Control of Leishmania major infection in mice lacking TNF receptors

TNF participates in the induction of nitric oxide (NO) production and macrophage activation, leading to the elimination of intracellular pathogens. We previously found that TNF receptor p55-deficient mice (TNFRp55-/-) control replication of Leishmania major in vivo but fail to resolve their lesions. Here we report that mice lacking the p75 receptor (TNFRp75-/-) or both receptors (TNFRp55p75-/-), also control parasite replication, albeit mice lacking the p55 receptor (either TNFRp55-/- or TNFRp55p75-/-) are delayed in their elimination of L. major compared with controls. All TNF receptor-deficient mice developed a Thl-type immune response and up-regulated inducible NO synthase (iNOS) mRNA gene expression in lesions during infection. Thus, neither TNF receptor appears to be absolutely required for NO production or elimination of L. major in vivo. In vitro, however, while macrophages from naive TNFRp75-/- mice could be activated to produce NO and kill L. major, we observed a defect in NO production and parasite killing by resident peritoneal macrophages from naive TNFRp55-/- or TNFRp55p75-/- mice. However, when macrophages were elicited with leishmanial Ag from 4-wk-infected TNFRp55-/- or TNFRp55p75-/- mice, they produced NO and were leishmanicidal. These data suggest that the TNFRp75 plays no essential role in L. major infection in mice and that the p55 receptor may be required for optimal macrophage activation. However, the results also show that a mechanism exists by which macrophages can be primed in vivo during L. major infection to produce NO and kill L. major in the absence of signaling through either of the TNF receptors.     

Class II major histocompatibility complex-deficient mice initially control an infection with Leishmania major but succumb to the disease

Class II major histocompatibility complex (MHC)-deficient (H-2b) mice do not express I-A or I-E molecules and, as a result, do not develop CD4 cells. Thus, they represent the ideal model for examining the importance of CD4 cells and MHC class II molecules in resistance to infection with Leishmania major and the capacity of MHC class I-restricted T cells to mediate resistance to L. major. Class II MHC-deficient mice and control (C57BL/6, normal and nude) mice were infected with L. major. Although MHC class II-deficient mice were able to control infection more effectively than nude mice, cutaneous lesions on the mice eventually progressed, and parasite replication became uncontrolled. These results suggest that CD4 cells and MHC class II molecules are essential for resistance to L. major and that in the absence of these cells and molecules, such mice can transiently control infection with L. major but are unable to resolve such infections.     

IL-4-deficient Balb/c mice resist infection with Leishmania major

Mice with a genetically engineered deficiency for either IL-4 or IFN-gamma R1 (single mutants), and IL-4/IFN-gamma R1 (double mutants) on the Balb/c and 129Sv background were used to study the course of infection with Leishmania major. In contrast to genetically resistant 129Sv wildtype mice, IL-4/IFN-gamma R1 double mutant mice developed fetal disease with parasite dissemination to visceral organs similar to mice lacking IFN-gamma R1 only. Balb/c mice, which are exquisitely susceptible to L. major, were rendered resistant to infection by disruption of the IL-4 gene. As compared to homozygous IL-4+/- mice, heterozygous IL-4+/- mice, heterozygous IL-4+/- animals consistently developed smaller lesions with less ulceration and necrosis, indicating the likelihood of gene-dosage effects. This implicates that the magnitude of the IL-4 response determines the severity of disease. CD4+ T cells of IL-4-deficient mice showed impaired Th2 cell development, as assessed by quantitative RT-PCR of characteristic cytokines. Development of resistance is not explained by default Th1 development, because this was observed only at very late stages of infection. Moreover, the induction of inflammatory cytokines (e.g., IL-1 alpha, IL-1 beta, TNF-alpha, IL-12) together with iNOS in the lesion and draining lymph nodes was not altered in the absence of IL-4.     

