The contribution of neutrophils to HSPC mobilization by G-CSF is controversial. Neutrophils are a major source of proteases that have been implicated in HSPC mobilization, including neutrophil elastase, cathepsin G, and MMP-9 (Kjeldsen et al., 1994
; Borregaard and Cowland, 1997
). However, the importance of these proteases is controversial, as mice lacking these proteases exhibit a normal mobilization response to G-CSF (Robinson et al., 2003
; Levesque et al., 2004
; Pelus et al., 2004
). There is strong evidence showing that HSPC mobilization by the chemokine IL-8 is dependent on neutrophils. Csf3r−/−
mice, which are profoundly neutropenic, fail to mobilize in response to IL-8 (Liu et al., 1997
). Moreover, antibody-mediated depletion of neutrophils (using anti-CD11a or anti–Gr-1 antibodies) abrogated IL-8–induced HSPC mobilization (Pruijt et al., 1998
). With respect to G-CSF, Pelus et al. (2004)
reported that neutrophil depletion using anti–Gr-1 antibodies attenuated G-CSF–induced HSPC mobilization. However, Gr-1 is expressed on both neutrophils and a subset of monocytes; thus, a role for reduced monocytes in this phenotype is possible. In the current study, we demonstrate, using G-CSFR–deficient bone marrow chimeras, that the mobilization response to G-CSF is poorly correlated with the number of wild-type neutrophils. Moreover, CD68:G-CSFR mice, which are neutropenic and have barely detectable expression of G-CSFR on neutrophils, exhibit a normal mobilization response to G-CSF. These data, although not excluding a role for neutrophils, strongly suggest that G-CSF signals in neutrophils are not sufficient to induce normal HSPC mobilization.
The contribution of lymphocytes to G-CSF–induced HSPC mobilization is also controversial. Reca et al. (2007)
reported that G-CSF–induced mobilization is impaired in Rag2−/−
, SCID, and Jh mice and that this deficit can be reversed through administration of complement-inducing immunoglobulin. In contrast, Katayama et al. (2006)
reported that Rag1−/−
mice and IL-7R−/−
mice exhibited a normal mobilization response to G-CSF. Consistent with the latter findings, we observed that Rag1−/−
mice exhibit a normal mobilization response to G-CSF. The basis for these discrepancies remains unclear. Although all of these mouse lines share deficits in B and T lymphopoiesis, there are subtle differences. For example, natural killer activity is normal in Rag1−/−
mice but is absent in NOD/scid/IL-2
mice. In any case, our data strongly suggest that neither B and T lymphocytes nor NK cells are required for a normal mobilization response to G-CSF.
There is accumulating evidence that monocyte lineage cells in the bone marrow contribute to osteoblast homeostasis and HSPC trafficking. Chang et al. (2008)
demonstrated that macrophages are anatomically juxtaposed with endosteal osteoblasts, forming a canopy over the osteoblasts at sites of bone formation. Moreover, they showed that ablation of monocytic cells using the MAFIA transgenic mouse model resulted in a loss of osteoblasts (Chang et al., 2008
). Similarly, Winkler et al. (2010)
recently showed that macrophage ablation using the MAFIA transgenic mouse model or through administration of clodronate-loaded liposomes resulted in a loss of osteoblasts and HSPC mobilization. Finally, Chow et al. demonstrated in a companion paper in this issue that depletion of monocytic lineage cells using a variety of methods is sufficient to induce mobilization of HSPCs. Together, these data strongly suggest that monocytic cells produce trophic factors required for osteoblast maintenance and HSPC retention. Consistent with this conclusion, we show that macrophages support osteoblast growth in vitro, at least in part, through production of a soluble factor. The identity of these factors is currently unknown.
In this study, we provide novel evidence that G-CSFR signaling in monocytic cells is sufficient to induce HSPC mobilization. We generated transgenic mice in which expression of the G-CSFR is mainly limited to cells of the monocyte lineage and showed that G-CSF–induced HSPC mobilization, osteoblast suppression, and decrease in CXCL12 expression are similar to that control mice. In the bone marrow, there are at least four distinct monocytic cell populations: inflammatory monocytes/macrophages, resident monocytes/macrophages, myeloid dendritic cells, and osteoclasts. Because the CD68 transgene used in our study is expected to direct G-CSFR expression in each of these cell populations, all of them are candidates to mediate HSPC mobilization. There is considerable (though conflicting) data on the role of osteoclasts in HSPC mobilization. Kollet et al. (2006)
reported that activation of osteoclasts by injection of RANKL (RANK ligand) was associated with moderate HSPC mobilization, and inhibition of osteoclasts, either genetically by knocking out PTPε
or by injecting mice with calcitonin, blunts the mobilization response to G-CSF. It is of note that osteoclasts produce the protease cathepsin K, which can cleave CXCL12 in vitro (Drake et al., 1996
; Kollet et al., 2006
). In contrast, other studies indicate that osteoclasts may actually inhibit mobilization, as mice that were given pamidronate, an osteoclast-inhibiting bisphosphonate, exhibit increased mobilization in response to G-CSF (Takamatsu et al., 1998
; Winkler et al., 2010
). In contrast to osteoclasts (Takamatsu et al., 1998
; Winkler et al., 2010
), we show that inflammatory and resident monocytes/macrophages decrease after G-CSF treatment. It is of note that the timing of the decrease in these cell populations during G-CSF treatment is similar to that reported for the decrease in osteoblasts (Christopher and Link, 2008
) and precedes HSPC mobilization. Definitive identification of the monocytic cell population that mediates G-CSF–induced HSPC mobilization will require further study.
In summary, we provide evidence that monocytic cells in the bone marrow are sufficient to elicit HSPC mobilization and osteoblast suppression by G-CSF. These data suggest a model in which monocyte lineage cells in the bone marrow produce trophic factors required for the maintenance of osteoblasts. G-CSF–induced suppression of monocytic cells and/or signaling in these cells results in decreased production of the putative trophic factors, suppression of osteoblast lineage cells (and CXCL12 expression), and ultimately HSPC mobilization. The precise monocytic cell population and factors produced by these cells that regulate osteoblast lineage cells are areas of active investigation.