The involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.     

Leishmania mexicana: effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse

Effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse. Experimental Parasitology 65, 269-276. Symptoms of human leishmaniasis vary greatly, ranging from cryptic infections to cases with fatal sequelae. Factors regulating the severity of the disease are largely undetermined. Malaria coincides geographically with leishmaniasis in many areas and the immunosuppressive effects of malaria are well documented. It is therefore plausible that malaria could enhance the course of concomitant leishmaniasis. Interactions between Leishmania mexicana and Plasmodium yoelii were examined in BALB/c mice. Percentage of blood cells infected with P. yoelii and diameter of footpad lesions caused by L. mexicana were the criteria used to assay for disease severity. L. mexicana and P. yoelii infections were each significantly enhanced in dually infected mice when compared to mice infected with either parasite alone. Mortality rates due to the normally nonlethal P. yoelii were high during concurrent infections.     

Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea

Susceptibility of inbred mouse strains to Metagonimus yokogawai, Metagonimus miyatai, and Metagonimus takahashii infections was studied using BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J mice, with H-2 haplotypes d, s, b, k, and a, respectively. Two hundred metacercariae were orally fed to each mouse, and the worm recovery rates (WRR), worm dimensions, and intrauterine egg numbers were measured at days 3, 7, 14, 21, and 28 postinfection (PI). On day 14 PI, the WRR of M. yokogawai was highest in ddY mice (average, 62.2%); those of M. miyatai and M. takahashii were highest in ddY (19.5%) and BALB/c mice (10.4%), respectively; worm maturation was best in C3H/HeN (M. yokogawai), C57BL/6J (M. miyatai), and ddY mice (M. takahashii). All mouse strains showed higher susceptibility to infection with M. yokogawai than with M. miyatai or M. takahashii. The results show that susceptibility of mice to Metagonimus infection varies according to mouse strain and parasite species but is suggested to be independent of the mouse H-2 haplotype.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.     

Susceptibility of several strains of mice to Echinostoma hortense infection

Susceptibilities of 5 different mice strains, including C3H/HeN, BALB/c, C57BL6, FvB and ICR, to Echinostoma hortense infection, was evaluated. The worm expulsion rate, worm size and egg production were observed from 1 to 8 weeks after infection with 30 metacercariae. C3H/HeN and ICR mice showed the highest worm maturation rates. The worm recovery rate and the number of eggs per gram (EPG) of feces was also higher in C3H/HeN and ICR mice than in BALB/c, C57BL6, and FvB mice. It is suggested that E. hortense is highly infectious to ICR and C3H/HeN mice, but not to the other strains of mice. Based on the results obtained, we believe that the susceptibility of different mouse strains to E. hortense infection is dependent on the genetic and immunologic background of mice.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Influence of long-term treatment with pravastatin on the survival, evolution of cutaneous lesion and weight of animals infected by Leishmania amazonensis

The high toxicity of current drugs for treatment of leishmaniasis is a major hindrance for controlling the disease. Pravastatin is a well-known drug with anti-inflammatory and immunomodulatory properties that may modulate host defense mechanisms against Leishmania. We evaluated the influence of prolonged pravastatin treatment on the survival of Leishmania amazonensis-infected animals (BALB/c, C57BL6 mice and Syrian hamsters), including weekly measurement of cutaneous lesions (footpad thickness) and weight. Pravastatin improved survival of Leishmania-infected BALB/c mice but not of infected C57BL6 mice or hamsters. On the 50th week of follow-up, 71% of pravastatin-treated Leishmania-infected BALB/c mice were alive against 29% of control group (p < 0.01). Low footpad thickness was found on BALB/c pravastatin treated mice from the 14th week (p < 0.05), and 20th week onward for C57BL6 treated mice. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL6 mice and Syrian hamsters, but not infected BALB/c mice. Our results points to beneficial effects of pravastatin on the evolution of the disease in the murine leishmaniasis model.     

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

Therapeutic effect of anti-L3T4 monoclonal antibody GK1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice

The effect of in vivo treatment with anti-L3T4 monoclonal antibody (mAb) on the course of experimentally-induced cutaneous leishmaniasis was studied in genetically susceptible BALB/c mice. Administration of anti-L3T4 mAb resulted in a pronounced therapeutic effect as reflected by both resolution of lesions and a dramatic reduction in the parasite load in treated mice. This effect was specific for anti-L3T4 mAb and correlated with a selective depletion of L3T4+ T cells in the lymphoid tissues of treated mice. Moreover, both the therapeutic effect of anti-L3T4 mAb on cutaneous leishmaniasis and the concurrent depletion of L3T4+ T cells in the lymphoid tissues of treated mice were dependent upon the isotype of the anti-L3T4 mAb employed.     

