Immunology

Great strides have been made toward understanding the immunology of leishmanial infections during the past two decades (Locksley and Louis, 1992; Reiner and Locksley, 1993; Scharton-Kersten and Scott, 1995). The life cycle has been reproduced entirely in vitro, genetically susceptible and resistant inbred strains of mice have been used to explore the immunogenetics of murine infection, and the immunology of human disease has been studied in a number of endemic areas. The findings indicate that spontaneous resolution of leishmanial infection and protection against reinfection are controlled by cell-mediated immune responses.

Leishmania in Macrophages

The interactions of promastigotes with mono-nuclear phagocytes have been characterized in vitro using axenically cultured promastigotes and murine peritoneal or human monocyte-derived macrophages (Wilson et al., 1992) (Figure 13.3). Promastigotes can attach to several macrophage receptors, including the mannose-fucose receptor, the complement receptor for C3bi (CR3) (Blackwell et al., 1985; Wozencraft et al., 1986; Wilson et al., 1992) and the receptor for advanced glycosylation end-products (Russel and Wilhelm, 1986). Two parasite surface molecules are known to be involved in attachment; a 63 kDa neutral protease (GP63) and a lipopho-sphoglycan (LPG). The glycosylation of these molecules is dependent on the Leishmania sp. and the stage of promastigote development. In the presence of serum, complement is activated by either the alternative or the classical pathway, depending on the Leishmania sp., and promasti-gotes are opsonized for binding to CR1 or CR3. With metacyclic promastigotes, complement activation occurs at a distance from the cell membrane, the membrane attack complex (C5b-C9) is not inserted and the parasites are not lysed (Puentes et al., 1990). Phagocytosis by macrophages follows attachment. Once inside a parasitophorous vacuole, promastigotes convert to amastigotes and lysosomes fuse with the vacuole. Amastigotes are adapted to survival under acidic conditions.

In landmark studies Murray et al. (1983) demonstrated that interferon-y (IFNy) could activate human monocyte-derived macrophages to kill intracellular Leishmania. Oxidative

Wright Giemsa Monocytes

Fig. 13.4 Leishmania sp. amastigotes (stained with Wright-Giemsa) are seen in a pulmonary macrophage in a cytocentrifuge preparation of pleural fluid from a patient with AIDS, fever and a pleural effusion. The patient had traveled extensively in the Mediterranean littoral (Chenoweth et al., 1993). Photograph by courtesy of David M. Markovitz MD

Fig. 13.4 Leishmania sp. amastigotes (stained with Wright-Giemsa) are seen in a pulmonary macrophage in a cytocentrifuge preparation of pleural fluid from a patient with AIDS, fever and a pleural effusion. The patient had traveled extensively in the Mediterranean littoral (Chenoweth et al., 1993). Photograph by courtesy of David M. Markovitz MD

microbicidal mechanisms were initially thought to be responsible, but subsequent studies with murine macrophages have pointed to the importance of the L-arginine-dependent nitric oxide microbicidal system. This results from tumor necrosis factor-a (TNFa)-dependent sustained induction of nitric oxide synthase (iNOS) by IFNy, resulting in the production of large amounts of nitric oxide and associated metabolites that are lethal for amastigotes (Green et al., 1990; Liew et al., 1991). Anti-leishmanial microbicidal mechanisms can also be activated in an antigen-specific manner by direct contact with CD4+ cells bearing surface-bound TNFa (Sypek and Wyler, 1991).

Cell-mediated Immune Responses in the Murine Model

The activation of macrophages to kill intra-cellular amastigotes is dependent on complex and only partially understood cell-mediated immune responses. Early studies in rodents revealed that ablation of T helper cells by radiation or chemotherapy resulted in progressive disease in animals that were otherwise capable of mounting protective immune responses against Leishmania. Immunity was restored by the transfer of syngeneic lymphocytes from immune animals. Anti-leishmanial antibodies were not protective.

