Pathophysiology of experimental leishmaniasis: pattern of development of metastatic disease in the susceptible host.

A clear understanding of the etiology of the various forms of leishmaniasis will require knowledge of how physiological properties of the parasite and host immunity influence the pattern of development of the disease. Of particular importance are how these factors affect the growth rate of Leishmania spp. at the site of inoculation in the skin, their capacity to disseminate to visceral and distant cutaneous sites, and their capacity to multiply once there. This paper details the pattern of development of disseminated Leishmania major infection in susceptible BALB/c nu/+ and BALB/c nu/nu mice. It was found that the parasite disseminates from the hind footpad to distant cutaneous sites soon after metastatic foci are established in the liver and spleen. Both mononuclear phagocytes and neutrophils may be the vehicles for the transport of the parasite in the blood. Once visceral and cutaneous metastases are established, the parasites in those foci increase in number progressively. L. major has the capacity to multiply at visceral and cutaneous sites at the same rate. Despite the presence of viable parasites in a number of skin sites, cutaneous metastatic lesions developed almost exclusively on the feet and the tail. Furthermore, these lesions appeared to develop preferentially at sites near joints, suggesting that factors other than temperature may influence the development of cutaneous metastatic lesions.     

Reduced numbers of CD4+ suppressor cells with subsequent expansion of CD8+ protective T cells as an explanation for the paradoxical state of enhanced resistance to Leishmania in T-cell deficient BALB/c mice.

Compared to their normal, T-cell competent counterparts, BALB/c mice that have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells (TXB) were found to be resistant to Leishmania major. Even though TXB mice possess less than 30% of the normal number of T cells in their lymphoid organs, they generated a protective immune response that prevented the progressive multiplication of the parasite in the primary cutaneous lesion and its dissemination to distant visceral sites. Studies of TXB mice depleted of residual CD4+ or CD8+ T cells by systemic monoclonal antibody (mAb) treatment showed that this protective immunity depends on a residual, radio-resistant CD8+ T-cell population, and that it develops in the virtual absence of CD4+ T cells. This immune response can be negated by an infusion of CD4+ T cells from normal susceptible donors, provided that the donor cells are infused before the recipients' response is generated. It is therefore apparant that TXB BALB/c mice are more resistant than T-cell competent BALB/c mice because the former mice lack a threshold number of CD4+ suppressor T cells necessary to down-regulate the induction and expansion of CD8+ protective T cells.     

Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major

To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L. major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L. major-betaGAL. BALB/c and C3H mice responded to infection with L. major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection with L. major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL. Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL. Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L. major.     

Susceptibility of germfree or antibiotic-treated adult mice to Cryptosporidium parvum.

Adult mice are more resistant than neonatal mice to intestinal colonization with the protozoan parasite Cryptosporidium parvum. Development of a mature intestinal flora may play a role in this resistance. We compared susceptibilities to colonization with C. parvum in adult conventional mice, adult germfree mice, and adult conventional mice treated with oral antibiotics to deplete the intestinal flora. Germfree mice of both CD1 and BALB/c strains were colonized at day 7 following inoculation with C. parvum oocysts isolated from the feces of an infected, diarrheic calf. Age-matched conventional mice of the same strains were comparatively resistant to colonization. Conventional mice treated with antibiotics remained resistant to colonization. These results suggest that the microflora in the intestine was not the sole determinant of resistance or susceptibility to colonization. The germfree adult mouse as an experimental model of cryptosporidiosis is discussed.     

Ineffective elimination of Leishmania major by inflammatory (MRP14-positive) subtype of monocytic cells