Prophylactic treatment of BALB/c mice with cyclosporine A and its analog B-5-49 enhances resistance to Leishmania major

The effect of cyclosporine A (Cs A) and its analog B-5-49 on Leishmania major in vitro and in vivo in the highly susceptible BALB/c mouse strain has been investigated. In vitro, both of these drugs showed significant toxicity toward L. major, but only at relatively high levels (greater than 25 micrograms/ml). However, at 5 and 10 micrograms/ml, levels which correspond more closely to physiologically achievable concentrations, no growth-inhibitory effect in vitro was observed. On administration of the drugs to animals with established lesions, no beneficial effect was observed and, in fact, some exacerbation of lesion development and disease progression was noted. Surprisingly, a majority of the mice treated prophylactically with Cs A for a period of 7 consecutive days beginning 1 day before infection with L. major did not develop ulcerated cutaneous lesions, although some footpad swelling was observed 10 days to 2 wk after infection. These resistant animals displayed a sustained DTH after infection, and were resistant to further challenge with virulent L. major. Prophylactic treatment with the B-5-49 analog of Cs A was also effective in enhancing resistance to L. major infection in BALB/c mice, although to a somewhat lesser degree. Because the cyclosporines tested do not appear to be directly toxic nor inhibitory in vivo for established L. major infections, it appears that these drugs may be effective in modulating the induction stage of the immune response toward the parasites in the BALB/c mouse in such a way as to allow a protective immunity to develop.     

Host and parasite genetics shape a link between Trypanosoma cruzi infection dynamics and chronic cardiomyopathy

Host and parasite diversity are suspected to be key factors in Chagas disease pathogenesis. Experimental investigation of underlying mechanisms is hampered by a lack of tools to detect scarce, pleiotropic infection foci. We developed sensitive imaging models to track Trypanosoma cruzi infection dynamics and quantify tissue‐specific parasite loads, with minimal sampling bias. We used this technology to investigate cardiomyopathy caused by highly divergent parasite strains in BALB/c, C3H/HeN and C57BL/6 mice. The gastrointestinal tract was unexpectedly found to be the primary site of chronic infection in all models. Immunosuppression induced expansion of parasite loads in the gut and was followed by widespread dissemination. These data indicate that differential immune control of T. cruzi occurs between tissues and shows that the large intestine and stomach provide permissive niches for active infection. The end‐point frequency of heart‐specific infections ranged from 0% in TcVI‐CLBR‐infected C57BL/6 to 88% in TcI‐JR‐infected C3H/HeN mice. Nevertheless, infection led to fibrotic cardiac pathology in all models. Heart disease severity was associated with the model‐dependent frequency of dissemination outside the gut and inferred cumulative heart‐specific parasite loads. We propose a model of cardiac pathogenesis driven by periodic trafficking of parasites into the heart, occurring at a frequency determined by host and parasite genetics.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.     

Susceptibility of five strains of mice to Babesia microti of human origin.

One outbred (CF1) and four inbred (BALB/c, C57, CBA and C3H) strains of mice were tested for susceptibility to Babesia microti of human origin. Of these, intact C3H mice developed higher parasitemia than all other intact mice, while BALB/c mice developed the highest parasitemia among splenectomized mice. Susceptibility was not related to H-2 haplotype in any obvious way. Because C3H and BALB/c mice developed relatively high initial peak parasitemias, the parasite was serially passaged in both of these mouse strains in an attempt to increase parasite virulence. After 30 passages in BALB/c and 49 passages in C3H mice over a period of 12 months, maximum parasitemias were 50 times higher than those observed initially. After the peak parasitemias of these two mouse-adapted parasites had stabilized, the relationship between onset and level of maximum parasitemia and number of parasites inoculated was determined. With both C3H- and BALB/c-adapted parasites, as inoculum size increased, the time required to reach maximum parasitemia decreased and the level of maximum parasitemia increased. Studies involving infection of either mouse strain with parasites adapted to the heterologous mouse strain indicated that C3H mice were more susceptible than BALB/c mice to homologous or heterologous parasites. These data suggest that the virulence of B. microti to the mouse can be increased by prolonged passage in this host. Once adaptation to this host species has occurred, virulence appears to be more dependent on the innate susceptibility of the mouse strain than on adaptation of the parasites to a particular strain of mouse.     

Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response

Host resistance to the intracellular protozoan Leishmania major is highly dependent on IL-12 production by APCs. Genetically resistant C57BL/6 mice develop IL-12-mediated Th1 immune response dominated by IFN-gamma and exhibit only small cutaneous lesions that resolve spontaneously. In contrast, because of several genetic differences, BALB/c mice develop an IL-4-mediated Th2 immune response and a chronic mutilating disease. Myeloid differentiation marker 88 (MyD88) is an adaptator protein that links the IL-1/Toll-like receptor family to IL-1R-associated protein kinase. Toll-like receptors recognize pathogen associated molecular patterns and are crucially implicated in the induction of IL-12 secretion by APC. The role of MyD88 protein in the development of protective immune response against parasites is largely unknown. Following inoculation of L. major, MyD88(-/-) C57BL/6 mice presented large footpad lesions containing numerous infected cells and frequent mutilations. In response to soluble Leishmania Ag, cells from lesion-draining lymph node showed a typical Th2 profile, similar to infected BALB/c mice. IL-12p40 plasma level collapses in infected MyD88(-/-) mice compared with infected wild-type C57BL/6 mice. Importantly, administration of exogenous IL-12 rescues L. major-infected MyD88(-/-) mice, demonstrating that the susceptibility of these mice is a direct consequence of IL-12 deficiency. In conclusion, MyD88-dependent pathways appear essential for the development of the protective IL-12-mediated Th1 response against the Leishmania major parasite. In absence of MyD88 protein, infected mice develop a nonprotective Th2 response.