Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin

TNF and lymphotoxin-alpha (LT alpha) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LT alpha, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF-/-). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF-/- mice, and by 28-35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF-/- mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF-/- and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF-/- mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF-/- and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF-/- is not compensated for by the presence of LT alpha, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas.     

基因打靶突变小鼠的早期T细胞发育

早期Ｔ细胞的发育是一个受到多种分子精确调控的过程,基因打靶技术的建立和发展 内研究上述分子的作用提供了有效的手段。对ＴＣＲ、ＣＤ３基因打靶小鼠的研究表明,ＣＤ４４－ＣＤ２５阶段是早期Ｔ细胞发育的重要调控点,在此发育阶段,由ＴＣＲβ、ＴＣＲα和ＣＤ３成分组成的ｐｒｅ－ＴＣＲ复俣体的表达或其与未知配体的结合通过ｐ５６ｌｃｋ传递信号,介导ＣＤ４４－ＣＤ２５细胞的进一上发育,该复全体任何成分的缺失都将使Ｔ     

Surfactant metabolism in SP-D gene-targeted mice

Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(-/-)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice. (125)I-SP-D was cleared with a half-life of 7 h from SP-D(-/-) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [(3)H]palmitic acid and [(14)C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(-/-) mice. Clearance rates of intratracheal [(3)H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(-/-) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.     

Mice with a targeted mutation in lymphotoxin-alpha exhibit enhanced tumor growth and metastasis: impaired NK cell development and recruitment.

Mice deficient in lymphotoxin (LT)-alpha lack peripheral lymph nodes and Peyer's patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTalpha is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTalpha for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTalpha-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTalpha-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTalpha-/- mice. Although LTalpha-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTalpha-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTalpha signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.     

Fbxw7 in cell cycle exit and stem cell maintenance: Insight from gene-targeted mice

Regulation of the exit of cells from the cell cycle is important in the development of multicellular organisms and is also implicated in the maintenance of stem cells. Furthermore, defects in cell cycle exit are thought to be a major cause of cancer. However, the mechanisms responsible for regulation of cell cycle exit have remained largely unknown. Fbxw7 is the F-box protein subunit of an SCF-type ubiquitin ligase complex that targets positive regulators of the cell cycle—including cyclin E, c-Myc, Notch, and c-Jun—for ubiquitylation and subsequent degradation by the 26S proteasome in order to promote cell cycle exit. Consistent with such a function, mutations of the Fbxw7 gene have been detected in various human malignancies. We have recently generated conventional and conditional Fbxw7 knockout mice and examined stem cells, progenitor cells, and differentiated cells in the mutant animals for cell cycle defects. Here we summarize the pleiotropic phenotypes of Fbxw7 deficiency in various cell types including T cells, hematopoietic stem cells, and embryonic fibroblasts. Such phenotypes have provided insight into the biological roles of Fbxw7 in cell cycle exit, stem cell maintenance, and oncosuppression.     

Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD^−/−) by gene targeting in embryonic stem (ES) cells. The MCAD^−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD^−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD^−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation.     

Loss of lymphotoxin-alpha but not tumor necrosis factor-alpha reduces atherosclerosis in mice.

Inflammatory processes are involved with all phases of atherosclerotic lesion growth. Tumor necrosis factor-alpha (TNFalpha) is an inflammatory cytokine that is thought to contribute to lesion development. Lymphotoxin-alpha (LTalpha) is also a proinflammatory cytokine with homology to TNFalpha. However, its presence or function in lesion development has not been investigated. To study the role of these molecules in atherosclerosis, the expression of these cytokines in atherosclerotic lesions was examined. The presence of both cytokines was observed within aortic sinus fatty streak lesions. To determine the function of these molecules in regulating lesion growth, mice deficient for TNFalpha or LTalpha were examined for induction of atherosclerosis. Surprisingly, loss of TNFalpha did not alter lesion development compared with wild-type mice. This brings doubt to the generally held concept that TNFalpha is a "proatherogenic cytokine." However, LTalpha deficiency resulted in a 62% reduction in lesion size. This demonstrates an unexpected role for LTalpha in promoting lesion growth. The presence of LTalpha was observed in aortic sinus lesions suggesting a direct role of LTalpha in modulating lesion growth. To determine which receptor mediated these responses, diet-induced atherosclerosis in mice deficient for each of the TNF receptors, termed p55 and p75, was examined. Results demonstrated that loss of p55 resulted in increased lesion development, but loss of p75 did not alter lesion size. The disparity in results between ligand- and receptor-deficient mice suggests there are undefined members of the TNF ligand and receptor signaling pathway involved with regulating atherogenesis.     

Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.     