Myeloid-related protein (MRP) 14, an intracellular protein involved in calcium-dependent activation of myeloid cells, presents a differentiation marker for a subtype of macrophages. In experimental leishmaniasis, BALB/c mice succumb to visceral dissemination after infection with L. major, due to a Th2 cell response, while C57Bl/6 mice develop protective immunity associated with a Th1 cell response. We have previously shown that resistance in (C57Bl/6 mice was also associated with a significantly lower percentage of MRP14-positive cells in the infiltrate than in susceptible BALB/c mice. In C57Bl/6 mice, weekly injections of bone marrow (BM) cells enriched with MRP14-positive cells (d1 of culture) did not reverse, but prolonged the course of infection, associated with increased local parasite spread. In BALB/c mice a single dose of an antiphlogistic agent (dexamethasone or lipoxygenase inhibitor) was associated with reduction of infiltrating MRP14-positive cells and also with a decrease of parasite loads in footpads, lymph nodes as well as spleens, and with delayed progression of disease, Double labeling experiments in vitro revealed that at least 43.1% of MRP14-positive mononuclear cells in BM cultures (8h) had phagocytosed parasites after 4 h of co-incubation. Activation by IFN-gamma (20 U/ml) for 24h and 48h did not significantly reduce parasite load in these cells. In contrast, 77.0% of F4/80-positive macrophages (6d of culture) were infected with L. major parasites and these cells responded to activation with IFN-gamma (20 U/ml) with significant reduction of parasite load (25.3%). The protein MRP14 did not have an effect on parasite survival in vitro. Thus, the impaired capability of MRP14-positive cells to kill L. major upon stimulation may be one reason for the adverse course of infection observed with their increased appearance.     

Increased sensitivity of Corynebacterium parvum-treated mice to toxic effects of indomethacin and lipopolysaccharide.

Female BALB/c and C3H/HeJ mice develop increased sensitivity to the toxic effects of indomethacin after injection of nonviable Corynebacterium parvum. The increased sensitivity developed within 4 days of intraperitoneal injection of the organisms and started to resolve 14 days after injection. The development of increased sensitivity was dependent on the quantity of organisms injected and the concentration of indomethacin utilized. The effect was not observed when C. parvum-treated animals were injected with aspirin. C. parvum-treated BALB/c mice also developed increased sensitivity to E. coli lipopolysaccharide (LPS). Although increased sensitivity to LPS and indomethacin paralleled each other in BALB/c mice, the experiments with the LPS-resistant C3H/HeJ mice indicated that the two phenomena could be separated. The pyridine extract residue of C. parvum was as effective as C. parvum whole cells in inducing indomethacin and LPS sensitivity. Therefore, activation of the reticuloendothelial system is probably a critical element in the induction of sensitivity to these agents.     

Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen

BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.     

BCG, in contrast to C. parvum, does not induce increased sensitivity to the toxic effects of indomethacin in mice

Treatment of mice with killed Corynebacterium parvum (also designated Propionibacterium acnes, P. acnes) or Bacille Calmette Guerin (BCG) leads to modification of several of the same host systems. However, BCG, in contrast to C. parvum, did not induce increased sensitivity to the toxic effects of indomethacin in BALB/c or C57B1/6 mice. In addition, treatment of mice with BCG did not interfere with the induction of sensitivity by C. parvum. Therefore C. parvum must uniquely induce changes in host systems which alter the sensitivity to this anti-inflammatory drug. Additional experiments with splenectomized animals revealed that the presence of this organ, which undergoes hypertrophy following C. parvum treatment, was not necessary for the induction of indomethacin sensitivity. Presentation of C. parvum via the subcutaneous route versus the intraperitoneal route revealed that the two routes were equally efficient in inducing sensitivity in C57B1/6 mice but the former route was less effective (50% deaths) than the intraperitoneal route (95% deaths) in BALB/c mice. These results indicate that host related factors (genetic) may be important in the generation of enhanced sensitivity to the toxic effects of indomethacin.     

T-cell responses to immunodominant LACK antigen do not play a critical role in determining susceptibility of BALB/c mice to Leishmania mexicana

Although BALB/c mice develop lesions when infected with Leishmania mexicana, the mechanisms which are responsible for susceptibility to this parasite have not been elucidated. In contrast, susceptibility of BALB/c mice to Leishmania major has been shown to depend on the early production of interleukin-4 (IL-4) by T cells which react to the parasitic LACK antigen. Here, we demonstrate that the lesions induced by L. mexicana are delayed compared to those induced by L. major but rapidly develop at later time points. Interestingly, while LACK-tolerant BALB/c-derived IE-LACK transgenic mice were resistant to L. major, they were susceptible to L. mexicana and developed lesions similar to those observed in wild-type BALB/c mice. The latter result was observed despite the fact that (i) LACK was expressed by L. mexicana, (ii) splenocytes from BALB/c mice were able to stimulate LACK-specific T-cell hybridoma cells when incubated with live L. mexicana promastigotes, and (iii) LACK-specific T cells contributed to IL-4 production in L. mexicana-infected BALB/c mice. Thus, in contrast to what was observed for L. major-infected mice, LACK-specific T cells do not play a critical role in determining susceptibility to L. mexicana. Although BALB/c mice are susceptible to both L. major and L. mexicana, the mechanisms which are responsible for susceptibility to these parasites are likely to be different.     

