Cvtokines and Paf Release from Human Monocytes and Macrophages: Effect of Hemoglobin and Contaminants

Abstract

Monocytes [M] were isolated from venous blood of healthy volunteers and activated macrophage-leukocytes (MØ-L] were obtained from peritoneal fluid of patients with mild endometriosis. The M were incubated with pyrogen free CELLGRO culture medium [Control], and with 0.2 mM of [A] unmodified bovine hemoglobin (UHb), [B] Hb crosslinked to form polymers with M.W.< 400 kDa (LMWHb), [C] Hb crosslinked to form large polymers (< 1,020 kDa) (HMWHb), and MØ-L additionally with [D] UHb contaminated with endotoxin (Hb+E) (2.5 EU/mL), and [E] UHb contaminated with phospholipids (Hb+PLs). The MØ-L medium of incubation was tested for TNFα, IL-lα, IL-6, GM-CSF and PAF after 6 and 24 hours, but M for TNFα and GM-CSF at 12, 24 and 36 hours. MØ-L were found more responsive than M colonies. The strongest reaction of MØ-L was to Hb+E, which produced levels of cytokines and PAF higher than Controls (p < 0.001). Hb+PLs induced smaller increases of TNF and IL-6, and a decrease in the levels of IL-1 and GM-CSF. However, the release of PAF was much greater with this Hb than with Hb+E. UHb caused an increase in TNF, as compared to control (p < 0.01). LMWHb generated a similar increase in TNF, but also a decrease in IL-1. Both polymerized Hb forms inhibited expression of GM-CSF. HMWHb induced high levels of TNF, IL-1 and PAF. UHb, LMWHb and HMWHb significantly increase levels of TNF in M cultures after 36 hours of incubation.