Strain difference in the induction of T-cell activation-associated, interferon gamma-dependent hepatic injury in mice

A single intravenous injection of concanavalin A (Con A) induces T-cell activation-associated inflammatory injury selectively in the liver. This study investigated the strain difference in the development of Con A-induced hepatic injury. Normal C57BL/6 and BALB/c spleen cells produced comparable levels of T-cell-derived lymphokines (interferon gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], and interleukin-2 [IL-2]) following in vitro stimulation with Con A. A single intravenous injection of Con A to C57BL/6 mice induced the plasma levels of TNF-alpha and IL-2 comparable with or slightly higher than those observed in BALB/c mice, whereas the same treatment resulted in an apparently lower level of IFN-gamma production in C57BL/6 mice. RNA from livers of Con A-treated C57BL/6 mice exhibited lower levels of IFN-gamma mRNA than RNA of BALB/c livers. Unexpectedly, a dramatic difference in the severity of hepatic injury was observed between C57BL/6 and BALB/c. Namely, the peak alanine transaminase (ALT) level was more than 15,000 U/L and inducible as early as 8 hours after injection of 0.2 mg Con A per mouse in the C57BL/6 strain, whereas the peak was approximately 3,000 U/L and induced as late as 24 hours after Con A injection in the BALB/c strain. The increase in plasma ALT levels was limited to less than 10% by injection of anti-IFN-gamma monoclonal antibody (mAb) in both strains. The C57BL/6 strain inducing lower levels of IFN-gamma exhibited higher IFN-gamma responsiveness as exemplified by the intrahepatic expression of an IFN-gamma-inducible gene, an inducible type of nitric oxide (NO) synthase (iNOS). These results indicate that, while IFN-gamma produced in vivo by activated T cells induces hepatic injury, there exists a striking strain difference in the induction of IFN-gamma-dependent hepatic injury.     

Ambiguous role of interleukin-12 in Yersinia enterocolitica infection in susceptible and resistant mouse strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-gamma) production in NK and CD4+ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-gamma-receptor-deficient (IFN-gamma R-/-) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55-/-) mice. IFN-gamma R-/- mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-gamma R+/+ mice. Administration of IL-12 was protective in IFN-gamma R+/+ mice but not in IFN-gamma R-/- mice, suggesting that IFN-gamma R-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor beta (TGF-beta). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-beta. While administration of IL-12 alone increased TNF-alpha levels, administration of TGF-beta or TGF-beta plus IL-12 decreased both TNF-alpha and IFN-gamma levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55-/- mice, suggesting that TNF-alpha accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis

In response to stimulation with immobilized anti-CD3 antibody, splenocytes from C57BL/6 and BALB/c mice principally produced INF-gamma and IL-4, respectively. However, both splenocytes equally proliferated in response to ConA. We compared the changes after inoculation with BCG (1 mg/mouse) in their capacity to produce IL-4 or IFN-gamma in response to anti-CD3 antibody and to proliferate in response to ConA. Splenocytes from C57BL/6 and BALB/c mice, that had been inoculated with BCG 4 weeks before, produced IFN-gamma with diminished IL-4 production in response to anti-CD3 antibody. Furthermore these splenocytes became anergic to ConA stimulation and died due to cell apoptosis in stead of proliferation. However, we observed the strain difference at 12 weeks after BCG-infection. BCG-primed C57BL/6 splenocytes, that continuously produced IFN-gamma in response to anti-CD3 antibody, failed to proliferate in response to ConA. In contrast, BCG-primed BALB/c splenocytes, that increased IL-4 production but decreased IFN-gamma production when stimulated with anti-CD3 antibody, could proliferate well in response to ConA. Since the splenocytes of BALB/c mice became ConA responsive along with their shifting from Th1 dominant immune response at 4 weeks to Th2 dominant immune response at 12 weeks after BCG-inoculation, IL-4 was assumed to play a crucial role in activation of anergic T cells. Therefore, we stimulated splenocytes from both strains of mice infected with BCG 4 weeks before with ConA in the presence or absence of IL-4. Splenocytes from BCG-infected BALB/c mice showed marked proliferation, while those from BCG-infected C57BL/6 mice failed. We found that IL-4 protected against ConA-induced cell apoptosis in BALB/c splenocytes but not C57BL/6 splenocytes.     

