Characterization of tumor necrosis factor-deficient mice

Abstract

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF-/- mice) and have begun to examine the multiple functions attributed to TNF. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF-/- mice. (ii) TNF-/- mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF+/+ mice, similarly treated TNF-/- mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process-i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF+/+ mice and TNF+/- mice would have identical phenotypes, TNF+/- mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.

Figure 1

Schematic diagram of the targeted disruption of the TNF gene in mice. (A) Structure of the linked wild-type LT-α, TNF, and LT-β genes. Solid boxes indicate exons and restriction sites are indicated as follows: A, AvrI; E, EcoRI; B, BamHI; H, HindIII; S, StuI. (B) Targeting construct and (C) targeted TNF gene. Targeting results in the insertion of a PGK-neo cassette and acquisition of a novel BamHI site as indicated. The location of the Southern probes for TNF (TNF), PGK-neo (NEO), and LT-α (LT) genes are indicated. (D) Southern blot analysis of BamHI-digested genomic DNA probed with a TNF-specific probe demonstrates that the TNF gene (TNF) is disrupted by the insertion of the PGK-neo cassette yielding the wild-type 12-kb band only in TNF^+/+ mice, the shorter 11-kb fragment only in TNF^−/− mice, and both fragments in TNF^+/− mice. (E) Southern blot analysis of BamHI-digested genomic DNA using a PGK-neo-specific probe demonstrates the predicted gene dosage effect (NEO) in TNF^+/+, TNF^+/−, or TNF^−/− mice, and no other sites of integration. (F) Southern blot analysis of StuI-digested genomic DNA using an LT-α-specific probe demonstrates that the LT-α gene is not affected by integration of the targeting construct in TNF^+/+, TNF^+/−, or TNF^−/− mice.

Figure 2

Serum levels of inflammatory mediators after i.p. injection of 100 μg LPS. TNF^+/+ mice (▪), TNF^−/− mice (□); ∗, P < 0.05 compared with TNF^+/+ mice.

Figure 3

Survival after treatment with the indicated doses of (A) LPS alone, (B) LPS + D-gal (20 mg), (C) TNF. TNF^+/+ mice (▪), TNF^+/− mice (▴), TNF^−/− mice (□); ∗, P < 0.05 compared with TNF^+/− mice.

Figure 4

(A) Survival after infection with C. albicans. TNF^+/+ mice (▪), TNF^+/− mice (▴), TNF^−/− mice (□). (B) Colony counts recovered from organs at the indicated times after infection. TNF^+/+ mice (solid bars), TNF^−/− mice (open bars); ∗, P < 0.05 compared with TNF^+/− mice.

Figure 5

Liver and spleen weight after C. parvum. TNF^+/+ mice (solid columns), TNF^+/− mice (shaded columns), TNF^−/− mice (open columns); ∗, P < 0.05 compared with TNF^+/+ mice.

Figure 6

Histology of liver and spleen from C. parvum-treated TNF^+/+ and TNF^−/− mice (hematoxylin/eosin, ×200). (A–D) Liver and spleen sections from mice 10 days after C. parvum treatment showing characteristic inflammatory responses in liver (A) and spleen (C) of TNF^+/+ mice and the absence of these responses and the preserved architecture of the liver (B) and spleen (D) of TNF^−/− mice. (E–H) Liver and spleen sections from mice 40 days after C. parvum treatment showing the almost normal morphology typical of the down-regulated inflammatory response in liver parenchyma (E) and spleen (G) of TNF^+/+ mice, and the late inflammatory infiltration in liver (F) and spleen (H) of TNF^−/− mice.