POLYMYXIN-B STIMULATES TUMOR-NECROSIS-FACTOR-ALPHA PRODUCTION BY HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS

Gram-negative bacterial lipopolysaccharide (LPS) is a well known stimu lus for cytokine production, particularly interleukin-1 (IL-1) and tum or necrosis tactor alpha (TNF alpha). Polymyxin B (PMX-B) is a cationi c polypeptide that binds to LPS, neutralizing its biological effects. PMX-B also disrupts gram-negative bacterial cell membrane phospholipid s but is highly toxic to mammalian cells, therefore is of limited use. PMX-B is used as additive to media, as a way to handle LPS contaminat ion. To derive benefit from the ability of PMX-B to neutralize lipid A in vivo while avoiding its systemic toxicity, PMX-B was covalently bo und to polystyrene-derivative fibers, creating a hemoperfusion column (PMX-F) for the selective removal of circulating ET: In vitro PMX-F he moperfusion studies have demonstrated effective ET removal, using eith er the Limulus amebocyte lysate assay or TNF alpha production by perip heral blood mononuclear cells (PBMC) as an index of ET removal. Howeve r, the question whether PMX-B itself could stimulate human PBMC to pro duce cytokines has not been adequately addressed. We examined the effe ct of increasing concentrations of PMX-B on cytokine production by PBM C in vitro. PBMC harvested from healthy volunteers were incubated for 24 hours at 37 degrees C with control (tissue culture media RPMI), or 5 mu g/ml, 10 mu g/ml, 20 mu g/ml or 100 mu g/ml PMX-B. At the end of 24 hours, PBMC were subjected to three freeze-thaw cycles, and total T NF alpha production (pg/2.5x10(6) PBMC) was measured by radioimmunoass ay. Total TNF alpha production by PBMC was 163 +/- 3 pg, 171 +/- 9 pg 164 +/- 4 pg 323 +/- 63 pg and 331 +/- 58 pg, in the control, PMX-B 5 mu g/ml, 10 mu g/ml, 20 mu g/ml and 100 mu g/ml conditions, respective ly Compared to controls (RPMI), the percentage increase in TNFa produc tion by PBMC was 5 +/- 6% (P=0.23), 1 +/- 3% (P=0.45), 99 +/- 40% (P=0 .03) and 103 +/- 36% (P=0.02) in the presence of 5 mu g/ml, 10 mu g/ml , 20 mu g/ml and 100 mu g/ml of PMX-B, respectively. Furthermore, tota l TNF alpha production correlated significantly with increasing concen trations of PMX-B (R=0.53, P=0.007). We conclude that the use of PMX-B in in vitro studies as an LPS-neutralizing agent, or in the experimen tal treatment of endotoxic or septic shock can lead to erroneous inter pretations of cytokine production by PBMC, and should be used cautious ly in in vitro systems at high concentrations.