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

Cryptosporidium parvum infection in gene-targeted B cell-deficient mice

The importance of B cells in host resistance to, and recovery from, Cryptosporidium parvum infection was examined in gene-targeted B cell-deficient (muMT-/-) mice. Neonatal muMT-/- mice infected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1-/- adult mice with spleen cells from muMT-/- donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 donors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice.     

Multiple developmental processes underlie sex differentiation in angiosperms

The production of unisexual flowers has evolved numerous times in dioecious and monoecious plant taxa. Based on repeated evolutionary origins, a great variety of developmental and genetic mechanisms underlying unisexual flower development is predicted. Here, we comprehensively review the modes of development of unisexual flowers, test potential correlations with sexual system, and end with a synthesis of the genetics and hormonal regulation of plant sex determination. We find that the stage of organ abortion in male and female flowers is temporally correlated within species and also confirm that the arrest of development does not tend to occur preferentially at a particular stage, or via a common process.     

Novel Sxr(a) ES cell line offers hope for Y chromosome gene-targeted mice

A mouse targeted for a Y Chromosome gene has not been reported. Because the Y Chromosome is present in only one copy, and most of its genes are critical for germ cell development, such a mouse would likely be infertile. Thus, we describe a new reproductive strategy to enable transmission of targeted Y Chromosome genes to subsequent generations. The strategy uses two segregating copies of Y Chromosome genes to mimic the autosomal condition. To achieve this, we developed a new embryonic stem cell line from the XYSxr(a) mouse, which carries a duplication of the gene-rich Y Chromosome short arm. Importantly, we demonstrate germ line transmission of the YSxr(a) chromosome and describe this significant new tool as a practical solution to enable reproduction in mice targeted for Y Chromosome genes.     

B cell selection defects underlie the development of diabetogenic APCs in nonobese diabetic mice

One mechanism whereby B cells contribute to type 1 diabetes in nonobese diabetic (NOD) mice is as a subset of APCs that preferentially presents MHC class II-bound pancreatic beta cell Ags to autoreactive CD4 T cells. This results from their ability to use cell surface Ig to specifically capture beta cell Ags. Hence, we postulated a diabetogenic role for defects in the tolerance mechanisms normally blocking the maturation and/or activation of B cells expressing autoreactive Ig receptors. We compared B cell tolerance mechanisms in NOD mice with nonautoimmune strains by using the IgHEL and Ig3-83 transgenic systems, in which the majority of B cells recognize one defined Ag. NOD- and nonautoimmune-prone mice did not differ in ability to delete or receptor edit B cells recognizing membrane-bound self Ags. However, in contrast to the nonautoimmune-prone background, B cells recognizing soluble self Ags in NOD mice did not undergo partial deletion and were also not efficiently anergized. The defective induction of B cell tolerance to soluble autoantigens is most likely responsible for the generation of diabetogenic APC in NOD mice.     

Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Defective activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies

Nonobese diabetic (NOD) is an inbred mouse strain susceptible to development of T cell-mediated autoimmune diabetes. The strain is characterized by high percentages of T lymphocytes in lymphoid organs. The syngeneic mixed lymphocyte reaction (SMLR), a T cell response to self MHC class II Ag, is reportedly involved in the generation of a number of immunoregulatory cells, including suppressor inducers. A severely depressed SMLR characteristic of certain other autoimmune strains was found in NOD but not in nonautoimmune SWR/Bm mice. Moreover, IL-2 produced by NOD T cells at day 6 in an SMLR was at least one hundredfold reduced compared with SWR, and NOD T cells harvested from an SMLR at day 6 were functionally defective when tested for ability to induce suppression of an allogeneic MLR. However, functionally competent suppressor T cells were generated in NOD splenic leukocyte cultures in response to Con A, and IL-2 release from these was equivalent to that released by Con A-stimulated SWR splenocytes. A deficiency in cytokine release was not limited to IL-2, because peritoneal exudate cells from NOD exhibited a greatly diminished sensitivity to LPS-stimulated IL-1 release in comparison to SWR mice. IL-2 supplementation both in vitro and in vivo restored the ability of NOD T cells to respond in a SMLR, with production of cells capable of inducing suppression. Like SMLR-activated T cells from untreated SWR controls, SMLR blasts from IL-2-treated NOD mice were enriched for the L3T4 phenotype. IL-1 supplementation in vitro resulted in partial restoration of T suppressor activation in a SMLR. The depressed SMLR exhibited by NOD mice was apparently a stimulator cell dysfunction, because NOD stimulator cells failed to activate T cells from (SWR x NOD)F1 mice, whereas stimulators from SWR or F1 mice were capable of doing so. Collectively, these results suggest a defect in suppressor cell activation rather than an absence of this immunoregulatory cell population.     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.