Cytokines and PAF release from human monocytes and macrophages: effect of hemoglobin and contaminants
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1Texas Tech University Health Sciences Center, Department of Surgery, Lubbock, 79430.
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 TNF alpha, IL-1 alpha, IL-6, GM-CSF and PAF after 6 and 24 hours, but M for TNF alpha 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.
Insights
This study investigated how different forms of hemoglobin (Hb) affect immune cells. Hemoglobin contaminated with endotoxin or phospholipids strongly activated macrophages, while polymerized Hb forms modulated cytokine release and increased TNF-alpha levels.
Area of Science:
- Immunology
- Biochemistry
Background:
- Monocytes and activated macrophage-leukocytes are key immune cells.
- Hemoglobin (Hb) is abundant in red blood cells and can be released during various conditions.
- The immunomodulatory effects of different Hb forms require further investigation.
Purpose of the Study:
- To evaluate the in vitro immune response of human monocytes and macrophage-leukocytes to unmodified, polymerized, and contaminated hemoglobin.
- To assess the release of key inflammatory mediators including TNF-alpha, IL-1 alpha, IL-6, GM-CSF, and PAF.
Main Methods:
- Human monocytes and macrophage-leukocytes were incubated with various hemoglobin preparations: unmodified Hb (UHb), low molecular weight Hb (LMWHb), high molecular weight Hb (HMWHb), Hb with endotoxin (Hb+E), and Hb with phospholipids (Hb+PLs).
- Culture supernatants were analyzed for cytokine and PAF levels at different time points (6, 12, 24, and 36 hours).
Main Results:
- Macrophage-leukocytes showed a stronger response than monocytes.
- Hb+E induced the highest levels of cytokines and PAF.
- Hb+PLs increased TNF and IL-6 but decreased IL-1 and GM-CSF, while significantly increasing PAF.
- UHb and LMWHb increased TNF levels, with LMWHb also decreasing IL-1.
- Both polymerized Hb forms (LMWHb and HMWHb) inhibited GM-CSF expression.
- HMWHb induced high levels of TNF, IL-1, and PAF.
Conclusions:
- Hemoglobin's immune-stimulating properties depend heavily on its modification and contamination.
- Endotoxin and phospholipid contamination significantly enhance the inflammatory potential of Hb.
- Polymerized Hb forms exhibit complex immunomodulatory effects, including TNF-alpha induction and GM-CSF inhibition.