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The most widely studied model for cutaneous leishmaniasis is infection of mice with Leishmania major, wherein it has been observed that certain mice (e.g., C3H) develop a parasite-specific Th1 response (high levels of gamma interferon [IFN-] and low levels of interleukin-4 [IL-4]), which often is associated with cure, while other mice (BALB/c) develop a Th2 response (low levels of IFN- and high levels of IL-4), which leads to disease progression (reviewed in references 4, 8, 13, 14, and 21). In contrast, although Leishmania braziliensis induces a disease that is a serious health problem in South America, relatively little experimental work has been done to characterize the immune response to this parasite, probably because the parasite is weakly infectious for mice (6, 12). To examine why L. braziliensis is weakly infectious, we infected the same mouse strain (BALB/c) with either L. major (which kills BALB/c mice) or L. braziliensis (which BALB/c mice kill) and compared the development of cutaneous lesions and the immune responses to the two species of parasites.

The course of cutaneous lesion development following infection with L. major or L. braziliensis in BALB/c mice. BALB/cBy mice (National Cancer Institute, Frederick, Md.) were injected with either 10⁶ L. major (R/SU/59/Neal P) (22) or 10⁷ L. braziliensis (MAN/BR/LTB-111) (10) organisms in a hind footpad. Because L. braziliensis is weakly infectious for mice, a dose of 10⁷ parasites was required to obtain consistent results in the assay systems used. On the other hand, because L. major is quite infectious for BALB/c mice, a lower dose of L. major (10⁶ organisms) was used; a dose of 10⁷ L. major organisms would have overwhelmed the mice so rapidly that there would not have been sufficient time to compare the courses of infection with the two species of parasites. Using this approach, we found that the majority of both species were killed within the first 3 days of infection. At this time the numbers of parasites remaining in the developing footpad lesions were similar for the two species (parasites were enumerated by limiting-dilution analysis; for techniques, see reference 11): 1.4 × 10⁴ L. major organisms (95% confidence limits, 0.2 × 10⁴ to 2.6 × 10⁴) and 5.6 × 10⁴ L. braziliensis organisms (confidence limits, 0.4 × 10⁴ to 10.7 × 10⁴). Such massive destruction of L. major within the first few days of infection has been reported before (22). Beyond day 3 postinfection, L. major replicated to achieve a 712-fold expansion by day 42 postinfection. In contrast, L. braziliensis doubled its numbers by day 7 postinfection and thereafter was gradually destroyed, so that beyond day 42 postinfection the parasite could not be detected. Cutaneous lesion development directly correlated with lesion (footpad) parasite burden. L. major induced a rapid increase in footpad size, such that by day 42 postinfection, footpad thickness had tripled and lesions had become ulcerated and necrotic (at which point the animals were sacrificed). L. braziliensis induced modest (never more than a 50% increase in footpad thickness) lesions that were nodular and never ulcerated.

Production of Th1- and Th2-associated cytokines by BALB/c mice infected with L. major or L. braziliensis. Cytokines play counteracting roles in the control (e.g., IFN-) or exacerbation (e.g., IL-4) of L. major infection. Therefore, we determined the cytokines produced by lymph node cells taken from BALB/c mice infected with either L. major or L. braziliensis. Three to five mice from each group were killed for evaluation at various times postinfection. Single-cell suspensions were prepared from the lymph nodes draining the lesion, and the cells were placed into culture as described elsewhere (20). To assess IL-4, IL-10, and IFN- production, culture supernatants were harvested 72 h later (a time when peak production of the cytokines was achieved) and analyzed for the presence of the cytokines by using enzyme-linked immunosorbent assays (ELISA) described elsewhere (5, 20). For tumor necrosis factor alpha (TNF-) production, culture supernatants were harvested 6, 24, 48, and 72 h later and analyzed for TNF by ELISA (capture and detection antibodies and recombinant TNF standard were obtained from PharMingen [San Diego, Calif.]).

The course of cutaneous lesion development following infection with L. braziliensis in BALB/c mice treated with either anti-IFN- or anti-IL-4 antibody. Because IL-4 can inhibit the protective effects of IFN- in mice infected with L. major (7, 9), and because L. braziliensis-infected BALB/c mice produced significantly less IL-4 than L. major-infected BALB/c mice (Table 1), we hypothesized that the amount of IFN- produced by L. braziliensis-infected mice might be sufficient to control infection. Therefore, we treated L. braziliensis-infected mice with a neutralizing anti-IFN-.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Course of cutaneous lesion development following infection with L. braziliensis in BALB/c mice treated with anti-IFN- antibody. Each value represents the mean (± standard error of the mean) lesion size of five animals per group. The results are representative of two independent experiments. Control animals were treated with an isotype-matched antibody (1.0 mg of anti--galactosidase [GL113; DNAX]/week) prepared in a fashion identical to that for the anti-IFN- antibody. The asterisk indicates that from day 56 of infection and beyond, there was a significant difference (P < 0.05) in lesion size between the groups as determined by analysis of variance followed by Student-Newman-Keuls posthoc t tests. Numbers of parasites per foot in control and IFN--treated animals are given for day 144. (Inset) BALB/c and C3H mice were infected with 106 L. major organisms and left untreated or treated with anti-IFN-, as indicated.