Skin Dendritic Cells in Murine Cutaneous Leishmaniasis

Immunobiol. (2001) 204, pp. 590 – 594 © 2001 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol

1 Dermatology Branch, National Cancer Institute and 2Laboratory of Parasititic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD USA, and 3 Department of Dermatology, Johannes Gutenberg University, Mainz, Germany

Skin Dendritic Cells in Murine Cutaneous Leishmaniasis MARK C. UDEY 1, ESTHER VON STEBUT 3, SUSANA MENDEZ 2, DAVID L. SACKS 2, and YASMINE BELKAID 2

Abstract Studies of the immunopathogenesis of Leishmania major-induced murine cutaneous leishmaniasis provide a framework for understanding the evolution of L. major infection of skin in humans and the foundation for rationale vaccine design. Experiments in which infection is initiated with “supraphysiologic” numbers of parasites clearly identify Th-derived type I cytokines as essential participants in macrophage activation and macrophage nitric oxide production as prerequisite for parasite control. Dendritic cells, rather than macrophages, appear to be responsible for L. major-specific Th priming in these studies. Recent studies of murine cutaneous leishmaniasis in a model system in which infection is initiated with lower, more physiologic numbers of parasites confirm many of the important findings obtained in “high dose” inoculation models, but important differences have been noted. The low dose inoculation model should ultimately provide insights into mechanisms that are responsible for dendritic cell recruitment into leishmania lesions, mechanisms that facilitate parasite acquisition by skin dendritic cells and cellular interactions that eventuate in T cell priming and lesion involution.

Cutaneous leishmaniasis results when female sandflies disgorge flagellated L. major promastigotes into the dermis of humans or mice during a blood meal (1). Experiments in mice (and supporting studies in humans) indicate that parasites that are introduced into the dermis are rapidly and efficiently taken up by macrophages where they transform into amastigotes with proliferative potential within hours. Infection of macrophages with L. major does not result in the elaboration of proinflammatory cytokines, and the response of parasitized cells to strong proinflammatory stimuli such as endotoxin is attenuated (2, 3). In the absence of T cell-derived IFN-g, infected macrophages are not able to kill L. major and parasite accumulation occurs. Engagement of adaptive immune mechanisms that result in the elaboration of type I cytokines by T cells, arming of macrophages and parasite control or eradication likely depends on active participation by dendritic cells (DC). Langerhans’ cells (LC), the epidermal contingent of the dendritic cell lineage, take up L. major (4) and have been implicated as relevant antigen presenting cells in cutaneous

Abbreviations: DC = dendritic cells; LC = Langerhans’ cells; L. major = Leishmania major 0171-2985/01/204/05-590 $ 15.00/0

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leishmaniasis. Interestingly, and in contrast to what is observed with macrophages, LC and LC-like cells propagated in vitro preferentially internalize amastigotes (4, 5). Internalization of amastigotes by LC-like DC is associated with expression of increased cell surface levels of MHC class I and II antigens and costimulatory molecules, and elaboration of IL-12 p40 (5). Preferential ingestion of amastigotes is not observed with peripheral blood monocyte-derived DC (6), perhaps reflecting a species difference (human vs. mouse) or the extent to which these cells incompletely model LC. DC that harbor parasites and/or L. major antigens have been recovered from lymph nodes draining L. majorinfected murine skin (7, 8). In addition, LC or LC-like cells loaded with L. major antigens or amastigotes restimulate L. major-specific T cells (8, 9) and effectively vaccinate susceptible (BALB/c) mice against infection (10, 11). These data are compatible with the following scenario leading to T cell priming in cutaneous leishmaniasis. Macrophages are initially parasitized after inoculation of L. major into the dermis. Extracellular amastigotes that accumulate in lesional tissue encounter LC that are trafficking through dermis constitutively, or that are recruited by chemokinedependent mechanisms that are incompletely defined, and are ingested and transported to draining lymph nodes. In the lymph nodes, L. major-derived antigens are presented to naïve T cells by LC or by DC that acquire parasites or L. major antigens from LC. Priming and expansion of IFN-g-producing T cells eventuates in lesion involution after macrophage killing of parasites is enabled. CD4 priming, CD40:CD40L interactions, IL-12 and IFN-g production and iNOS-dependent nitric oxide synthesis are all required for disease control in experimental cutaneous leishmaniasis in mice (12). Results described thus far were obtained in models in which (usually) C57BL/6 mice were inoculated with 106 (or more) parasites and disease progression was followed. Inoculae consisted of L. major promastigotes that were propagated in vitro and (generally) introduced into the subcutis of footpads. An alternative model employs inoculation of small numbers (∼100) of highly infectious metacyclic promastigotes into ear dermis (13). Results obtained with this low dose model, which may more closely recapitulate natural infection, confirm some of the results obtained in the high dose model, but also offer several new insights that may be relevant to understanding DC involvement in naturally occuring cutaneous leishmaniasis. The most striking feature of the low dose model is a prolonged “silent phase” in which organisms proliferated in skin in the absence of an inflammatory response (14). Parasite numbers increased ~1000-fold in the first 4–5 weeks after inoculation and begin to decline coincident with appearance of clinically identifiable lesions. Lesion development reflected a dramatic accumulation of macrophages and neutrophils and more modest increases in tissue dendritic cell and T cell numbers. Interestingly, numbers of lesional DC increased as cutaneous lesions involuted. Initial increases in parasite burden and early phases of lesion development were indistinguishable in wild type mice, anti-CD4-treated mice, SCID mice and C57BL/6 mice deficient in IL-12 p40, IFN-g, iNOS and IL-4. Parasite accumulation and lesion development was somewhat delayed in CD40L-deficient C57BL/6 animals. Lesion involution in both the low dose and high dose models required CD4 T cells and the ability of infected animals to synthesize IL-12, IFN-g, CD40L and iNOS. Quantitation of cells containing type I cytokines in epidermis overlying low-dose inoculation sites revealed the accumulation of IL-12 and IFN-g-producing cells in conjunction with lesion development, and L. major-specific IFN-g-producing T cells appeared in draining lymph nodes in parallel (14). Although the identity of the intraepidermal IL-12-

