) and may also act concertedly in vivo. Activated monocytes/ macrophages also produce the antiinflammatory cytokine IL-10. This cytokine mediates important autoregulatory effects by downregulating inflammation, and its maximal production is delayed compared with the initial burst of proinflammatory cytokines (45). IL-10 production was aug- mented by stimulation with LPS or PHA, but not with IgA1 protease (50). Thus, IgA1 protease seems unique in stimu- lating an asymmetric synthesis of proinflammatory cyto- kines from monocytes and fails to induce strong regulatory feedback loops. Besides the neisserial IgA1 protease reported here, other bacterial products have been found to induce proinflamma- tory cytokines. These components have been classified as a separate class of bacterial virulence factors termed modulins (51, 34). Examples include the listeriolysin from Listeria mono- cytogenes (52), the E. coli hemolysin (53), and the streptococ- cal pneumolysin (54). Other proteins with enzymatic activ- ity, such as the urease of Helicobacter pylori (55) and the sphingomyelinase from Staphylococcus aureus (56), also modu- late cytokine production. Only a few modulins, like the an- thrax lethal toxin from Bacillus anthracis (34) and the clostrid- ium toxin from Clostridium difficile (57), act in the attomolar or femtomolar range, while most other modulins including IgA1 protease exhibit their activity in the nanomolar range. Some modulins, such the staphylococcal toxic shock syn- drome toxin 1 (TSST-1) from S. aureus or the erythrogenic exotoxin (ET) from Streptococcus pyogenes possess superanti- genic activity, thereby inducing T cells to proliferate and to produce cytokines (58). IgA1 protease is different from those superantigenic factors because it is not a T cell mitogen.