Immunoregulation in experimental murine Trypanosoma congolense infection: anti-IL-10 antibodies reverse trypanosome-mediated suppression of lymphocyte proliferation in vitro and moderately prolong the lifespan of genetically susceptible BALB/c mice

We infected highly susceptible BALB/c and relatively resistant C57BL/6 mice with cloned Trypanosoma congolense and followed the effects of these infections on the circulating parasite numbers, mouse mortality and cytokine expression. C57BL/6 mice controlled their parasitaemia and survived for up to 163 +/- 12 days, while BALB/c mice could not control their parasitaemia and succumbed to the infection within 8.4 +/- 0.5 days. Susceptible BALB/c mice had dramatically higher plasma levels of IL-10 than the resistant C57BL/6 mice from day 7 forward. This was preceded by an earlier and higher level induction of splenic IL-10 messenger RNA (mRNA) expression in the infected BALB/c mice. There was a strong negative correlation between the splenocyte proliferative responses to Concanavalin-A (Con-A) and their production of IL-10 in these infected BALB/c mice. Co-treatment of the Con-A-stimulated spleen cell cultures with monoclonal anti-IL-10 antibodies, but not isotype-matched control antibodies, could completely reverse this suppression of the splenocyte proliferative response. Finally, in three experiments, anti-IL-10 antibody treatment in vivo reduced the peak circulating parasitaemia of infected BALB/c mice by 43% and increased their median survival periods by 38% relative to isotype-matched control antibody-treated mice.     

IL-18 (IFN-gamma-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice

IL-12-induced IFN-gamma production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-gamma-inducing factor) is produced by macrophages and induces IFN-gamma production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-gamma production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-gamma production was not IL-18 dependent. IL-18-induced IFN-gamma production was to a higher degree dependent on IFN-gammaR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-gamma levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-gamma production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.     

Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice

Recent evidence suggests that T cells and their associated cytokines critically influence outcome in mice experimentally infected with Borrelia burgdorferi (Bb), the causative agent of human Lyme disease. In vivo T cell subset and cytokine depletion studies suggest that CD4+ T cell-derived IL-4 plays a critical role in control of spirochete growth in vivo, whereas CD8+ T cell-derived IFN-gamma appears to promote disease, particularly in susceptible mouse strains. To further investigate the immunologic basis of protection and the role of IL-4, we have examined the effects of early rIL-4 treatment on outcome in susceptible mice infected with Bb. In this study, we show that administration of rIL-4 to susceptible C3H mice during the first week of infection with Bb leads to early control of their infections, as evidenced by significant reductions in joint swelling at wk 5, 6, and 7 postinfection, and in the numbers of spirochetes recovered from their joints and skin at wk 7 when compared with sham-treated mice. Increased resistance in rIL-4-treated mice was accompanied by significant reductions in their in vitro splenic Bb-specific IFN-gamma responses and in serum levels of specific IgG2a and IgG3 Abs and significant increases in specific IgG1 Abs. We also show that the inherent susceptibility of Ab-deficient, C57BL/6-IgM knockout (B6-MKO) mice to Rh infection is intermediate relative to C57BL/6 severe combined immunodeficient (B6-SCID) mice (susceptible) or normal C57BL/6 mice (resistant), confirming the importance of both Ab-dependent and Ab-independent, T cell-dependent immune mechanisms in control of Bb infections. The additional finding that early treatment with rIL-4 significantly reduced the severity of Bb infections in B6-MKO mice indicates that IL-4 may augment anti-spirochetal immunity via an Ab-independent mechanism.     

Analysis of the role of CD4+ T-cells during murine cytomegalovirus infection in different strains of mice

We examined the role of CD4+ T-cells in peritoneal exudate cells (PECs) during the course of acute murine cytomegalovirus (MCMV) infection in two strains of mice. Cell counts of PECs and cytofluorometric analysis showed that C57BL/6, a resistant strain, had more CD4+ T-cells than BALB/c, a susceptible strain, after intraperitoneal infection of 3 x 10(3) PFU of the Smith strain of MCMV, though both strains had an equivalent number of CD8+ T-cells. CD4+ T-cells of both strains expressed mRNA of IFN-gamma, IL-2, and IL-4 on days 5 and 7 after infection, with much higher expression of these cytokines in C57BL/6 than in BALB/c. At the same time point after infection, macrophages were shown to express mRNA of IL-1 alpha and TNF-alpha with higher expression of IL-1 alpha in C57BL/6 than in BALB/c. Production of nitric oxide, recently shown to be one of the antiviral effector mechanisms of macrophages, by macrophages of both strains was examined showing more production of nitric oxide on day 7 after infection in C57BL/6 than in BALB/c. From these findings, we suggest the possibility that CD4+ T-cells contribute to the protection against MCMV infection via the secretion of cytokines and the resultant activation of macrophages to produce nitric oxide.     

