By carefully studying the kinetics and distribution of the innate and adaptive T cell immune response after i.p. injection of OVA in alum, we have uncovered a previously unappreciated role for monocyte-derived DCs in mediating the adjuvant effects of alum on cellular and humoral immunity. This is underscored by the fact that inflammatory monocytes and DCs were attracted to the peritoneum after injection of OVA-alum; that they took up and processed the Ag on their way to the MLNs; that they acquired a functional phenotype of mature DCs once in the LN; and, finally, that removal of CD11c+ DCs abolished T cell proliferation in OVA-alum–immunized mice, an effect that was restored by adoptive transfer of Ly6Chi monocytes.
One of the aspects of our study that allowed us to uncover this new mechanism of action of alum was the assessment of the precise localization where Ag presentation occurred after i.p. injection. The peritoneal route is easily accessible and often used as a site for immunization to test the protective effect of novel vaccines against subsequent infection. The good resorption of drugs from the peritoneal cavity has mislead the immunological community, as it is often assumed that i.p. administration of a protein Ag leads to rapid systemic resorption into the bloodstream, leading to the common notion that i.p. administered Ags are presented by APCs in the spleen, similar to i.v. injected Ags. Therefore, i.p. immunization is often equalled to “systemic immunization,” and investigators studying the immunogenicity of alum have focused on the spleen as a site where immune activation might occur (22
). We show that i.p. injection of the OVA Ag in the right lower quadrant of the peritoneal cavity leads to Ag presentation and Ag-induced T cell proliferation in the MLN and the ipsilateral ILN, but not in the spleen. After T cell divisions over time, it was clear that only after 4 d, when T cells had undergone at least 3–4 divisions, could we detect divided cells in nondraining LNs and spleen, very similar to what we and others described for immunization with Ags via the skin or respiratory mucosa (23
). Therefore, the Ag-specific reactivity that can be measured in the spleen ex vivo is the result of recirculating effector and/or memory cells. This finding does not exclude that there is immune activation occurring in the spleen before day 3–4 of the response. Within 24 h of injection of OVA-alum, there was induction in the spleen of an IL-4–producing Gr1+
myeloid cell, as described before by others 6 d after injection of alum (5
). The induction of divisions in the ipsilateral ILN was unexpected, but was caused by an artifact induced by skin puncture. One important lesson is that ILN nodes should not be taken as “control nondraining nodes,” as is often done because of their easy accessibility for Ags that are injected i.p.
It was less surprising that Ag was presented in the MLN as previous studies in rat, mice, and sheep have shown that the peritoneal cavity has a lymphatic drainage consisting of stomata that cross the diaphragm and drain into the parathymic LN and MLN (35
). The transport of Ag could be either through free-flowing lymph, gaining access to the subcapsular sinus and conduit system of MLNs and thus to resident DCs and to follicular B cells (29
), or it could be mediated by DCs or other APCs that pick up Ag in the peritoneal cavity and migrate to these nodes (38
). Both scenarios might come into play. By visualizing unhydrated LN slides, we could detect a massive amount of fluorescent Ag in the MLN within 2 h after i.p. injection, irrespective of whether alum was added or not, which would never be caused by cell transport alone. Cell-mediated transport by inflammatory Ly6Chigh
monocytes and DCs occurred especially when alum was added.