Infectivity and neutralization of Cryptosporidium parvum sporozoites.

Cryptosporidiosis, a diarrheal disease of calves and humans caused by the coccidian parasite Cryptosporidium parvum, is terminated in hosts with normal immune systems. To assess the mechanisms of immunity in cryptosporidiosis, it is necessary to isolate and quantitate sporozoites, the infective stage of Cryptosporidium spp. Here we report the (i) separation of infective C. parvum oocysts from calf feces by ether extraction, sieving, and hypochlorite treatment; (ii) separation of viable C. parvum sporozoites from intact and excysted oocysts by anion-exchange chromatography; and (iii) quantitation of sporozoite infectivity in vivo by direct intraintestinal injection of isolated sporozoites in 7-day-old BALB/c mice. When isolated sporozoites were incubated with heat-inactivated immune bovine serum, 25 times the 50% infective dose for 7-day-old mice was completely neutralized. Sporozoites incubated with preimmune bovine serum were infectious for 7-day-old mice.     

Membrane glycoprotein M-2 protects against Leishmania amazonensis infection.

Previous passive antibody transfer experiments have indicated that immunity to a 46-kilodalton membrane glycoprotein (M-2) of Leishmania amazonensis may protect against infection with this parasite. In the studies described in this paper, we investigated the ability of the purified M-2 molecule to elicit a protective immune response in conjunction with Freund incomplete and complete adjuvants, saponin, and Corynebacterium parvum. Both relatively susceptible (BALB/c and CBA) and resistant (C57BL/6) strains of mice were examined. C. parvum appeared to be the most effective adjuvant in the three mouse strains tested. The level of protection varied with the mouse strain, although all animals received identical preparations of antigen and adjuvant. Immunization of CBA mice with the M-2 glycoprotein and C. parvum resulted in complete protection against a challenge infection of 10(4) and 10(6) late log-phase promastigotes of L. amazonensis. In the BALB/c strain, complete protection was observed in some of the immunized animals (28 to 50%); in the rest of the mice the onset of infection was significantly delayed. Protective immunity for C57BL/6 mice was observed only at the low infecting dose (10(4) L. amazonensis organisms). The level of protection observed is reflected by increased antibody response (immunoglobulins G1 and G2) developed to the M-2 molecule. The relationship of pure T-cell (nonantibody) immunity to this protection remains to be elucidated.     

Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice.

Adult murine models of Cryptosporidium infection involving Cryptosporidium muris and C. parvum were used to study immunity to cryptosporidiosis in the mammalian host. Immunocompetent BALB/c or C57BL/6 mice developed a highly patent infection with the RN 66 strain of C. muris but overcame the infection and were immune to reinfection. In contrast, severe combined immunodeficiency (SCID) mice or nude mice had a chronic infection lasting at least 109 days. The development of the C. muris infection appeared to be confined to the gastric epithelium in immunocompetent and immunocompromised mice. SCID mice injected intraperitoneally with histocompatible spleen or mesenteric lymph node cells from uninfected BALB/c mice were able to recover from the C. muris infection. The protective effect of donor spleen cells was not reduced by depletion of the B cell population but was significantly reduced by depletion of Thy.1 cells. Treatment of C57BL/6 or BALB/c mice during infection with a gamma interferon-neutralizing monoclonal antibody, but not a tumor necrosis factor-neutralizing monoclonal antibody, resulted in a significant increase in oocyst production. In the C. parvum model, a severe and eventually fatal chronic infection with a cervine isolate was established in SCID mice, with parasitization occurring in the ileum, cecum, and colon. SCID mice injected with unprimed BALB/c spleen cells prior to inoculation of C. parvum oocysts were resistant to infection. These results suggested that the two animal models should be valuable in the study of immunity to cryptosporidial infection.     

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.     

Generalized infection and lack of delayed hypersensitivity in BALB/c mice infected with Leishmania tropica major.