In vivo blocking of L-selectin rescues BALB/c mice from fatal Leishmania major infection

Susceptibility and resistance to experimental Leishmania major (L. major) infection in mice are associated with a Th2- or Th1-type response, respectively. We have previously shown that immunological events occurring within the first 24 h after infection in the lymph node (LN) draining the site of parasite challenge are critical for the development of either type of T-cell responses. In the present study we manipulated these events by preventing the entry of naive lymphocytes into the draining LN by injecting BALB/c mice with a single dose of the anti-L-selectin mAb MEL-14 one day prior to infection with L. major. In contrast to control BALB/c mice, in MEL-14 treated animals the primary lesion healed 12 weeks after infection. The parasite load in the spleen and lymph nodes of MEL-14 treated mice was significantly reduced. The healing was found to be associated with an increased production of IFN-gamma and with a decrease in IL-4 production by LN cells. We observed a dramatic decrease in cellularity in the draining LN in Mel-14 treated L. major-infected mice within the first week of infection. Moreover, the cells in the LN of MEL-14 treated mice were highly enriched in activated cells as well as in cell influx in the draining LN after local L. major infection of BALB/c mice prevents fatal disease. The data suggest the MEL-14-induced enrichment of the draining LN in memory and activated cells is fundamental for the initiation of a protective Th1-type response.     

Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and renders them capable of healing Leishmania major infection

C.B-17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)-like response characterized by delayed-type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon-gamma (IFN-gamma), but no interleukin-4 (IL-4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN-gamma during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti-IFN-gamma antibody prior to infection and reconstitution prevented healing and permitted the development of a Th-2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL-4, but very little IFN-gamma, after antigen stimulation in vitro. As few as 10(4) transferred T cells led to a Th1-like response, suggesting that the IFN-gamma is of host rather than donor origin. The transfer of very high numbers (7.5 x 10(7)) of BALB/c spleen cells overcame the effects of the IFN-gamma and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measurable effect upon the development of a healing response in reconstituted scid mice.     

Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires the development of a resistance-promoting CD4+-mediated Th1 response. Epidermal Langerhans cells (LC) are critically involved in the induction of the primary immune response to Leishmania infection. They are able to ingest the parasites, to express MHC class II molecules with extraordinarily long half-life and to activate naive L. major-specific Th cells. Considering these unique properties, we studied the capacity of LC to mediate resistance to L. major in vivo. A single i.v. application of LC that had been pulsed with L. major antigen in vitro induced the protection in susceptible BALB/c mice against subsequent challenges with L. major parasites. Resistance could neither be induced by unpulsed LC, nor by L. major antigen alone or by L. major-pulsed macrophages. Development of resistance was paralleled by a reduced parasite burden and by a shift of the cytokine expression towards a Th1-like pattern. In contrast, control mice developed a Th2 response. In vitro exposure of LC to L. major antigen induced the expression of IL-12 (p40) mRNA. In conclusion, our data demonstrate that LC are able to serve as a natural adjuvant and to induce a protective immune response to L. major infection. This effect is based on the initiation of a Th1-like response that is likely to be mediated by IL-12.     

An in vitro model for infection with Leishmania major that mimics the immune response in mice

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).     

Selective expansion of activated V delta 4+ cells during experimental infection of mice with Leishmania major

Previous work from this laboratory has revealed that infection of mice with Leishmania major leads to an expansion of gamma delta+ T cells in the spleen. Further examination of the gamma delta+ T cells expanding in infected mice has shown that the majority of these cells in the spleen, lymph nodes, blood and liver expressed the V delta 4 gene segment. Cell cycle analysis, using propidium iodide incorporation, demonstrated that while only 1% of alpha beta+ T cells in the spleen were in either S + G2/M phase, up to 10% of the gamma delta+ T cells were in cycling phase 8 weeks after infection. Comparison of the state of activation of the two populations in different organs after infection, confirmed that gamma delta+ T cells are actively dividing in lymph nodes, liver and blood, but not in the thymus or among intraepithelial lymphocytes. Examination of the expression of different activation markers on the surface of gamma delta+ T cells in the spleen of both normal and chronically infected BALB/c mice by FACS analysis, revealed increased expression of LFA-1, CD25, CD44, 4F2, CD28 and the heat-stable antigen, whereas Thy-1 and CD5 decreased. Collectively, these results suggest an oligoclonal expansion and activation of gamma delta+ T cells in response to L. major infection.     

Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice

PURPOSE: Cataract surgery is often followed by a posterior capsule opacification, usually treated with YAG laser capsulotomy, however, there are huge variations in the incidence figures available in the literature, from 18 to 50% (Sterling & Wood 1986). We have therefore analyzed the incidence of secondary cataracts in a population-based cohort of patients, as revealed by the number of YAG laser capsulotomies performed postoperatively. METHODS: Data for all patients undergoing cataract surgery from 1986 up to and including 1990 in the Lund Health Care District were prospectively recorded, and 4722 patients were retrieved for analysis, using only one eye per patient. The patients had been operated on with extracapsular extraction (phacoemulsification or planned large incision procedure) or a combined trabeculectomy and cataract extraction procedure leaving an intact capsule after surgery. Death dates for each patient were obtained from the Swedish Bureau of Census up to and including 1991. Different risk factors were considered such as sex, age, preoperative axial length, preoperative average keratometry, preoperative intraocular pressure, glaucoma history, diabetes history, uveitis history (including both anterior and posterior uveitis), history of age related macular degeneration and a history of rheumatoid arthritis. We also considered the influence of factors connected to the operation itself on the incidence of secondary capsular haze: extraction mode (ordinary ECCE versus phacoemulsification or trabeculectomy) and the type of implant and the surgeon's surgical activity. RESULTS: Besides age, four variables significantly influenced the risk of having postoperative YAG laser treatment. They were gender, iris sphincterotomy, operation date, and whether the patient came from a rural or an urban region. After about four to five years, the percentage of patients not having had a YAG laser capsulotomy was reduced to around 50% for women and 60% for men. These percentages were based on a survival analysis, minimizing the confounding effect of the limited life span of these elderly patients. CONCLUSIONS: In this material, the most important predisposing factors for YAG laser capsulotomy after extracapsular cataract surgery are: young age, female gender, if the patient was operated late in the period observed, and if the patient came from an urban area.     

Anti-TGF-beta treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production

CB6F1 mice display intermediate susceptibility to Leishmania major infection compared with the highly susceptible BALB/c and resistant C57BL/6 parental strains. During early weeks of infection, these mice develop dominant Th2 type responses to L. major, although they eventually exhibit a Th2 to Th1 switch and spontaneously resolve their infections. In this study, we have examined the effects of either IL-12 or anti-TGF-beta therapy on the immune response and course of disease in chronically infected CB6F1 mice. Local treatment with IL-12 inoculated into the parasitized lesion at 4 wk of infection induced a marked increase in IFN-gamma production but did not result in a significant reduction in numbers of parasite or promote more rapid healing. However, local treatment with an Ab to TGF-beta led to both a decrease in parasite numbers and more rapid healing, despite the fact that such treatment did not significantly alter the pattern of IL-4 and IFN-gamma production. Immunohistochemical studies showed that anti-TGF-beta treatment resulted in increased nitric oxide production within parasitized lesions. Our results suggest that TGF-beta may play an important regulatory role during chronic stages of a L. major infection by suppressing macrophage production of nitric oxide and that, in the absence of TGF-beta, even the relatively low levels of IFN-gamma observed in mice with dominant Th2-type responses are sufficient to activate macrophages to destroy amastigotes within parasitized lesions.     

Offspring of xenogeneically-reconstituted scid/scid mice are capable of a primary xenogeneic immune response to DNP-KLH

The distribution of [125I]SRIF-28 ([Leu8,D-Trp22,125I-Tyr25]somatostatin-28), [125I]204-090 ([Tyr3]octreotide) and [125I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) labelled recognition sites was studied by autoradiography in rat brain at embryonic day 18 (E 18) and postnatal day 5 (P 5). These results were compared with mRNA expression of somatostatin receptors SSTR1-5 (named sst1-5 now) as studied by in situ hybridization. [125I]SRIF-28, [125I]204-090 and [125I]CGP 23996 binding displayed different although partially overlapping distributions, and showed an increase between E 18 and P 5, which was less marked for [125I]204-090 binding. -125I-204-090 binding and sst2 receptor mRNA were similarly distributed, whereas [125I]CGP 23996 binding did not correlate with any single somatostatin receptor mRNA. The data suggest that most SRIF receptor subtypes in rat brain are present before birth, but evolve differently.     