Genetics of resistance to Trypanosoma congolense in inbred mice: efficiency of apparent clearance of parasites correlates with long-term survival

To study the genetic parameters of resistance to Trypanosoma congolense infection, highly susceptible BALB/c and relatively resistant C57BL/6 mice were crossed to produce reciprocal F1 and F2 offspring. Mice were infected with T. congolense and monitored for parasitemia within the first 2 wk and also for their survival periods. BALB/c mice showed unrestrained parasite growth to the time of death (median survival period, msp = 12.0 days), whereas in C57BL/6 mice, parasitemia reached an initial peak on day 6 and was followed by a rapid apparent clearance of the parasites in an average period of 3 days. Their msp was 163.0 days. The F1 mice cleared the parasites, following the first peak of parasitemia, in an average period of 4 days and had an msp of 69.5 days. Thus, the F1 offspring displayed an intermediate phenotype between susceptible and resistant parents in terms of parasite clearance and survival period. Resistance in F2 mice, as measured by survival times, was inherited as a polygenic trait. Among F2 mice, there was an inverse correlation between the time taken to clear the initial wave of parasitemia and the survival period, r = -0.58; P < 0.05. Thus, the pattern of control of the parasites following the first peak of parasitemia appears to be a good predictive factor for the survival period of mice infected with T. congolense.     

Interferon-gamma antagonizes transforming growth factor-beta2-mediated immunosuppression in murine Toxoplasma encephalitis

The in vivo modulating activity of recombinant transforming growth factor (TGF)-beta2 on acute toxoplasmosis was evaluated in both Toxoplasma gondii susceptible C57BL/6 and resistant BALB/c mice. TGF-beta2 lethally exacerbated Toxoplasma encephalitis in C57BL/6, but not in BALB/c mice. In C57BL/6 mice, TGF-beta2 induced a profound dose-dependent increase of the intracerebral parasitic load as well as a reduction of IFN-gamma levels in serum and cerebrospinal fluid with a coincident decrease of MHC class II antigen expression of macrophages, microglial cells, and B cells. Furthermore, TGF-beta2-treated C57BL/6 mice showed a reduced activation of CD4+ and CD8+ T cells and a diminished recruitment of immune cells to the brain. The TGF-beta2-mediated development of lethal toxoplasmosis in C57BL/6 mice was abolished by treatment with recombinant interferon (IFN)-gamma.     

The in vitro expression patterns of individual type I interferon genes in Newcastle disease virus infected murine splenocytes and fibroblasts

Murine type I interferon levels present in mice sera following Newcastle disease virus infections are influenced by the If-1 locus. Sera interferon levels in C57BL/6 mice (If-1h allele) are 10- to 15-fold higher than in BALB/c mice (If-1(1) allele). The B6.C-H-28c strain, which carries BALB/c If-1(1) allele on C57BL/6 genomic background, has low interferon levels in sera. This study examined the expression of interferon alpha 1, alpha 4, alpha 5, alpha 6, alpha 9 and beta mRNAs at 7 hr after Newcastle disease virus infection of primary cells (splenocytes and mouse embryo fibroblasts) from C57BL/6, B6.C-H-28c and BALB/c mouse genotypes. Total RNA from these cells was reverse transcribed and all known type I interferon subtypes were amplified. The products were identified by differential hybridization to a panel of subtype specific oligonucleotides. The results show that the pattern of interferon subtypes examined in splenocytes did not differ between If-1h and If-1(1) allele carrying C57BL mice. However, when the genotype was different (BALB/c splenocytes) the pattern of type I interferon mRNAs seen was altered. This genotype-dependent expression was also seen in newcastle disease virus infected fibroblasts. Within a given mouse strain, there were also differences in the subtype response patterns detected in fibroblasts compared with those seen in splenocytes. In conclusion, the present study indicates that mouse genotype appears to be a major determinant of the subtype response pattern seen and tissue specific pattern differences are present within a given mouse genotype.     

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.     

