Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha.

Circulating peripheral blood polymorphonuclear neutrophils (PMNs) have long been considered terminally differentiated cells that do not synthesize or secrete protein. However, work of others and ourselves has shown that PMNs can secrete the cytokine interleukin 1. In the present study we investigated whether circulating PMNs are capable of synthesizing and secreting another cytokine, tumor necrosis factor alpha (TNF-alpha). Highly purified (greater than 99% granulocytes) PMNs were isolated from normal human volunteer blood and cultured with or without bacterial lipopolysaccharide (LPS) for up to 24 hr. Cell culture supernatants were collected and tested for TNF-alpha, and total RNA was isolated from cells at various times after stimulation and assessed for TNF-alpha mRNA by Northern blot techniques. The results showed that message for TNF-alpha was produced after 60 min of in vitro stimulation with LPS and was maximal at about 4 hr. TNF-alpha was secreted into the supernatant of unstimulated PMNs from two different donors during 24 hr of culture (35-50 pg/ml), but significantly more (160-190 pg/ml) was secreted by PMNs when stimulated with LPS. PMNs from six other normal volunteers showed significant LPS-stimulated secretion of TNF at 60-180 min of culture. The secreted product also had biological activity against the TNF-sensitive L-M cell line, confirming that PMNs can make and secrete immunologically and biologically active TNF. Since it is also possible for monocytes to synthesize and secrete TNF, the amount of TNF secreted by a monocyte population equal to 20% of the PMNs cultured was measured. The results showed that monocytes at a concentration 20 times that potentially contaminating the PMN populations cultured could not produce as much TNF (unstimulated, 26-65 pg/ml; stimulated, 32-87 pg/ml). The PMN must now be considered a cell capable of altering the acute inflammatory response and modulating the immune response through the synthesis and release of cytokines.

Effect of low dose recombinant interleukin 2 plus indomethacin on mortality after sepsis in a murine burn model.

Under anaesthesia, 129 8-week-old male A/J mice were subjected to a 25 per cent scald or sham burn and then resuscitated. They were divided at random into two groups. Mice from the first group were allocated into two groups. Mice from the first group were allocated into four subgroups to receive 6 days intraperitoneal (I.P.) injections as follows: (i) recombinant human interleukin 2 (rhIL-2) (250 units day-1); (ii) saline; (iii) indomethacin (5 micrograms-1 day-1); or (iv) rhIL-2 (250 units) + indomethacin (5 micrograms). Sham burned mice served as no treatment controls. All animals were subjected to peritonitis induced by caecal ligation and puncture 10 days after the burn and mortality was assessed. Mice from the second group were allocated to two subgroups to receive 6 days intraperitoneal injections of: (i) rhIL-2 + indomethacin; or (ii) saline. Animals in this group did not undergo septic challenge. They were randomly killed on days 7, 9 or 10 after the burn. Their splenocytes were harvested and assayed for response to the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A), and for production of interleukin 2. Mortality rate in animals subjected to burn and septic challenge without treatment was 75 per cent; in mice receiving rhIL-2 alone it was 68 per cent, in mice receiving indomethacin alone it was 62 per cent (no significance) and in mice receiving rhIL-2 + indomethacin it was reduced to 38 per cent (P less than 0.02). Splenocytes from animals receiving combination therapy had markedly improved responses to PHA on days 7 (P = 0.01), 9 (P = 0.02), and 10 (P = 0.008), and to Con A on days 7 (P = 0.001), 9 (P = 0.002) and 10 (P = 0.001), after burn injury. Interleukin 2 production was also significantly (P = 0.004) improved by therapy with rhIL-2 + indomethacin. These data suggest that low dose rhIL-2 in combination with indomethacin may have potential use in the therapy of burn victims.