Accelerated thymic atrophy as a result of elevated homeostatic expression of the genes encoded by the TNF/lymphotoxin cytokine locus.

TNF, lymphotoxin (LT)-alpha, LT-beta and LIGHT are members of a larger superfamily of TNF-related cytokines that can cross-utilize several receptors. Although LIGHT has been implicated in thymic development and function, the role of TNF and LT remains incompletely defined. To address this, we created a model of modest homeostatic overexpression of TNF/LT cytokines using the genomic human TNF/LT locus as a low copy number Tg. Strikingly, expression of Tg TNF/LT gene products led to profound early thymic atrophy characterized by decreased numbers of thymocytes and cortical thymic epithelial cells, partial block of thymocyte proliferation at double negative (DN) 1 stage, increased apoptosis of DN2 thymocytes and severe decline of T-cell numbers in the periphery. Results of backcrossing to TNFR1-, LTbetaR- or TNF/LT-deficient backgrounds and of reciprocal bone marrow transfers implicated both LT-alpha/LT-beta to LTbetaR and TNF/LT-alpha to TNFR1 signaling in accelerated thymus degeneration. We hypothesize that chronic infections can promote thymic atrophy by upregulating LT and TNF production.

Novel lymphotoxin alpha (LTalpha) knockout mice with unperturbed tumor necrosis factor expression: reassessing LTalpha biological functions.

Lymphotoxin alpha (LTalpha) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTalpha and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTalpha-deficient mouse strain (neo-free LTalpha KO [LTalphaDelta/Delta]). Unlike the "conventional" LTalpha-/- mice, new LTalphaDelta/Delta animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/D-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTalphaDelta/Delta mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTalpha KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTalpha KO mice displayed several features resembling those of LTbeta KO mice rather than conventional LTalpha KO animals. The phenotype of the new LTalphaDelta/Delta mice indicates that LTalpha plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression.

Inhibitory effects of tumor necrosis factor on hematopoiesis seen in vitro are translated to increased numbers of both committed and multipotent progenitors in TNF-deficient mice.

OBJECTIVES: The effects of TNF deficiency on myelopoiesis were evaluated in long-term (LTBMC) and short-term bone marrow cultures (STBMC) and compared to hematopoietic activity in vivo in TNF-deficient mice. METHODS: LTBMC and STBMC were established from bone marrow of TNF-deficient mice in the presence or absence of soluble TNF. Total cell production was measured over time, as well as the number of colony-forming units in culture (CFU-C). Morphology of nonadherent (NA) cells in LTBMC was assessed after 10 weeks. Bone marrow cells (BMC) and peripheral blood (PB) cells were used to determine lineage distribution within the hematopoietic system. BMC were sorted to obtain Lin(-)c-kit(+)Sca-1- and Lin(-)c-kit(+)Sca-1+ cells, which were plated in semisolid media to determine CFU-C numbers or injected into irradiated recipients to determine colony formation in the spleen (CFU-S). RESULTS: TNF-deficient LTBMC and STBMC show increased proliferative capacity, which can be inhibited by exogenous TNF to wild-type levels. Morphological analysis of NA cells from TNF-deficient LTBMC revealed increased numbers of cells at early stages of granulocytic differentiation (myeloblasts/promyelocytes) paralleled by a sharp decrease in the number of terminally differentiated polymorphonuclear neutrophils. Slightly elevated numbers of leukocytes, mainly neutrophils, were detected in PB of TNF-deficient mice. In bone marrow of TNF-deficient mice a significant increase in the number of both CFU-GM within Lin(-)c-kit(+)Sca-1- population and CFU-S within Lin(-)c-kit(+)Sca-1+ population was observed. CONCLUSIONS: TNF has inhibitory effects on granulocyte-macrophage progenitors in vitro and on committed and primitive hematopoietic progenitors in vivo. However, in adult organism TNF deficiency is mostly compensated and controlled.

Distinct and nonredundant in vivo functions of TNF produced by t cells and macrophages/neutrophils: protective and deleterious effects.

Tumor necrosis factor (TNF, TNFalpha) is implicated in various pathophysiological processes and can be either protective, as in host defense, or deleterious, as in autoimmunity or toxic shock. To uncover the in vivo functions of TNF produced by different cell types, we generated mice with TNF ablation targeted to various leukocyte subsets. Systemic TNF in response to lipopolysaccharide was produced mainly by macrophages and neutrophils. This source of TNF was indispensable for resistance to an intracellular pathogen, Listeria, whereas T-cell-derived TNF was important for protection against high bacterial load. Additionally, both T-cell-derived TNF and macrophage-derived TNF had critical and nonredundant functions in the promotion of autoimmune hepatitis. Our data suggest that T-cell-specific TNF ablation may provide a therapeutic advantage over systemic blockade.

Novel tumor necrosis factor-knockout mice that lack Peyer's patches.

We generated a novel tumor necrosis factor (TNF) null mutation using Cre-loxP technology. Mice homozygous for this mutation differ from their "conventional" counterparts; in particular, they completely lack Peyer's patches (PP) but retain all lymph nodes. Our analysis of these novel TNF-knockout mice supports the previously disputed notion of the involvement of TNF-TNFR1 signaling in PP organogenesis. Availability of TNF-knockout strains both with and without PP enables more definitive studies concerning the roles of TNF and PP in various immune functions and disease conditions. Here, we report that systemic ablation of TNF, but not the presence of PP per se, is critical for protection against intestinal Listeria infection in mice.