We herein show that various pro-inflammatory molecules can generate distinct activation profiles in DC and that members of the IL-1 family of proteins play a biologically relevant role in the regulation of the functional differentiation of DC.
Multiple activation signals appear to be required to induce the production of high levels of cytokines by DC. The production of IL-12 which is a pivotal event in the development of type-1 cellular immunity is effectively induced in vitro
by treating DC with CD40L+IL-1β and even more so by CD40L+IL-1β+IFN-γ, in agreement with recent findings by Luft et al. [15
]. Such synergistic concept is consistent with in vivo
studies showing the dependence on both microbial signals and endogenous CD40L to induce effective immunity [13
]. Exposure to multiple pro-inflammatory mediators is likely to occur under biological circumstances as these mediators can be transcriptionally induced in a contemporary manner and are regulated by autocrine loops, commonly described in inflammation [24
]. In combination with CD40L, IL-1 induces the production of IL-12, IL-1, IFN-γ and IL-18 in DC. IL-18, acting in combination with IL-12 could induce large amounts of IFN-γ by lymphocytes, further sensitizing DC to produce extremely high levels of IL-12. The role played by DC-derived IFN-γ remains unclear. Neutralization studies suggest that IFN-γ is not responsible for IL-12 production in response to IL-1β and CD40L [14
]. As IFN-γ is produced approximately 24 hours after stimulation it seems that its role would be to contribute to T cell activation rather than to DC activation itself. DC-derived IFN-γ has been reported in the mouse [21
], albeit under distinct activating conditions. Based on these studies it remains to be tested if IFN-γ production by human DC might also be indirectly due to the presence of IL-12 and IL-18.
Here, we report distinct pro-inflammatory stimuli that all induce high levels of IL-12 but appear to provide otherwise distinct responses. In contrast to IL-1, co-stimulation with IFN-γ induced relatively little IL-10, IL-6, IL-18 and IFN-γ. LPS induced higher IL-10, higher IL-1Ra and strikingly less IL-23. The difference in the ability to produce IL-10 was confirmed at the protein level (data not shown and [14
]). While IL-12 is important for naïve CD4+
T cell priming, IL-23 is reportedly important for memory CD4+
]. Differences between IL-12 and IL-23 production suggest that DC may display distinct cytokine profiles that correspond to distinct phases of an immune response. Another interpretation is that DC may interpret distinct pathogen signals, for instance IL-1 or LPS, to mount different types of cytokine reponses [19
]. This would be desirable to optimize host defense against particular aggressions or to be effective in particular tissues. The biological consequences of these differences will have to be determined in future studies.
An important aspect of the autocrine regulation of cytokines in DC is the production of negative regulators such as IL1Ra. Genetic invalidation of IL-1Ra not only up-regulates inflammation but also affects the immune system, causing spontaneous development of auto-immune arthritis in certain strains of mice and rendering other strains more susceptible to immunogens causing this disease [27
]. The precise mechanisms underlying the role of IL-1Ra in immune responses are complex, but implicate antigen presentation. APCs of mice lacking IL-1Ra induce higher proliferation and higher activation of wild-type CD4+
T cells than control APCs, resulting in the up-regulation of CD40L, OX40 and IL-2Rα, and thus have the opposite effects of APCs lacking IL-1 [28
]. Our own data provide a potential mechanistic explanation clearly showing for the first time that IL-1Ra can have a direct role on APC function by regulating the secretion of IL-12. Neutralization experiments in DC showed that IL-1Ra inhibited the secretion of IL-12 in response to CD40L. This is most likely through negative regulation of endogenous IL-1 responses as well as CD40L-induced IL-1 in DC. While the RPA technique did not reveal IL-1 transcripts in unstimulated immature DC, it has been reported that IL-1β mRNA are detected by the more sensitive RT-PCR analysis in freshly-isolated Langerhans cells, generally considered a paradigm for unstimulated immature DC [29
]. Furthermore, direct evidence of IL-1β protein secretion has been shown in monocyte-derived DC [15
]. This demonstrates that autocrine cytokine regulation via IL-1 family members is a biologically relevant mechanism in DC. As IL-1Ra appears to be a negative regulator of DC activation, it is notable that the LPS+IFN-γ stimulation induces the highest levels of IL-1Ra mRNA in DC. As high levels of IL-1α, IL-1β are also induced, IL-1Ra may provide a negative balance to prevent the establishment of an IL-1-mediated autocrine stimulation cascade and to curtail the effects of LPS. Thus, IL-1Ra may be one significant built-in mechanism in the APC to regulate its immuno-stimulatory properties and to prevent the development of uncontrolled immunity. The biological relevance of these conclusions seems further supported by genetic analyses studies in humans. Polymorphisms in the IL-1Ra gene such as the IL1RN*2 allele cause quantitative differences in both IL-1Ra and IL-1β production and associate with the severity of inflammatory and auto-immune diseases such as ulcerative colitis and Crohn's disease, lupus erythematosus and possibly with coronary artery disease. Conversely, these polymorphisms negatively associate with various infections such as vaginal colonization with mycoplasmas, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and with the occurrence of ovarian cancer (recently reviewed in [30
Our results therefore identify IL-1 as an effective co-activator of DC in conjunction with CD40L. This is consistent with the fact that IL-1 has been recognized as an adjuvant for almost 2 decades [31
] and may refine our understanding of the effects of IL-1 in the context of DC-initiated immunity. Thus, IL-1 which is produced locally and systemically in response to inflammation or infection may play an important role as a T cell-independent factor complementing the effects of T cell-derived CD40L. Upon encountering these two molecules, DC would become more effective APCs, producing IL-12, IL-23, IL-18 and IFN-γ and stimulating the differentiation of CD4+
T cells. Besides implications in inflammatory, infectious or auto-immune diseases, our data are also consistent with the identification of IL-1 as an important adjuvant of CTL immunization in vivo
in conjunction with IL-12, IL-18 or IFN-γ [32
]. Like IL-1, IL-12 is not critically required for the generation of CTL, yet the addition of exogenous IL-12 or the inhibition of IL-12 respectively improve or inhibit the development of CTL [33
]. Our results confirm that IL-12 is not essential for priming against fluMP since neutralizing Abs do not block the development of specific T cells. However, we show that enhanced production of IL-12 or IL-23 by CD40L+IL-1-activated DC mediates the enhancing effects on the priming of CD8+
T cell responses.