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Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, which is capable of inducing apoptosis in many cell types, including tumour and virus-infected cells, but rarely in normal cells. Expression of TRAIL mRNA and TRAIL receptors has previously been detected in neutrophils; however, the expression of TRAIL protein and the regulation of TRAIL and TRAIL receptor expression in these cells remain unknown. Here we report, for the first time, that neutrophils constitutively express TRAIL protein on their cell surface and that the TRAIL protein is shed during culture. TNF-alpha is a down-regulator of TRAIL expression, whereas IFN-gamma up-regulates the expression of TRAIL. Neutrophils did not express a detectable level of TRAIL-R1 or -R4, but constitutively expressed a low, but substantial, level of TRAIL-R2 and a high level of TRAIL-R3. Although the level of TRAIL-R2 was not significantly altered during culture under different experimental conditions, approximately 30% of TNF-alpha-treated cells rapidly lost their high-level TRAIL-R3 expression, whereas the majority of IFN-gamma-treated cells retained a high level of TRAIL-R3 expression. Anti-TRAIL neutralizing antibody significantly inhibited neutrophil apoptosis during cultures in medium alone, or in the presence of TNF-alpha or IFN-gamma. Thus, our study identified human neutrophils as a cellular source of TRAIL and suggests that neutrophil-derived TRAIL may play a role in immune surveillance. Our results also suggest a role for the TRAIL/TRAIL receptor system in neutrophil apoptosis.

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.

Cattle, homozygous for the genetic disorder of factor XI (FXI) deficiency, exhibit less than 2% of normal plasma FXI activity, display an increased bleeding tendency and are more prone to infectious diseases. FXI is one of the protein components of the contact activation system of coagulation that assembles on the surface of circulating neutrophils. Because of the central role of neutrophils in inflammation, the in vitro responses of neutrophils from normal and FXI deficient cattle were compared. Neutrophil degranulation was evaluated by measuring the release of myeloperoxidase and alkaline phosphatase, and the respiratory burst was evaluated by determining superoxide anion production. Neutrophils from FXI deficient animals exhibited a significant increase (P < 0.05) in the spontaneous release of granule contents compared to the cells from normal cattle. Following stimulation with C5a complement derived from normal serum, the neutrophils from the FXI deficient animals exhibited a greater increase (P < 0.05) in both alkaline phosphatase release and superoxide production. In these neutrophils, following stimulation with C3b complement from normal serum, the relative increase in myeloperoxidase release compared to the unstimulated neutrophils was lower than that observed in the neutrophils from normal animals. There was minimal superoxide production in unactivated neutrophils from either normal or FXI deficient cattle and the response to phorbol ester stimulation was similar in both groups of animals. The C5a complement from FXI deficient serum was more effective (P < 0.05) in stimulating alkaline phosphatase release and superoxide production in normal neutrophils than the equivalent fraction from FXI deficient serum while the C3b complement from the FXI deficient serum was less effective than the normal serum fraction at inducing myeloperoxidase release from normal neutrophils. The results indicate that the differences in the in vitro neutrophil function are likely related to a variation in the function of the contact activation system on the neutrophil surface between normal and FXI deficient animals.