The effect of lipopolysaccharide (LPS) on cord-blood hemopoietic progenitors

Abstract

RATIONALE: The Hygiene Hypothesis has focused attention on the interaction of the innate immune system and the microbial environment. Since hemopoietic mechanisms are involved in the maintenance and development of atopy, we studied the effect of the gram-negative bacterial endotoxin, lipopolysaccharide, on cord-blood hemopoietic progenitors. METHODS: Non-adherent mononuclear (NAMNC) human cord blood cells were enriched for CD34+ hemopoietic progenitors using magnetic activated cell sorting (MACS), either via depletion of CD3+/CD16+ cells (n=9) or via positive CD34+ selection (n=14), then incubated for 24 hours in varying doses of LPS (0-, 0.01, 0.1, 1 and 10 mcg/ml), followed by washing and incubation in methylcellulose colony-forming assays (CFU) in the presence of IL-3, IL-5 or GM-CSF. Eosinophil/basophil (Eo/B) CFU were enumerated at 14 days, and analysis performed using Friedman's ANOVA. RESULTS: There was a significantly positive dose-response of Eo/B CFU to LPS using either enrichment method for CD34+ cord blood populations. For CD3/CD16 depleted NAMNC, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (2.5 compared to 11.5 CFU/2x105 cells plated, p= 0.018); overall LPS effect (p=0.047). For CD34-selected cells, there were more Eo/B CFU with the optimal LPS dose (10 mcg/ml) compared to control (3 compared to 8.25 CFU/104 cells plated, p= 0.033); overall LPS effect (p=0.048). CONCLUSIONS: CB NAMNC, specifically CD34+ cells, are capable of responding to LPS and hemopoietic cytokines functionally, by differentiating into mature eosinophils and basophils. The findings highlight a potential mechanism through which microbial stimuli could directly modulate atopy and/or allergic inflammation.