Published online before print January 2, 2004
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* Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, The Netherlands;
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands; and
Department of Medicine, Duke University Medical Center, Durham, North Carolina
1Correspondence: University Medical Center, Room KC03.068.0, Lundlaan 6, 3584 EA, Utrecht, The Netherlands. E-mail: c.heijnen{at}wkz.azu.nl
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Key Words: G protein-coupled receptor • knockout mice • migration • SDF-1 • mobilization
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In adult mice, the CXCR4/SDF-1 chemotactic axis is involved in the regulation of mobilization of hematopoietic progenitor cells and mature neutrophils from the bone marrow in response to granulocyte-colony stimulating factor (G-CSF), which plays a critical role in production and maturation of neutrophils [10 , 11 ] but is also known to be a potent stimulus for inducing neutrophil release from the bone marrow [12 13 14 ]. The release of immature and mature neutrophils in response to G-CSF from the bone marrow has been associated with the inactivation of the SDF-1/CXCR4 chemotactic pathway [15 , 16 ]. Upon treatment with G-CSF, the level of SDF-1 protein in the bone marrow is significantly decreased [14 , 15 , 17 ]. It is interesting that the degree of neutrophil mobilization in response to G-CSF is correlated with the magnitude of the decrease in bone marrow SDF-1 [14 ]. In addition, in mice reconstituted with CXCR4-deficient bone marrow cells, premature release of granulocytic precursors was observed [5 , 18 ]. These data suggest that CXCR4 activation by SDF-1 provides a key retention signal for hematopoietic cells in the bone marrow.
CXCR4 belongs to the class of G protein-coupled receptors (GPCRs). Members of a family of seven GPCR kinases (GRKs) can attenuate the responsiveness of GPCRs [19 20 21 ]. Phosphorylation of agonist-activated receptors by GRKs induces a rapid uncoupling of the receptor from G proteins, a process called homologous desensitization. GRK-mediated phosphorylation of the agonist-bound receptor induces uncoupling by promoting the binding of members of the arrestin family to the phosphorylated receptor, which then prevents further G protein activation and enhances receptor internalization [19 20 21 ]. The cellular levels of GRKs and arrestins are thought to be very important in this process, as this determines the extent of receptor desensitization and internalization [22 , 23 ].
GRK2, -3, -5, and -6 are particularly highly expressed in cells of the immune system [24 , 25 ]. Recently, it was demonstrated that GRK6 is an important regulator of CXCR4 activity in lymphocytes. T cells genetically deficient in GRK6 displayed changes in CXCR4 signaling and SDF-1-induced chemotactic responses [26 ]. It is interesting that expression levels of GRK6 were shown to be altered during differentiation of the promyelocytic cell line HL-60 toward the neutrophil lineage, suggesting a functional role for this GRK in regulating GPCR responsiveness in neutrophils as well [25 ].
In this study, we used mice with a targeted deletion of the GRK6 gene to investigate the possible role of GRK6 in regulation of CXCR4-mediated neutrophil chemotaxis in vitro. In addition, we assessed the effect of GRK6 absence on the G-CSF-induced rise in circulating neutrophils in vivo.
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G-CSF
Pegylated G-CSF (peg-G-CSF), synthesized by coupling recombinant G-CSF N-terminally to 20 kDa polyethylene glycol, was obtained from Amgen (Thousand Oaks, CA). Peg-G-CSF was administered subcutaneously (s.c.) at a dose of 15 µg in 150 µl phosphate-buffered saline (PBS). At different time points, peripheral blood was obtained via puncture of the orbital plexus or of the heart. Whole blood cell counts were performed using a Bürker counting chamber, and leukocyte differentials were performed on May-Grünwald/Giemsa-stained blood smears.
Isolation of bone marrow cells
Femurs and tibias were cleaned and subsequently crushed in a mortar. A total bone marrow suspension was obtained and resuspended in RPMI containing 5% fetal calf serum (FCS). The suspension was filtered and subsequently centrifuged for 5 min at 1200 rpm. Leukocyte differentials of the bone marrow were performed on May-Grünwald/Giemsa-stained cytospins of total bone marrow cell suspensions (in RPMI+5% FCS). For chemotaxis assays, erythrocytes and most primitive (blast) cells were removed using Percoll gradients (Amersham Biosciences AB, Uppsala, Sweden). Subsequently, cells were resuspended in RPMI + 0.5% FCS and were counted.
