Retro-orbital

Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Levesque, JP. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010 Aug 16;116(23):4815–28.

Abstract

In the bone marrow (BM), hematopoietic stem cells (HSC) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate regulation of these endosteal niches, we studied mobilization of HSC into the bloodstream in response to granulocyte colonystimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the i) loss of endosteal osteoblasts, ii) marked reduction of HSC-trophic cytokines at the endosteum, with iii) HSC mobilization into the blood as observed during G-CSF administration. Together these results establish that BM macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSC into the blood.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration	Total Lipid Concentration	Lipid Composition	Lipid Mole %	Liposome Type	Control Liposomes
5 mg/ml	23.4 mg/ml	EPC/Chol	84/16	MLV	PBS

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Animal Description	Clodronate Dose	Dosing Method/Site	Target Phagocytes	Local Dosing?	Local Results
C57BL/6, female mice, 8-12 w	250 µl/25 g	retro-orbital	F4/80+Ly-6G+CD11b+ osteomacs	no	N/A

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Notes

1. Liposome prep method cited — van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods. 1994 Sep 14;174(1–2):83–93.
2. Animals were dosed with clodronate (or control) liposomes on days 2 and 4.

Results

1. >95% of the F4/80+Ly-6G+CD11b+ osteomacs and other bone marrow macrophages were depleted by day 2; depletion continued throughout experiment (6 d).
2. Effects on endosteal niches began within 24 h of clodronate liposome dosing.
3. “Substantial reduction” in osteocalcin+ osteoblasts by 48 h presumably due to osteomac depletion rather than direct effect of clodronate liposomes on osteoblasts.
4. We are curious about the mechansim of osteomac depletion by liposomal clodronate since the osteomacs are shown to exclusively associate with osteoblasts which are remotely located from the sinusoid in the bone marrow. How does the liposome enter the marrow from sinusoid and migrate through the central bone marrow to the endosteum where the osteomacs are located? Since liposomes have been shown to predominantly remain near the capillaries rather than migrating into tissue (other than those carried by macrophages), we wonder if depletion of other macrophages located perisinally elicit migration of the osteomacs toward the sinusoid.