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 α (TNF-α). Highly purified (>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-α, and total RNA was isolated from cells at various times after stimulation and assessed for TNF-α mRNA by Northern blot techniques. The results showed that message for TNF-α was produced after 60 min of in vitro stimulation with LPS and was maximal at about 4 hr. TNF-α 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.