Chemotactic assay
Cell migration was assessed using a Transwell system with 5 µm pore filters (Costar, Cambridge, MA). Before use, the filters were coated overnight at 4°C with 20 µg/ml fibronectin (in PBS). Subsequently, cells were placed in the top well in a total volume of 100 µl and 600 µl SDF-1 (R&D Systems, Minneapolis, MN), or medium (RPMI 1640 with 0.5% FCS) was added to the lower chamber. After 2 h incubation at 37°C, cells in the bottom chamber were collected, and the number of migrated PMN was determined by flow cytometry, based on forward- and side-scatter characteristics (FACSCalibur, Becton Dickinson, San Jose, CA).
Calcium reponse
Bone marrow neutrophils were loaded with Fluo-3 AM(Molecular Probes, Eugene, OR) and stimulated with SDF-1 in RPMI 1640 supplemented with 0.05% bovine serum albumin (BSA). Changes in mean fluorescence intensity (MFI) were monitored using a FACSCalibur. Traces represent MFI for 200–300 cells/s sampled in 500-ms periods.
Colony assay
Bone marrow cells were plated in duplicate at densities of 1 x 104/ml in methylcellulose medium supplemented with 30% fetal bovine serum, 1% BSA, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, and G-CSF (100 ng/ml). Colonies (30 cells or more) were counted after 6 days of culture at 37°C.
Data analysis
Data are expressed as mean and SEM and were collected in independent experiments using three pairs of animals in each experiment. Wild-type (WT) and knockout samples were analyzed in parallel. Data were analyzed by Student’s t-test or two-way ANOVA. P < 0.05 was considered statistically significant.
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Figure 1. Chemotactic response of bone marrow-derived PMN from WT and GRK6−/− mice. Open bars, WT; solid bars, GRK6−/− animals. PMN chemotaxis was determined in a Transwell assay. Medium alone or increasing doses of SDF-1 were added to the lower well, and cells were placed in the upper well in medium for 2 h. Spontaneous cellular motility was determined with medium in upper and lower wells. To determine the chemokinetic response (kinesis), 100 ng/ml SDF-1 was added to the upper and the lower wells. Data represent percentage of cells from the total number used that reached the lower well. n = 5–10 per group; **, P < 0.01 (two-way ANOVA).
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Figure 2. Desensitization of the calcium response to SDF-1. Bone marrow-derived PMN were loaded with Fluo-3, and the change in MFI as an indicator of changes in intracellular calcium concentration was monitored by flow cytometry. Basal Fluo-3 fluorescence was recorded for 15 s, and cells were stimulated with 100 nM SDF-1. The response to SDF-1 was monitored for 60 s. Cells were kept in the presence of SDF-1 for 5 min, washed extensively on ice, and resuspended in warm medium, and a second baseline was recorded followed by resimulation with 100 nM SDF-1. As a control, cells were subsequently stimulated with formyl-Met-Leu-Phe (fMLP; 300 nM). Dotted line, Control; solid line, GRK6−/− PMN. Data are from one representative animal out of three in each group.
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Figure 3. (A) Neutrophil response to peg-G-CSF treatment. Open bars, WT; solid bars, GRK6−/−. Mice were given a single s.c. dose of peg-G-CSF (15 µg), and the number of blood PMN/ml was analyzed at the indicated times. Data represent mean ± SEM. (B) Short-term neutrophil response to peg-G-CSF. Circulating PMN/ml were determined before and 3 h after s.c. injection of 15 µg peg-G-CSF. Data are expressed as percentage increase over basal PMN/ml and represent mean ± SEM. n = 5–10 animals per group; *, P < 0.05; **, P < 0.01 (Student’s t-test).
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115% over baseline. However, the neutrophil response in GRK6−/− animals was significantly reduced, as compared with WT mice. PMN levels increased by only 38% in the GRK6−/− animals, suggesting that G-CSF-induced neutrophil mobilization is impaired in the absence of GRK6 (Fig. 3B) .
Cellular composition of the bone marrow before and after GCSF treatment
The observation that the peg-G-CSF-induced increase in blood PMN levels at day 3 is impaired in the absence of GRK6 might result from impaired mobilization but could also result from reduced neutrophil production or residence in the bone marrow. Therefore, we analyzed the cellular composition of bone marrow from WT and GRK6−/− animals. Under basal conditions, no differences were observed in percentages of immature myeloid cells, eosinophils, monocytes/macrophages, and lymphocytes between WT and GRK6−/− mice (data not shown). In addition, percentages of cells belonging to the granulocytic lineage, such as band neutrophils and polymorphonuclear neutrophils, did not differ between WT and GRK6−/− bone marrow either (data not shown). Thus, the reduced ability of G-CSF to mobilize bone marrow neutrophils does not result from a lower resident neutrophil population in the bone marrow of GRK6−/− animals.