Bradley, Blackwell and colleagues observed that inbred strains of mice differ in their susceptibility to Leishmania spp. (Plant et al., 1982; Blackwell et al., 1994). They found that the murine Nramp (natural-resistance-associated macrophage protein) locus on chromosome 1, previously known as LshjltyjBcg, governed susceptibility to L. (L.) donovani. Mice that were homozygous for the sensitive allele developed large parasite burdens, while those that were heterozygous or homozygous for the resistant allele spontaneously cleared infection. Among susceptible mice, alleles at H-2 loci determined whether the strain was able to reduce the parasite burden later in infection. The Nramp locus has pleiotrophic effects. It controls priming and activation of macrophages for antimicrobial activity and the differential expression of the early response gene KC. Low levels of nitric oxide synthase are involved in the signal trans-duction that controls Nramp expression. The murine model of L. (L.) donovani infection is limited in that susceptible mice do not develop signs of visceral disease. In addition, there does not appear to be an association between Nramp 1 and the development of human visceral leishma-niasis (Blackwell et al., 1997).

Leishmania (L.) major has emerged as the major model for visceralizing Leishmania infection. It disseminates to lymph nodes, liver and spleen in BALB/c and other susceptible strains of mice, which have been extensively studied (Moll and Mitchell, 1988; Locksley and Louis, 1992; Reiner and Locksley, 1993; Scharton-Kersten and Scott, 1995; Noben-Trauth et al., 1996; Scott and Farrel, 1998). In the case of L. (L.) major, multiple loci other than Nramp govern susceptibility to infection.

The outcome of L. (L.) major infection in mice is dependent on complex interactions between potentially protective and disease-enhancing cellmediated immune responses. The identification of morphologically similar, but functionally distinct CD4+ T helper cell populations in mice led to rapid advances. Leishmania-specific Th1 cells were found to dominate in mice with self-resolving infection and correlated with resistance to re-infection. Th2 cells were dominant in mice with progressive disease. Th1 cells from immune mice secreted IFNy and interleukin-2 (IL-2) in response to leishmanial antigens, whereas Th2 cells from mice with progressive infection produced IL-4, IL-5 and IL-10. As previously discussed, IFNy can activate macrophages to kill amastigotes. It can also inhibit expansion of Th2 cells. In contrast, IL-4 and IL-10 can inhibit proliferation of Th1 cells and activation of macrophages by IFNy.

A key question, which has not been fully resolved, is why Th1 responses become dominant in strains of mice with self-resolving infection and Th2 responses in those with progressive disease. mRNAs for Th1 and Th2 cytokines are present early in infection in both susceptible and resistant animals. Analysis of the T-cell receptor repertoires in mice infected with L. (L.) major suggests that the same parasite epitopes can drive Thl responses in immune animals and Th2 responses in animals with progressive disease (Reiner et al., 1993). IL-4 was initially hypothesized to be responsible for inhibiting the development of protective Thl cells in mice with progressive infection (Reiner and Locksley, 1993), but subsequent studies in which the IL-4 gene was knocked out demonstrated that infection progressed even in the absence of IL-4 (Noben-Trauth et al., 1996).

It now appears that macrophages may play a critical role in the development of Thl or Th2 responses. In immune animals, secretion of IL-12 precedes and supports the expansion of Leishmania-specific Thl cells and the production of IFNy (Heinzel et al., 1993, 1995). IL-12 stimulates natural killer (NK) cells, and they in turn produce IFNy, which can activate macrophage microbicidal mechanisms and prime macrophages to produce IL-1 and THFa when they encounter the parasite. The CD40-CD40 ligand signaling process appears to mediate IL-12 secretion (Campbell et al., 1996). In the murine model, CD8+ cytotoxic/suppressor cells may also contribute to protection by secreting IFNy (Murray et al., 1992).

In contrast, transforming growth factor-p (TGFp) stimulates Th2 expansion and inhibits the development of Th1 cells (Barral-Netto, 1992). It has been postulated to play an important role in the progression of disease in susceptible mice. In addition, intracellular amastigotes have been shown to inhibit the secretion of IL-1 and TNFa by infected macrophages and to decrease the expression of HLA Class I and Class II antigens (Reiner et al., 1987). Finally, Leishmania-infected macrophages produce prostaglandins and leukotrienes that may suppress development of protective cellmediated immune responses (Reiner and Malemud, 1985).