Distribution of protein kinase C isoforms after infection of macrophages with Leishmania major

We characterized the effects of Leishmania infection on activation-induced translocation of protein kinase C (PKC) isoforms in murine bone marrow-derived macrophages. Although PKC-dependent gene expression was attenuated by infection, the distribution and translocation of PKC isoforms were unaffected. However, subsequent dissociation from membranes was substantially delayed for some isoforms, particularly PKCbeta.     

Non-MHC-linked Th2 cell development induced by soluble protein administration predicts susceptibility to Leishmania major infection

Continuous administration of soluble protein Ag followed by immunization with the same Ag in adjuvant results in the selective development of Ag-specific CD4+ Th2 cells in both normal and beta2-microglobulin-deficient BALB/c mice. In addition to chronic administration by mini-osmotic pump, single bolus i.p., but not i.v., injection of protein Ag induces Th2 cell expansion. Strong Th2 cell priming depends on a non-MHC-linked genetic polymorphism. It is observed in all congenic strains on BALB background tested, BALB/c, BALB/b, and BALB/k, but not in MHC-matched strains on disparate genetic background, B10.D2, C57BL/6, and C3H. DBA/2 mice appear to have an intermediate phenotype, as shown by their weaker capacity to mount Th2 responses as compared with BALB/c mice after soluble Ag administered by either mini-osmotic pumps or single bolus i.p. Conversely, induction of Th1 cell unresponsiveness by soluble protein is observed in any mouse strain tested, following any mode of Ag administration. These data demonstrate that non-MHC-linked genetic polymorphism controls the priming of Th2 but not the inhibition of Th1 cells induced by administration of soluble protein. The pattern of Th2 responses in these different strains is predictive of disease outcome following Leishmania major infection and supports the hypothesis that systemic Ag presentation in the absence of strong inflammatory signals may represent an important stimulus leading to selective Th2 cell development in susceptible mouse strains.     

Leishmania major infection in C57BL/10 mice differing at the Lps locus: a new non-healing phenotype

The course of cutaneous leishmaniasis was examined in mice from two genetically closely related strains, C57BL/10ScCr (Cr) and C57BL/10ScSn (Sn). Sn mice are able to heal Leishmania major infections, while Cr mice are unable to heal. The cutaneous lesions of the Cr mice progressed continuously and the increase in lesion size was paralleled by an unrestricted growth of the parasites in vivo. Cr mice, in contrast to their Sn counterparts, are highly resistant to all effects of lipopolysaccharide (LPS). The nonhealing L. major infection in Cr mice is in sharp contrast to the course of infection in another endotoxin-nonresponder mouse strain, C3H/HeJ, which heal infections with L. major. Cr mice exhibit, in addition to the defective LPS responsiveness, an impaired interferon-gamma (IFN-gamma) response after infection with a variety of microorganisms. The insufficient activation of parasitized macrophages to kill intracellular L. major could be due to the inability of splenocytes from infected Cr mice to secrete IFN-gamma upon restimulation with L. major. IFN-gamma is essential for the efficient activation of parasitized macrophages to kill intracellular L. major by producing nitric oxide (NO). Although bone marrow-derived Cr macrophage do not produce NO in response to LPS, both Sn and Cr macrophages release NO upon stimulation with IFN-gamma and tumor necrosis factor, indicating that they are responsive to activation by these cytokines.     

