One can use the term “authentic” for the Mo-DCs described here for several reasons, which have not previously been noted for inflammatory monocytes. The Mo-DCs are dendritic cells in terms of their motility, because they are nonadherent cells that continually form and retract processes in the living state, identical to the probing morphology of DCs in the T cell areas of living lymph nodes (Lindquist et al., 2004
). These Mo-DCs also concentrate in the T cell areas, again a classic feature of DCs and a location that facilitates clonal selection of antigen-specific T cells from the recirculating repertoire. The Mo-DCs are very similar in phenotype to DCs in lymphoid tissues including the loss of markers that were used previously to positively identify inflammatory monocytes in vivo, i.e., Ly6C and Gr-1 antigens, and CD115/c-fms receptor.
Importantly, when Mo-DCs are compared functionally to classical DCs from the same lymph nodes, the former are not only active but can be superior in stimulating the MLR and in presenting protein antigens, administered in vitro and also in vivo prior to testing as presenting cells. A large number of previous emphasis has been placed on the superior cross presenting activity of the CD8+
subset of DCs, but the Mo-DCs we describe can be equal or more effective than CD8+
DCs including for bacteria injected in vivo. Thus Mo-DCs are equivalent in many functional respects to DCs, except they are monocyte-dependent, whereas numerous prior studies show that classical DCs are monocyte-independent (Naik et al., 2006
) (Varol et al., 2007
) and derive from a committed pre-DC in the bone marrow (Liu et al., 2009
). None of these new functional features of Mo-DCs have been described before for monocyte derived cells in various inflammatory conditions.
The finding that permitted our research was the derivation of mAbs to DC-SIGN or CD209a that recognized this lectin in tissue sections, much of which is intracellular in location (Cheong et al., 2010
). The new anti-DC-SIGN/CD209a mAbs allowed us to visualize the LPS-induced mobilization of Mo-DCs in the T cell areas and distinguish them from the resident DCs there. Previously, a combination of CD11b and CD11c markers were used to help identify inflammatory monocytes with some features of DCs (Leon et al., 2007
) (Serbina et al., 2003
) (Nakano et al., 2009
) (Hohl et al., 2009
) (Siddiqui et al., 2010
) (Kool et al., 2008
), but these integrins are not sufficient to permit localization in situ, and the Mo-DCs actually have lower levels of CD11c than classical DCs. Previous isolations also used antibodies to Ly6C or Gr-1, but these markers are lost from the Mo-DCs described here.
While DC-SIGN/CD209a was critical to identify authentic Mo-DCs in vivo, functions for this lectin needs research. We showed for example that DC-SIGN−/−
monocytes become Mo-DCs (marked by MMR/CD206) in the T cell areas, just like WT monocytes, when the mice are given LPS (Figure S2E
). Therefore DC-SIGN seems not to be involved in Mo-DC mobilization and differentiation. Also Mo-DCs cultured from DC-SIGN−/−
mice still present antigens to OT-I and OT-II transgenic T cells comparably to WT (not shown). DC-SIGN/CD209 can play pathogenic roles, either in transmitting infectious agents like HIV and CMV in the case of cultured human Mo-DCs (Geijtenbeek et al., 2000a
) (Halary et al., 2002
), or in transducing inhibitory signals as seen when human DC-SIGN/CD209 interacts with mycobacteria (Geijtenbeek et al., 2003
) (Tailleux et al., 2003
). DC-SIGN/CD209 could also have protective functions for capture and presentation of glycan-modified antigens (Tacken et al., 2005
). Also the pathway described here to mobilize DC-SIGN/CD209a+
DCs could generate new vaccination strategies, given the powerful antigen presentation and immune stimulatory consequences of this full DC differentiation pathway.
We have identified one molecular pathway to produce Mo-DCs in vivo, which is rapid differentiation from blood monocytes upon administration of TLR4 agonists to mice. The classical method to produce DC-SIGN/CD209+
Mo-DCs from human (Romani et al., 1994
) (Sallusto and Lanzavecchia, 1994
) and mouse (Schreurs et al., 1999
) (Agger et al., 2000
) blood monocytes takes several days of culture in GM-CSF and IL-4, but here we show that LPS and LPS+
living and dead bacteria act rapidly within hours. Blood monocytes drop to 20% of their normal levels 6–12 h after i.v. LPS, and at the same time, cells move into lymph nodes and differentiate into DC-SIGN/CD209a+
Mo-DCs. This influx requires CCR7 and CD62L, both expressed by bone marrow and blood monocytes. Amongst the agonists for Toll-like receptors that we studied, only LPS via TLR4 had this capacity to induce Mo-DCs. In spite of hundreds of studies of the response of mice to LPS, this mobilization of antigen presenting cells was not previously appreciated.
To explain the peculiar role of TLR4 agonists, we first examined gene expression for several TLRs. While monocytes expressed many TLRs, only TLR4 increased markedly when monocytes differentiated into Mo-DCs in culture. This was also the case for the CD14 coreceptor for TLR4, which mediates MyD88 independent and Trif dependent, TLR4 signaling (Jiang et al., 2005
). Xu et al have shown previously that GM-CSF/IL-4 derived DCs produce cytokines in response to several agonists, e.g., Pam3Cys and ODN1826 (Xu et al., 2007
), which we found did not mobilize Mo-DCs from monocytes in vivo. However, a key feature of the Mo-DCs that are mobilized by LPS is that they express CD14, which not only proved to be an independent marker for Mo-DCs but was also essential for their generation.
We would like to propose that the mobilization of Mo-DCs described here has two roles. One is part of the innate response to gram negative bacteria and other agents that contain agonists for the TLR4-CD14 complex, although this will require additional studies of the functional properties of MoDCs such as the production of cytokines and chemokines. A second is as a segway to the adaptive immune response. During the TLR4 based response, Mo-DCs increase while classical DCs decrease, so that Mo-DCs become the dominant cell for induction of effective and combined CD4+ and CD8+ T cell immunity, with or without the requirement for bacterial replication in this newly mobilized DC reservoir.