Background
Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and supports the survival, proliferation, and maturation of myeloid progenitor cells along the neutrophil (PMN) lineage [
1]. G-CSF also activates certain functions of mature PMN and stimulates hematopoietic stem cell mobilization [
2‐
6]. The growth of neutrophilic granulocytes
in vitro from progenitor cells committed to neutrophils and monocytes (CFU-GM) is absolutely dependent upon G-CSF and sigmoidally increases with increasing G-CSF concentrations [
2,
5,
7,
8]. A critical role for G-CSF in regulating granulopoiesis
in vivo has been demonstrated in G-CSF null mice who have chronic neutropenia and severely impaired granulopoietic responses to infection [
6].
The biological activities of G-CSF are mediated by the G-CSFR receptor (G-CSFR), a transmembrane protein predominantly expressed on the surface of cells of the neutrophil lineage [
7]. Like other cytokine receptors, the extracellular portion of the G-CSFR binds ligand and the cytoplasmic tail transduces intracellular signals [
3,
4,
7]. Studies of mice with knock-out or knock-in mutations in their G-CSFR gene have suggested the G-CSFR generates unique signals required for PMN production and marrow egress to maintain homeostatic levels of circulating PMN during basal and stress granulopoiesis [
9‐
12].
G-CSFR null mice have chronic neutropenia, a uniform decrease in myeloid cells in the bone marrow, and defects in PMN activation [
6,
10]. Competitive repopulation assays in these mice indicate G-CSF drives nearly all of granulopoiesis under basal conditions and that G-CSFR signals regulate the
in vivo production and maintenance of both committed-myeloid progenitor cells and primitive multipotential progenitors [
12]. Additional insights have come from mice expressing a chimeric G-CSFR (GEpoR) comprised of the extracellular ligand-binding domain of the G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor (EpoR) [
13]. GEpoR mice retain the ability to produce PMN but have chronic neutropenia, and despite near normal bone marrow PMN levels, G-CSF treatment fails to mobilize significant numbers of PMN into the peripheral blood.
The regulated manner in which PMN are produced and released into the circulation suggests that positive regulation of granulopoiesis via G-CSF/G-CSFR interactions must be balanced by negative feedback loops [
14,
15]. However, little is known about the mechanisms downregulating G-CSFR surface expression to negatively regulate granulopoiesis. An
in vivo role for neutrophil granule enzymes in both modulating PMN and stem cell mobilization and in downregulating G-CSFR surface expression on PMN was previously suggested by Jilma [
16]. Subsequent studies by Levesque
et al identified neutrophil elastase (NE) as a neutrophil granule enzyme that promotes stem cell mobilization by cleaving chemokines and chemokine receptors [
17,
18], such as stem cell derived factor-1 (SDF-1) and its corresponding receptor, CXCR4 [
19].
Recent studies by our laboratory and others indicate that NE also degrades G-CSF and inhibits G-CSF-stimulated proliferative responses
in vitro[
20,
21]. The paradigm of both ligand and receptor cleavage provided by SDF-1 and CXCR4 prompted us to investigate whether NE also cleaves the G-CSFR to modulate its expression and signaling and whether it might be the putative granule enzyme in PMN reported by Jilma that decreases G-CSFR surface expression [
16]. Here, we show that NE proteolytically cleaves the G-CSFR to downregulate its expression on PMN and that it also inhibits G-CSFR-mediated granulopoiesis
in vitro. These results suggest a novel role for NE as a negative regulator of granulopoiesis.
Discussion
A remarkable feature of granulopoiesis is the regulated production and release of PMN to maintain homeostatic levels in the circulation during basal granulopoiesis and to rapidly increase numbers during environmental stress [
15]. G-CSF is the major cytokine regulating granulopoiesis [
3,
6,
7], and its regulatory capacity depends upon its ability to bind to the G-CSFR. Thus, both G-CSF concentration and G-CSFR numbers modulate myeloid cell responsiveness and, hence, PMN numbers. The physiologic processes that regulate G-CSF levels have been well-characterized [
7], but little is currently known about the mechanisms modulating G-CSFR surface expression.
Ligand-binding has been shown to trigger endocytosis and internalization of most growth factor receptors, which are then either recycled back to the cell surface or degraded intracellularly [
3,
24]. Ligand-induced internalization decreases the number of surface receptors and thereby serves to attenuate growth factor-induced signals [
25‐
27]. In the case of the G-CSFR, ligand binding has been shown to modulate G-CSFR surface expression
in vitro[
23,
28]. Following ligand binding, the G-CSFR on immature myeloid cells, U937 cells, and PMN is rapidly internalized and degraded [
28]. More than 70% of specifically bound G-CSF is internalized after 5 min. Treatment of PMN with GM-CSF, TNF, LPS, fMLP, TPA, or C5a also downregulates G-CSFR numbers, while only TPA significantly reduces G-CSFR numbers on immature cells. There is no evidence the G-CSFR is recycled back to the cell surface.
Studies of naturally-occurring G-CSFR deletion mutants isolated from patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have provided evidence for the importance of downregulation of G-CSFR expression [
29,
30]. A critical cytoplasmic domain that mediates G-CSFR internalization and degradation has been shown to be deleted in cells from these patients, which exhibit enhanced growth and survival signals to G-CSF [
21,
23,
31,
32]. G-CSFR surface expression is prolonged and G-CSF-mediated activation of Stat5 and Akt are sustained in these cells, indicating that receptor downregulation plays a critical role in extinction of G-CSFR signals.
