Summary When the tumour necrosis factor-alpha (TNF-a) gene was cloned the protein became available for use in clinical trials as an antineoplastic agent. However, side effects have severely limited its application in cancer treatment. Studies on the species specificity of TNF have indicated that the p75 TNF receptor (TNFR75) may play an important role in the generation of these side effects in humans. Using human TNF mutants with selective receptor-binding properties it has been demonstrated in neutrophils and endothelium that TNFR75 is involved in the mediation ofthe proinflammatory activity of TNF by facilitating the p55 TNF receptor {TNFR55). However, only TNFR55 appears to be involved in mediating TNF cytotoxicity. Therefore the potential exists for the successful reintroduction of TNF-a. in the form of TNFR55-selective mutants, into the clinical arena with the promise of reduced side effects.

Human tumour necrosis factor alpha (TNF‐alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF‐alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF‐alpha can be dissociated. The TNFR55‐selective mutants (R32W, E146K and R32W‐S86T) which bind poorly to TNFR75 displayed similar potency to wild‐type TNF in causing cytotoxicity of a human laryngeal carcinoma‐derived cell line (HEp‐2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55‐selective mutants exhibited lower proinflammatory activity than wild‐type TNF. Specifically, TNF‐alpha's priming of human neutrophils for superoxide production and antibody‐dependent cell‐mediated cytotoxicity, platelet‐activating factor synthesis and adhesion to endothelium were reduced by up to 170‐fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E‐selectin expression, neutrophil transmigration and IL‐8 secretion were also reduced by up to 280‐fold. On the other hand, D143F, a TNFR75‐selective mutant tested either alone or in combination with TNFR55‐selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75‐transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-α (TNF-α), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-α inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4 , and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-α was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-α neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-α to accelerate apoptosis was lost if neutrophils were primed with 1 μmol/L PAF or aged for 6 hours before TNF-α addition. The TNFR55-selective TNF-α mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-α. Although the TNFR75-selective mutant (D143F ) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-α–stimulated apoptosis. Hence, TNF-α has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.