Abstract

Previously, we have shown that IL-8 and G-CSF-induced hematopoietic stem cell (HSC) mobilization is inhibited in mice that underwent low dose (0.5 Gy) total body irradiation (TBI), whereas the number of progenitor cells in the bone marrow remained similar in all groups. The mechanism underlying this inhibition remains unknown. Since the release of granular proteases by neutrophils is well known to play a role in HSC mobilization, we also considered a possible role for serine protease inhibitors in the induction of HSC mobilization. Serine proteases, such as elastase and cathepsin G, are irreversibly inhibited by serine protease inhibitors including alpha-1 antitrypsin (alpha-1 AT) and alpha2-macroglobulin. In-vitro tests revealed that addition of bone marrow extracellular extracts, that were obtained from murine femurs 24 hours following low dose (0.5 Gy) TBI, inhibited the activity of exogenous elastase in a chromogenic substrate conversion assay up to 78.1 % compared to extracts obtained from sham irradiated controls (p<0.05). Since elastase inhibition by alpha2-macroglobulin cannot be detected in a chromogenic substrate conversion assay, alpha-1 AT was considered as the primary candidate serine protease inhibitor to inhibit elastase activity in our in-vitro system. Quantitative PCR of total bone marrow cells revealed that alpha-1 AT mRNA was 20-fold increased relative to the housekeeping gene ß-actin and 7-fold relative to the housekeeping genes HPRT and GAPDH at 24 hours following low dose (0.5 Gy) TBI. In addition, Western blot analysis indicated that alpha-1 AT protein concentrations were significantly (p<0.01) increased in bone marrow extracellular extracts derived from low dose (0.5 Gy) irradiated mice, compared to extracts obtained from sham-irradiated controls (5.1 ± 0.6 scanning units [SU] vs. 3.9 ± 0.7 SU for 0.5 Gy;n=8 vs. 0 Gy; n=6 respectively). To further substantiate a possible in-vivo role of alpha-1 AT in the inhibition of HSC mobilization, we administered alpha-1 AT (300 μg/mouse i.p.) at 2 hours and at 5 minutes prior to IL-8 injection (30 μg/mouse i.p.). Administration of alpha-1 AT prior to IL-8 injection completely (p<0.05) inhibited IL-8-induced HSC mobilization (472.9 ± 289.5 CFU-GM per ml blood for IL-8; n=5 vs. 44.8 ± 35.5 CFU-GM per ml blood for alpha-1 AT/IL-8; n =11). These results indicate that 1) alpha-1 AT is a potent inhibitor of IL-8-induced HSC mobilization and 2) in-vivo induced alpha-1 AT contributes to the inhibition of HSC mobilization after low-dose (0.5 Gy) TBI. We hypothesize that a critical balance between serine proteases and serine protease inhibitors plays an important role in cytokine-induced HSC mobilization.

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