Increased numbers of OCPs have been reported in the peripheral blood of mice in several animal models of arthritis [4
] and in patients with arthritis [3
], but the mechanisms that mediate this increase have not been elucidated. In the present study, we investigated whether the SDF-1/CXCR4 axis is involved in TNF-mediated OCP mobilization because this chemokine system plays an essential role in hematopoietic stem cell and progenitor homing [6
]. We found that TNF directly inhibits bone marrow stromal cell production of SDF-1 and reduces SDF-1 levels in the bone marrow, which is accompanied with an increase in the egress of OCPs from the marrow. Decreased SDF-1 production by bone marrow stromal cells in response to TNF overexpression may therefore be one of the mechanisms mediating release of OCPs to the peripheral blood in mice with TNF-induced arthritis or in patients with inflammatory arthritis.
SDF-1-regulated cell mobilization is determined by local SDF-1 gradients and/or CXCR4 expression on target cells. Although alternation of either of these could lead to impaired cell mobilization and homing, external factor regulation of SDF-1 expression levels appears to be the major mechanism. For example, hypoxia [15
], DNA damage [14
], proteases [21
] and cytokines – including TGFβ [16
] and G-CSF [17
] – all reduce SDF-1 levels and stimulate hematopoietic stem cell release from bone marrow. Regulation of CXCR4 expression by external factors has been studied less and the results have been inconsistent. This inconsistency may be related to small numbers of CXCR4-expressing cells and low expression levels by these cells, making it difficult to reliably detect a change in the number of CXCR4-positive cells.
Our findings that TNF significantly decreases SDF-1 levels but has little effect on OCP CXCR4 expression suggest that, like most hematopoietic cell mobilizers, TNF also promotes OCP mobilization through regulation of SDF-1 rather than through CXCR4 expression. TNF-mediated OCP mobilization, however, is different from stem cell and precursor mobilization induced by SDF-1, G-CSF or other agents because TNF also has a strong stimulatory effect on OCP generation. This represents a unique pathologic situation in chronic inflammatory arthritis, in that the entire process of generation of OCPs and their egress from the bone marrow is accelerated in response to TNF. This situation leads to increased numbers of OCPs in both bone marrow and blood, whereas SDF-1 or G-CSF administration triggers a rapid release of cells from the bone marrow – and the total bone marrow cell number is consequently reduced.
We do not currently know the molecular mechanisms by which TNF inhibits SDF-1 production. SDF-1 is regulated at both transcriptional and post-translational levels [16
]. We found that TNF induced massive apoptosis of ST2 cells when a transcription or translation inhibitor was used with TNF (data not shown). In these circumstances it is therefore difficult to investigate the mechanism of action for TNF. Protease degradation is one of the major mechanisms to reduce SDF-1 protein levels [21
], and protease release from neutrophils and other myeloid cells can be stimulated by TNF. However TNF may also inhibit SDF-1 expression at the RNA level within 8 hours of treatment as shown by our data (Figure ).
TGFβ at concentrations as low as 0.01 ng/ml decreases SDF-1 mRNA expression in stromal cells [16
], implying that a small change in TGFβ could alter SDF-1 concentrations. We found that TNF increases TGFβ mRNA expression in ST2 cells. TNF administration to wild-type mice had no effect on TGFβ expression, however, although it significantly decreased SDF-1 expression in bone marrow stromal cells. Therefore it is unlikely that TGFβ mediates TNF-induced bone marrow SDF-1 downregulation in vivo
. G-CSF is another cytokine that downregulates SDF-1 mRNA expression in osteoblasts [17
]. TNF did not increase G-CSF in ST2 cells (data not shown), however, suggesting that the reduction in SDF-1 induced by TNF in vitro
is not mediated by G-CSF. Furthermore, the SDF-1 promoter does not contain binding sites typically present in the other CXC chemokine promoters, especially for NF-κB, interferon regulatory factor recognition elements or NF-IL6, which are associated with transcriptional activation in response to proinflammatory extracellular signals, such as TNF, IL-6 or interferons [22
]. These data suggest that studying SDF-1 regulation may be more complicated than studying other CXC chemokines.
The present study did not provide a direct association between TNF-reduced SDF-1 production and OCP mobilization in vivo
. We have attempted to answer this question using mice with CXCR4 specifically deleted in OCPs by generating CXCR4 CKO mice via crossing CXCR4 floxed mice [18
] with CD11b-Cre mice [19
]. We injected TNF to these CXCR4 conditional mice to determine whether TNF-induced increased OCP release is altered when CXCR4 expression has theoretically been deleted in CD11b-expressing OCPs. Unfortunately, we found that only about 50% of bone marrow CD11b+
cells have no CXCR4 surface expression in these CXCR4 CKO mice (Figure ), suggesting a low excision frequency of the Cre recombinase in our system. With this leaky system, the blood OCP frequency was similar between CXCR4 CKO mice and wild-type mice (Figure ). Our results suggest that CD11b-Cre mice appear not a good system to delete the gene encoding cxcr4
in bone marrow CD11b-positive cells.
The importance of TNF-mediated reduction in SDF-1 production in increased OCP mobilization in vivo
needs to be further confirmed using a model where SDF-1 concentration in the bone marrow is maintained in the presence of TNF. Since rheumatoid arthritis and other forms of inflammatory bone disorders are chronic diseases, however, multiple factors may contribute to promote OCP release from the bone marrow. For example, we have demonstrated that TNF-stimulated OCP formation could increase the OCP pool in bone marrow and push cell egression [20
]. Kindle and colleagues reported that TNF activates endothelial cells and increases the attachment of OCPs to vascular endothelium in vitro.
They speculated that this could increase the ability of OCPs to enter the bloodstream [23
]. It has been reported recently that RANKL-stimulated osteoclastogenesis promotes the mobilization of hematopoietic progenitor cells by cleaving SDF-1 through bone-resorbing proteinase, cathepsin K [24
]. TNF stimulates osteoclastogenesis synergistically with RANKL [25
], and this mechanism may also apply to TNF-induced OCP mobilization. The regulation of OCP mobilization is therefore a complicated process, and decreased SDF-1 expression by bone marrow stromal cells may represent another important mechanism.