Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo

Tumor necrosis factor-alpha (TNFalpha) exists in two biologically active forms, a 26-kDa transmembrane form and a proteolytically cleaved and secreted form. We sequentially inactivated all three known cleavage sites of mouse TNFalpha by mutating the corresponding DNA sequences. A murine T cell hybridoma transfected with the nonsecretable mutant TNFalpha efficiently lysed L929 target cells in a cell contact-dependent manner and induced expression of vascular cell adhesion molecule-1 on mouse endothelioma cells. A genomic mouse TNFalpha clone encoding this mutant was subsequently introduced as a transgene into TNFalpha(-/-) lymphotoxin-alpha(-/-) mice. The 3' AU-rich regulatory elements of the TNF locus were maintained in the transgene to assure adequate gene regulation. Transmembrane TNFalpha transgenic mice were fully protected from endotoxic shock, and no TNFalpha bioactivity was detectable in the serum after stimulation with lipopolysaccharide. Activated CD4 T cells from these Animals, however, lysed L929 cells in a cell contact-dependent way. After administration of lipopolysaccharide, transmembrane TNFalpha transgenic mice produced significantly higher levels of interleukin-12 than wild-type mice or TNF-deficient mice. This indicates that transmembrane TNFalpha may greatly affect the course of a cellular immune responses in vivo and exerts quantitatively and qualitatively distinct functions from secreted TNFalpha in vitro and in vivo.