The importance of TGF-beta in murine visceral leishmaniasis

IFN-gamma is critical for the cure of leishmaniasis in humans and mice. BALB/c mice are genetically susceptible to infection with the visceralizing species of Leishmania, L. chagasi. We have evidence that a soluble factor(s) inhibits IFN-gamma production by cultured liver granuloma cells from BALB/c mice during L. chagasi infection. In contrast, liver granulomas from C3H.HeJ mice, which are genetically resistant to L. chagasi infection, produce abundant IFN-gamma. According to ELISAs and neutralization studies, there was not evidence that the Th2-type cytokines IL-10 or IL-4 contributed to IFN-gamma suppression. However, both Ab neutralization and immunohistochemistry showed that granuloma-derived TGF-beta was, at least in part, responsible for inhibiting IFN-gamma release by CD4+ cells in BALB/c liver granuloma cultures. Consistently, TGF-beta levels were high in liver granulomas from susceptible BALB/c mice but low in resistant C3H mice or in BALB/c mice that were immunized against L. chagasi disease. Administration of recombinant adenovirus expressing TGF-beta (AdV-TGFbeta) but not IL-10 (AdV-IL10) caused genetically resistant C3H mice to become significantly more susceptible to L. chagasi infection. In contrast, either AdV-TGFbeta or AdV-IL10 could abrogate the protective immune response achieved by immunization of BALB/c mice. We conclude that locally secreted TGF-beta inhibits Th1-associated cure of murine visceral leishmaniasis caused by L. chagasi, independently of Th2-type cytokines.     

Role of liver NK cells and peritoneal macrophages in gamma interferon and interleukin-10 production in experimental bacterial peritonitis in mice

Gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-gamma, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-gamma and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-alpha levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-gamma levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-gamma producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-gamma and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.     

Role of gamma interferon in natural clearance of Bordetella pertussis infection

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.     

Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes

The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.     

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.     

Allogeneic cell therapy for a murine mammary carcinoma

The effect of allogeneic cell therapy on tumor growth was studied in a murine model of mammary carcinoma (4T1) as an experimental model of solid tumors in humans. i.v. inoculation of 4T1 (H-2d) cells into syngeneic mice [BALB/c or (BALB/cXC57BL/6)F1] (F1) carrying the H-2d histocompatible antigens results in tumor colonies in the lungs that finally cause the death of all of the mice. Sublethally irradiated F1 mice were inoculated with 4T1 cells to simulate minimal residual disease and with immunocompetent splenocytes derived from naive donors of F1 (syngeneic), BALB/c (syngeneic to the tumor but semiallogeneic to the host), or C57BL/6 (allogeneic to the tumor and semiallogeneic to the host) mice. The survival of F1 tumor-bearing mice that were treated with allogeneic C57BL/6 splenocytes was significantly prolonged (P < 0.02) compared with hosts given F1 or BALB/c-derived splenocytes that are syngeneic to 4T1 tumor cells. Adoptive transfer of lung cells that were isolated from F1 primary mice inoculated with 4T1 cells and syngeneic BALB/c or F1 splenocytes led to local tumor growth and death in secondary recipients. In contrast, only 1 of 22 secondary recipients developed tumors when inoculated with lung cells derived from F1 mice given allogeneic C57BL/6 splenocytes. All of the 21 secondary hosts survived disease-free for a follow-up time of >200 days. These results indicate that immunocompetent cells allogeneic to the mammary carcinoma cells were able to inhibit tumor development in the primary hosts and to prevent tumor growth in the adoptive recipients, which suggests that allogeneic cell therapy may be an efficient antitumor tool to eradicate minimal residual disease in human solid tumors.     

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.     

Leishmania mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potentiate a Th1 response

Leishmania mexicana mutants lacking cysteine proteinase genes cpa (delta cpa), cpb (delta cpb), or both cpa and cpb (delta cpa/cpb) have been generated by targeted gene disruption. Delta cpa mutants produce a disease phenotype in BALB/c mice close to that of wild-type L. mexicana, but delta cpb mutants are much less infective, producing very slowly growing small lesions, and delta cpa/cpb double mutants do not induce lesion growth. Immunologic analysis of Ab isotype during infection and splenocyte IFN-gamma, IL-2, and IL-4 production following stimulation with Leishmania Ag or Con A indicates that there was a significant shift from a predominantly Th2-associated immune response in mice infected with wild-type L. mexicana to a Th1-associated response in mice inoculated with delta cpb or delta cpa/cpb. Significantly, delta cpa altered the balance of the immunologic response to a lesser extent than did the other mutants. Similar disease outcomes and switches in the Th1/Th2 balance were also observed when other L. mexicana-susceptible mouse strains were infected with the mutants. BALB/c and C57BL/6 mice vaccinated with delta cpa/cpb and CBA/Ca mice vaccinated with delta cpb or delta cpa/cpb were subsequently more resistant, to varying degrees, than were untreated mice to infection with wild-type parasites, as measured by development of lesions and parasite burden. These data implicate leishmanial cysteine proteinases not only as parasite virulence factors but also in modulation of the immune response and provide strong encouragement that cysteine proteinase-deficient L. mexicana mutants are candidate attenuated live vaccines.     

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.