DNA double-strand breaks, p53, and apoptosis during lymphomagenesis in scid/scid mice

The tumor-suppressing phenotype of p53 is thought to be due to its accumulation in response to DNA damage and resultant cell cycle arrest or apoptosis. scid/scid mice are defective in DNA double-strand break repair due to a mutation in DNA-dependent protein kinase (DNAPK). Treatment of scid/scid mice with gamma radiation or N-ethyl-N-nitrosourea resulted in approximately 86% incidence of T-cell lymphomas, compared with <6% in wild-type mice. The incidence of other tumor types was not increased in scid/scid mice, suggesting that the types of DNA double-strand break that are unrepaired in these mice are not strongly carcinogenic. To determine whether mutations in DNAPK and p53 interact, we examined mice deficient in both genes. Both scid/scid p53-/- and scid/scid p53+/- mice spontaneously developed lymphomas at shorter latency than did mice with either defect alone. Loss of the wild-type p53 allele was observed in 100% of tumors from scid/scid p53 +/- mice, indicating strong selection against p53. In contrast, p53 was not inactivated in lymphomas from scid/scid p53+/+ mice. Exposure of these tumor-bearing mice to gamma radiation resulted in p53 protein accumulation and high levels of apoptosis in all tumors that were not observed in tumors from scid/scid p53+/- mice. Thus, there was a bifurcation of molecular pathways to tumorigenesis. When p53 was heterozygous in the germ line, loss of the wild-type allele occurred, and the tumors became apoptosis resistant. When p53 was wild type in the germ line, p53 was not inactivated, and the tumors remained highly apoptosis sensitive.     

Effective and long-lasting immunity against the parasite Leishmania major in CD8-deficient mice

The results of earlier investigations that tested whether CD8(+) T cells are required in the defense against Leishmania major have been inconsistent. We used CD8-deficient mice to directly address this issue. After primary infection with L. major, CD8-deficient mice controlled the infection for over 1 year and mounted strong T helper 1 cell responses. Thus, CD8(+) T cells are not required for the long-term control of a primary infection with L. major.     

Increased capacity for interleukin-2 synthesis parallels disease progression in mice infected with Leishmania major

Lymph node cells of BALB/c mice with progressive leishmaniasis produced sixfold more interleukin-2 (IL-2) in culture than those of healing C57BL/6 mice. IL-2 synthesis also increased in C57BL/6 mice made susceptible by IL-12 or gamma interferon deficiency. However, IL-2 mRNA levels in vivo did not reflect IL-2 production in vitro. Because IL-2 contributes to the pathogenesis of progressive leishmaniasis, the functional significance of these findings should be further explored.     

Yersinia-induced apoptosis in vivo aids in the establishment of a systemic infection of mice

Pathogenic Yersinia cause a systemic infection in mice that is dependent on the presence of a large plasmid encoding a number of secreted virulence proteins called Yops. We previously demonstrated that a plasmid-encoded Yop, YopJ, was essential for inducing apoptosis in cultured macrophages. Here we report that YopJ is a virulence factor in mice and is important for the establishment of a systemic infection. The oral LD50 for a yopJ mutant Yersinia pseudotuberculosis increases 64-fold compared with wild-type. Although the yopJ mutant strain is able to reach the spleen of infected mice, the mutant strain seldom reaches the same high bacterial load that is seen with wild-type Yersinia strain and begins to be cleared from infected spleens on day 4 after infection. Furthermore, when in competition with wild-type Yersinia in a mixed infection, the yopJ mutant strain is deficient for spread from the Peyer's patches to other lymphoid tissue. We also show that wild-type Yersinia induces apoptosis in vivo of Mac-1(+) cells from infected mesenteric lymph nodes or spleens, as measured by quantitative flow cytometry of TUNEL (Tdt-mediated dUTP-biotin nick-end labeling)-positive cells. The levels of Mac-1(+), TUNEL+ cells from tissue infected with the yopJ mutant strain were equivalent to the levels detected in cells from uninfected tissue. YopJ is necessary for the suppression of TNF-alpha production seen in macrophages infected with wild-type Yersinia, based on previous in vitro studies (Palmer, L.E., S. Hobbie, J.E. Galan, and J.B. Bliska. 1998. Mol. Microbiol. 27:953-965). We conclude here that YopJ plays a role in the establishment of a systemic infection by inducing apoptosis and that this is consistent with the ability to suppress the production of the proinflammatory cytokine tumor necrosis factor alpha.