What is the reason for the dramatic difference in T cell outcome when alum adjuvant is added to an Ag? We demonstrated that in the absence of alum, Ag was presented predominantly by nonmigratory LN-resident DCs that acquired the Ag via afferent lymph, as evidenced in experiments in which these resident DCs were depleted locally in the mediastinal node before OVA administration (). Itano et al. demonstrated that after skin puncture, there is a rapid flux of cell-free Ag from the site of injection to the skin-draining node, leading to T cell divisions in Ag-specific T cells, without generation of T cell effector potential (37
). We speculate that the physiological drainage of the peritoneal cavity through the stomata in the diaphragm also leads to presentation of Ag in a tolerogenic form by immature resident DCs, inducing deletional T cell proliferation (39
). In contrast, when inflammation is induced by alum, there is additional recruitment of inflammatory monocytes and activation of already resident peritoneal DCs that migrate to the LN and arrive as CD11c+
mature cells expressing the necessary costimulatory molecules for naive T cell activation and generation of memory cells (41
). Several groups have recently shown that CCR2+
monocytes are the immediate precursors of inflammatory type DCs, also called “TIP”-DCs under conditions of Listeria monocytogenes
), with an enhanced potential to induce effector T cells (27
). We believe that our data support the notion that alum boosts immunity by inducing these “inflammatory” DCs. When the resident LN DCs were depleted in the MLN using lung application of a selective DC-depleting DT (32
), the induction of T cell division by OVA-alum was not suppressed, whereas when these inflammatory monocytes and DCs were depleted using peritoneal administration of the toxin (31
), almost all T cell division disappeared () and there was no longer any priming for humoral immune responses (). The effects of DC depletion on T cell division were, however, completely restored when we performed an adoptive transfer of bone marrow–derived Ly6C+
monocytes, cells that acquired a DC phenotype after arrival in the MLN. These data suggest that inflammatory DCs are strongly involved in mediating the enhancing effects of alum on adaptive immunity, and also demonstrate that uptake and processing by other APCs is not sufficient for generating immunity in the absence of DCs. This change of function in monocytes could be the result of their phagocytosis of particulate alum particles, as previously shown for phagocytosis of latex beads injected into the peritoneal cavity (26
). One striking feature was that all APCs contained more intracellular Ag when it was emulsified in alum ( and , and not depicted for B cells). Particularly in monocytes, the cells that had internalized Ag demonstrated the shift in CD11c, costimulatory molecules, and MHCII, suggesting that Ag uptake was indeed associated with DC differentiation. Inflammatory monocytes in the peritoneum contained fluorescent Ag by 6 h, whereas the same cells were found in the MLN only by 24 h, suggesting migration to these nodes, a finding that is also supported by adoptive transfer experiments of CD45.2 congenic donors.
As our own in vitro experiments () and experiments by others (20
) did not reveal a direct activation of monocytes and DCs by alum, we hypothesized that an endogenous danger signal might be released after injection of alum in vivo. We measured very high levels of the endogenous danger signal uric acid when alum was injected and more importantly, recruitment of neutrophils, inflammatory monocytes, and T cell activation induced by alum in the mediastinal LN was abolished when uric acid was neutralized by uricase treatment. Uric acid is released by necrotic cells and alum has been shown to induce a considerable degree of necrosis. It is well known that alum injection i.p. leads to cell death and, when injected into muscle alum, leads to myofascitis. The release of uric acid could explain the high degree of neutrophilic inflammation, as well as CXCL1 production, as a very similar response is seen when uric acid is injected i.p. (13
). Moreover, work by others (21
), along with our own unpublished work (unpublished data), demonstrated that alum, like uric acid, activates caspase-1 and leads to the release of IL-1β and -18 (33
). In support of a predominant role for this pathway in activating inflammatory DCs, we found that the alum response was abrogated in mice deficient in the signaling molecule MyD88, involved in transducing signaling from the IL-1 and -18 receptor. What we cannot presently explain, however, is the fact that the humoral immune response measured several weeks after injection of alum is variably dependent on MyD88 and/or IL1 (6
). Although these differences might depend on timing of analysis and contamination or addition of different TLR ligands to alum, it could also be that for induction of humoral responses, IL-1 signaling via Myd88 is redundant, whereas for T cell responses it is crucial (8
Whether uric acid is the only endogenous innate trigger for DC activation remains to be shown, but the fact that uricase was so effective points toward a predominant role for it. Just like uric acid, alum adjuvant can activate several other aspects of innate immunity, including activation of the coagulation and complement cascade, which is known to influence DC function (7
). As alum does not activate bone marrow–derived DCs in vitro, it is tempting to speculate that nonhematopoietic structural cells of the peritoneal cavity might undergo necrosis and subsequently release uric acid, although formal proof of this is lacking. The rapid recruitment of neutrophils and eosinophils within 6 h, along with DCs, could subsequently be responsible for the indirect activation of DCs. Indeed, neutrophils have been shown to activate DCs through CD11b–DC–SIGN interactions, leading to secretion of chemokines and cytokines (48
). Whether eosinophils could perform the same task is unclear at present, but they could certainly represent an early source of Th2-polarizing cytokines, which are necessary for Th2 induction by alum (28
). It has been shown that alum induces a Gr-1+
myeloid population (eosinophils and monocytes) in the spleen 10 d after injection (5
). We did see an increase in Gr1+
cells in the peritoneum and spleen, but not MLN, within 24 h after injection of alum, but do not know at present whether this population could be involved in activation of the monocytes and DCs.
In conclusion, through a series of in vivo experiments, we showed that alum adjuvant promotes adaptive immunity by releasing the endogenous danger signal uric acid, thus inducing the differentiation of nature's adjuvant, the inflammatory DC, from recruited monocytes.