Hypoxia-Inducible Factor 1α Expression and Activity in LAP PMN

Objective:

The oxidative burst in LAP PMN is significantly increased leading to PMN-mediated tissue injury. Hypoxia inducible factor-1α (HIF-1α) is a recently identified protein that has been negatively associated with activation of innate immunity. The aim of this study was to determine the relationship of HIF-1α to PMN O₂^- generation in LAP PMN.

Methods:

Human peripheral blood PMN from LAP (n=9) and control (n=10) subjects were isolated. Expression of HIF-1α was measured by FACS analysis, Western blotting, RT and quantitative PCR at protein and mRNA levels. HIF-1α is induced by hypoxia, which converts ferrous (Fe²⁺) to ferric ion (Fe³⁺). Ferrous ion was measured by ο-phenanthroline and ferric iron content was measured by hydroxylamine hydrochloride (0.33%w/w).

Results:

PMN from LAP had increased intracellular ferric ion (329.4%; p=0.019) and decreased ferrous ion. Both prolylhydroxylase domains, PHD-2 and PHD-3, which are involved in HIF-1α degradation, were significantly reduced in LAP PMN (p<0.05) at the mRNA level. In PMN from healthy individuals, deferoxamine (DFO; 1-100μM) chelated Fe²⁺ and increased HIF-1α expression. LAP PMN were resistant to DFO-induced elevation of HIF-1α and Fe³⁺.

Conclusion:

O₂^- generation is ferric ion dependent in normal cells. The increase in LAP O₂^- generation is associated with increased ferric ion suggesting pre-activation or priming of LAP PMN. Taken together, the data suggest that LAP PMN are primed and that HIF-1α plays a regulatory role maintaining ferrous ion steady state intracellular concentrations in resting PMN. Supported by USPHS grants DE16191 and RR00533.