It is likely that the manner in which leish-manial antigens are presented by macrophages, dendritic cells or other antigen-presenting cells and the presence of accessory molecules is important. Data from the murine model suggest that different T-cell subsets are activated when leishmanial antigens are presented by B

lymphocytes rather than macrophages (Rossi-Bergmann B et al., 1993). The balance of protective and disease-enhancing immune responses and the outcome of infection are affected by other factors. For example, a small promastigote inoculum was shown to result in the development of protective immune responses in genetically susceptible mice that developed progressive disease when infected with a larger inoculum (Bretscher et al., 1992).

Immune Responses in Humans

The majority of humans infected with L. (L.) donovani or L. (L.) chagasi have asymptomatic or mild, self-resolving infections and manifest Leishmania-specific Thl responses. Their peripheral blood lymphocytes proliferate and secrete IFNy and IL-2 in response to leishmanial antigens in vitro, and most develop delayed-type cutaneous hypersensitivity responses to leish-manial antigens in vivo, as demonstrated by a positive leishmanin (Montenegro) skin test. Antibodies are not protective. In general the antibody titer is inversely correlated with the parasite burden.

Persons with progressive visceral leishmaniasis do not manifest Leishmania-specific Thl responses. Their peripheral blood mononuclear cells neither proliferate nor secrete IFNy in response to leishmanial antigens in vitro, and the leishmanin skin test is non-reactive. mRNA for IL-lO has been demonstrated in the bone marrow of persons with visceral leishmaniasis, and elevated levels have been found in their serum (Karp et al., 1993; Holaday et al., 1993). IL-lO probably plays a role in inhibiting the development of protective T cell responses. The importance of Thl responses in defense against L. (L.) donovani and related species is further supported by the emergence of visceral leish-maniasis as an opportunistic infection among persons with concurrent HIV infection or other forms of T cell suppression.

Persons with chronic cutaneous or mucosal leishmanial lesions show evidence of Thl responses systemically. They have positive leish-manin skin tests, and their peripheral blood mononuclear cells proliferate and produce IFNy in response to leishmanial antigens in vitro. While the systemic immune response is Thl in character, there seems to be a mixture of potentially protective and disease-enhancing cellular elements and cytokines at the site of infection. Biopsies of lesions demonstrate mRNA for both Thl cytokines, including IFNy and IL-2, and Th2 cytokines such as IL-4, IL-5 or IL-lO (Caceres-Dittmar et al., l993; Pirmez et al., l993; Melby et al., l994). The result is a delay in eradication of amastigotes and the persistence of the lesions. Eventually, cutaneous lesions heal, and persons are left with high-level immunity against the infecting Leishmania sp.

Individuals with diffuse cutaneous leishmania-sis, like those with visceral leishmaniasis, have negative leishmanin skin tests and their lymphocytes fail to respond to leishmanial antigens in vitro. Recent attention has focused on the potential role of chemokines, the mediators that attract phagocytes and lymphocytes to lesions, in the human immune response. Monocyte chemo-attractant protein-l is secreted by persons with localized cutaneous leishmaniasis, while macrophage inflammatory protein-la is secreted by those with diffuse cutaneous leishmaniasis (Ritter et al., l996). The importance of these chemokines in immunopathogenesis remains to be defined.

In human leishmaniasis, antibodies and T cell responses are directed against an array of leish-manial antigens, and persons who are infected differ in the antigens that they recognize (Evans et al., l989; Jeronimo et al., l995). Several L. (L.) chagasi proteins that elicit T cell responses have been characterized. One is an abundant 7OkDa heat shock protein (HSP7O) (Jeronimo et al., l995). T cells from patients with mucosal leishmaniasis due to L. (V.) braziliensis recognize HSP83 as well as HSP7O (Skeiky et al., l995). It is notable that the cytokine profile of peripheral blood mononuclear cells in response to them represented both Thl and Th2 responses.

These observations and data from murine models illustrate the complexity of immune responses in leishmaniasis and the difficulty in generalizing from one Leishmania sp. to another or from animals to humans. Nonetheless, a great deal has been learned about the cell-mediated immune responses that determine the outcome of leishmanial infection, and it is likely that unifying explanations will emerge in time and become the basis for the design of effective approaches to immunoprophylaxis.

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