There is also evidence that the G-CSFR on PMN is downregulated
in vivo in response to G-CSF. Jilma
et al showed that a single injection of G-CSF decreased G-CSFR numbers on PMN in humans by ~75% [
16]. These effects were noted as early as 6 min, peaked at 90 min, and did not return to pre-treatment levels for 2 days. Notably, PMN numbers were transiently decreased and plasma levels of the neutrophil granule enzyme gelatinase b (also known as matrix metalloproteinase-9 or MMP-9) were increased 10-fold after G-CSF administration, implying a role for gelatinase b in both decreasing G-CSFR levels and PMN numbers. In related studies of the
in vivo effects of lipopolysaccharide (LPS) infusion on G-CSFR expression, a significant negative correlation between PMN activation and G-CSFR expression was found [
33]. The effects were reported to be independent of LPS-induced increases in G-CSF levels
in vivo, suggesting that PMN activation and G-CSFR expression are tightly co-regulated.
In the current paper, we have identified an alternative mechanism for modulating G-CSFR expression on PMN involving the primary granule enzyme NE. Our data provide the first evidence that NE cleaves the endogenously expressed G-CSFR on PMN and inhibits G-CSFR-mediated granulopoiesis in vitro. Treatment of PMN with NE induced a time-dependent reduction in G-CSFR surface expression and the appearance of G-CSFR cleavage fragments in conditioned media from treated PMN. Both serum and PMSF could prevent degradation of the G-CSFR, suggesting NE degrades the G-CSFR by enzymatic cleavage. The time-course for the appearance of G-CSFR cleavage fragments in conditioned media from NE-treated PMN correlated well with the decrease observed in G-CSFR surface expression on treated PMN as detected by flow cytometry. We also show that NE abrogates proliferative signals generated by the G-CSFR in myeloid progenitor cells, as indicated by the decreased number of CFU-GM arising from NE-treated marrow progenitor cells. Our data demonstrate that NE cleaves the G-CSFR at a site within its extracellular portion, within which lies the ligand-binding site for G-CSF. Notably, proteolytic cleavage of the G-CSFR within this region is predicted to modify the binding site for G-CSF and thereby affect the sensitivity of cells to G-CSF, consistent with our data.
We [
21]and others [
20] have previously reported that NE also cleaves G-CSF and antagonizes its
in vitro activity. However, unlike El-Ouriaghli
et al who could not demonstrate an effect of NE on the G-CSFR [
20], we reported that NE could also cleave the G-CSFR on transfected Ba/F3 cells [
21]. It is possible that El-Ouriaghli's group failed to observe an effect on the G-CSFR due to the significantly longer period of culture (up to seven days) in NE-containing media they used before analyzing G-CSFR expression or because of the lower pH (5.5 vs.7.2) of their reconstituted NE.
NE has been reported to cleave multiple substrates in addition to G-CSF and the G-CSFR including SDF-1, CXCR4, VCAM-1, CD14, CD23, and complement receptor 1 (CR1) [
17‐
19,
34‐
37]. For many cytokine and chemokine receptors that are cleaved by NE, the extracellular ligand binding region is the site of cleavage [
17‐
19,
36,
38]. Our findings with NE and the G-CSFR suggest a similar role for NE in regulating both cytokine and its respective receptor levels as reported for SDF-1 and CXCR4 [
19]. Inactivation of SDF-1/CXCR4 interactions by NE was shown to induce hematopoietic stem cell mobilization. In G-CSF-induced stem cell mobilization, NE levels increase
in vivo in the plasma and bone marrow microenvironment where PMN accumulate [
17,
36,
39].
More recently, NE was reported to downregulate expression of c-KIT (CD117), the receptor for SCF [
18]. Decreased c-KIT expression and increased NE levels were demonstrated in the marrows of mice receiving G-CSF for stem cell mobilization. Similar to our findings with the G-CSFR, a 2 h incubation with NE was required to induce a 50% reduction in c-KIT surface expression.
During G-CSF-induced stem cell mobilization, plasma levels of NE dramatically increase reaching levels of approximately 1 mg/ml. Within individual azurophilic granules from activated PMN, concentrations of NE in excess of 5 mM (150 mg/ml) have been measured. Thus, the concentrations of NE (0-150 μg/ml) and serum (a source of alpha-1 anti-trypsin) used in our experiments are well within the ranges reported
in vivo[
40‐
44].
The functional significance of our findings in vivo remains speculative, as we did not directly examine this. However, NE-induced downregulation of G-CSFR expression in vivo could promote cellular egress or inhibit further expansion of the myeloid compartment. A possible scenario is that during stem cell mobilization, release of NE from accumulating PMN in the bone marrow functions to inhibit granulopoiesis by degrading the G-CSFR and thereby preventing progressive and uncontrolled neutrophilia.
Our findings may have particular relevance to understanding the pathogenesis of SCN. In the majority of patients with this disease, mutations in the ELA2 gene encoding NE have been identified, some of which result in aberrant targeting of NE to the plasma membrane [
45]. It is possible that such NE mutants induce aberrant and/or accelerated cleavage of the G-CSFR in some cases of SCN.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MGP wrote the manuscript, designed and performed experiments. PM, LD, and TK assisted in experiments. ML and AC contributed to data analysis. BRA designed experiments and wrote the manuscript. All authors read and approved the final manuscript.