The susceptibility of a few strains of mice to a subcutaneous injection of Leishmania tropica major, the causative agent of cutaneous leishmaniasis in humans, was studied. The infection in six strains (CBA, AKR/J, AKR/cu, C57BL/6, A/J, and C3H) remained cutaneous, and the animals recovered within 3 to 4 months. In contast, the infection in BALB/c became generalized and killed 1005 of infected animals. Intraperitoneal injection of infected liver of BALB/c to A/J and syngeneic mice produced a lethal disease in BALB/c but no infection in A/J mice. Lower doses of the parasite produced a lethal infection in BALB/c but no apparent disease in A/J. Hence, the host rather than the parasite is responsible for the outcome of the disease. The peak antibody titer of BALB/c mice was not significantly higher than that of A/J mice. However, BALB/c failed to show any delayed hypersensitivity to leishmania tested by footpad reaction, whereas A/J mice showed a strong response.     

T cells that react to the immunodominant Leishmania major LACK antigen prevent early dissemination of the parasite in susceptible BALB/c mice

Susceptibility of BALB/c mice to Leishmania major depends on the early production of IL-4 by CD4(+) T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination of L. major to the internal organs.     

IL-10 mediates susceptibility to Leishmania donovani infection

Human visceral leishmaniasis (VL) results in a severe and potentially fatal systemic disease, accompanied by cellular immune depression. The production of IL-10 correlates with ongoing disease and it has been suggested that the cellular immune depression that accompanies active disease may be due to a predominance of IL-10 production rather than a lack of IFN-gamma production, which is essential for optimal macrophage activation and parasite elimination. To examine the role of IL-10 in resistance during L. donovani infection (a causative agent of VL), the course of infection was examined in mice lacking the gene for IL-10. BALB/c IL-10-/-, as well as C57BL/6 IL-10-/- mice, were highly resistant to L. donovani infection, as evidenced by liver parasite burdens which were tenfold lower than those in control mice after 14 days of infection. Enhanced resistance was accompanied by increased production of IFN-gamma and nitric oxide in BALB/c IL-10-/- mice. Susceptibility to infection in BALB/c IL-10-/- mice was enhanced following in vivo treatment with a neutralizing antibody to IFN-gamma or IL-12. Together these studies demonstrate for the first time that IL-10 is a critical component of the immune response that inhibits resistance to L. donovani.     

Leishmania major induces distinct neutrophil phenotypes in mice that are resistant or susceptible to infection

Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen-specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c-inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL-12p70 and IL-10. In contrast, L. major-infected BALB/c PMN transcribed and secreted high levels of IL-12p40 but did not secrete biologically active IL-12p70. Furthermore, IL-12p40 was shown not to associate with IL-23 p19 but formed IL-12p40 homodimers with inhibitory activity. No IL-10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL-12, and in BALB/c mice, secretion of IL-12p40 could contribute to impaired, early IL-12 signaling. These distinct PMN phenotypes may thus influence the development of L. major-specific immune response.     

MurineTaenia crassiceps cysticercosis: H-2 complex and sex influence on susceptibility

Several inbred strains of mice were infected by intraperitoneal injection of tenTaenia crassiceps cysticerci per mouse. Genes linked with the major histocompatibility complex (H-2) were found to influence parasite growth greatly, as demonstrated by the different parasite loads of H-2 congenic mice with BALB background: BALB/c (H-2d) mice were the most susceptible, whereas BALB/k (H-2k) and BALB/b (H-2b) animals were comparatively resistant. Non-H-2 genes had no significant effect on susceptibility in H-2d strains, as reflected by the similar parasite loads in BALB/c, DBA/2, and (BALB/cxDBA/2)F1 mice. Using the H-2b (BALB/b, C57BL/6J) and H-2k (C3H/HeJ, BALB/k, and C3HeB/FeJ) strains, we found that non-H-2 background genes caused a small but significant influence on parasite load. A recombinant mouse strain alleles (K^k, I^k, S^d, D^d) was also susceptible, indicating that S and/or D regions of the H-2d complex are probably involved in the control of resistance to murine cysticercosis. Females of all mouse strains were more susceptible than males. The same effects were observed for H-2 genes and sex, with two strains ofT. crassiceps differing in their rate of growth.