592 · M. C. UDEY et al. producing cells was not established with certainty, this population probably included LC and perhaps other cutaneous DC. Previous studies implicated TNF-a (15), IL-1 (15), MIP-1a (15, 16) and MCP-1 (17) in the recruitment of LC/DC into leishmania lesions. Because these studies utilized the “high dose” inoculation protocol, it will be important to verify that the same cytokines are involved in recruitment of DC into skin injected with limited numbers of parasites. In a recent study, the efficacy of protein- and DNA-based vaccines was tested in susceptible (BALB/c) and resistant (C57BL/6) mice challenged in the dermis with low doses of L. major (18). Administration of killed promastigotes in combination with IL-12 twelve weeks prior to challenge led to incomplete protection in C57BL/6 mice and vaccinated BALB/c mice were unable to control infection. In contrast, a triavalent plasmidbased genetic vaccine resulted in durable protection of both susceptible and resistant mice. Protection correlated with development of anti-L. major delayed type hypersensitivity in the skin of vaccinated mice, recruitment of CD8 T cells into lesions and accumulation of L. major-reactive IFN-g-producing CD8 cells, but not IFN-g-producing CD4 cells, in draining lymph nodes of vaccinated and challenged animals. Significantly, even larger numbers of IFN-g-producing CD8 cells were identified in C57BL/6 mice that spontaneously recovered from L. major infection. These findings are interesting in light of results obtained in earlier studies of the role that IFN-g-producing CD8 cells play in the immunophysiology of genetic vaccination against experimental cutaneous leishmaniasis resulting from high dose inoculation of BALB/c mice (19). Administration of anti-CD8 mAb to recipients of plasmid DNA encoding the LACK antigen at the time of vaccination or later at the time of challenge abrogated vaccine effectiveness. A subsequent study indicated that depletion of CD8 cells compromised the development of Th1-cells in LACK DNA vaccinated mice (20). This result, when interpreted in conjunction with previous results indicating that perforindeficient C57BL/6 mice are resistant to cutaneous leishmaniasis (21), suggests that CD8 T cells are functioning as regulatory cells rather than cytolytic cells and that CD8-derived IFN-g may be critically important. Although the precise role that CD8 cells play in the evolution of leishmania lesions in C57BL/6 mice remains to be determined, their accumulation in lesional skin and lymph nodes in healed mice clearly indicates that priming has occurred (18). Since CD8 priming does not seem to occur in mice treated with protein antigen (heat-killed L. major) (18), antigens derived from viable parasites appear to be metabolized differently than those in preparations of killed organisms. One possibility is that MHC class I molecules are loaded with relevant peptides via mechanisms that are responsible for “cross-priming” and that amastigotes are acquired by DC and metabolized like apoptotic cells or cell fragments. Alternatively, L. major-derived antigens may be shuttled from the parasitophorous vacuoles to the cytoplasm of dendritic cells and thus gain access to relevant antigen processing machinery (22). In summary, experimental cutaneous leishmaniasis initiated in mice by introduction of small numbers of highly infectious organisms into the dermis differs in important ways from that which follows inoculation with large numbers of parasites. The low-dose model may be of considerable use in identifying chemokines and other mediators that are responsible for the initial recruitment of inflammatory cells, including dendritic cells, into challenge sites prior to the onset of an inflammatory response. Studies in this model also suggest that CD8 cells play an important role in the immunopathophysiology of

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