Three days after peg-G-CSF injection, the percentage of neutrophils in the bone marrow had significantly and equally increased in WT and GRK6−/− mice. No differences were observed in the cellular composition of bone marrow from GRK6−/− mice and WT animals, suggesting that the G-CSF-induced production of neutrophils in the bone marrow in GRK6−/− mice is not impaired (data not shown). To further assess the possible role of GRK6 in the G-CSF-induced production of PMN, we determined the in vitro response to G-CSF in colony assays. G-CSF-induced colony formation by bone marrow progenitor cells from WT and GRK6−/− mice was analyzed. As is shown in Figure 4 , the number of colonies obtained after stimulation with G-CSF did not differ between GRK6−/− and WT cultures.
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Figure 4. Colony formation by bone marrow progenitors from WT and GRK6−/− mice. Colonies were grown in the presence of 100 ng/ml G-CSF. Data represent mean colony numbers + SEM. n = 5 per group.
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Figure 5. Assessment of neutrophil apoptosis. Neutrophils were isolated from bone marrow 3 days after injection of peg-G-CSF. Apoptosis was assessed by flow cytometric analysis of cells stained with fluorescein isothiocyanate (FITC)-labeled annexin-V and propidium iodine. In addition, the percentage of apoptotic cells was analyzed after a culture period of 24 or 48 h without growth factors. Percentage of positive cells is indicated in the dot plot. Four animals per group were analyzed. Shown are representative dot plots of one WT and one GRK6−/− animal.
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Figure 6. Motility, SDF-1-induced chemotaxis, and SDF-1-induced chemokinesis (kinesis) of PMN from G-CSF-treated WT and GRK6−/− mice. Bone marrow-derived PMN were obtained 3 days after s.c. injection of 15 µg peg-G-CSF. Chemotaxis and chemokinesis were assessed in response to 100 ng/ml SDF-1. Open bars, WT; solid bars, GRK6−/− animals. Data represent percentage of cells from the total number used that reached the lower well. n = 6 per group; **, P < 0.01 (two-way ANOVA).
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The impaired neutrophil response to G-CSF in GRK6−/− mice might be the result of altered G-CSF receptor (G-CSFR) signaling in the absence of GRK6. Growth factor receptors such as the G-CSFR do not belong to the GPCR family, which are substrates for desensitization by GRKs. However, previous studies have indicated that GRKs might affect signaling in response to growth factor receptors as well. For instance, overexpression of GRK2 has been shown to affect mitogenic signaling and proliferation of smooth muscle cells and osteoblastic cells in response to platelet-derived growth factor and insulin-like growth factor-1, respectively [31 , 32 ].
G-CSFR signals are essential for proliferation, maturation, and survival of neutrophils in the bone marrow, as well as for the maintenance of homeostatic/steady-state levels of neutrophils in the blood [11 , 14 , 33 , 34 ]. Thus, if G-CSFR signaling is altered in the absence of GRK6, we would expect to observe differences in steady-state blood PMN levels between WT and GRK6−/− animals. However, basal PMN counts were equal in WT and GRK6−/− mice. In addition, analysis of bone marrow cellular composition before and after G-CSF treatment revealed no differences between WT and GRK6−/− animals. Notably, percentages of bone marrow neutrophils had increased equally in both animals. Furthermore, G-CSF-induced colony formation by WT and GRK6−/− bone marrow progenitors did not differ, suggesting that neutrophil production in response to G-CSFR signals is normal. Moreover, there was no difference in percentages of apoptotic bone marrow cells as well as in cell survival between WT and GRK6−/− mice. Finally, Semerad et al. [14 ] demonstrated that in mixed chimeras reconstituted with WT and G-CSFR mutant bone marrow cells, G-CSFR-deficient and WT neutrophils were mobilized equally by G-CSF [14 ]. This finding indicates that neutrophil mobilization is not dependent on G-CSFR expression on neutrophils, suggesting that altered signaling of this receptor on GRK6−/− neutrophils is not the cause of the observed, impaired mobilization of these cells in response to G-CSF. Taken together, the data argue against a role of altered G-CSFR signaling in the observed deficient neutrophil response to G-CSF in GRK6−/− animals.
It is thought that G-CSF induces neutrophil mobilization indirectly through the generation of trans-acting signals. The nature of these secondary signals is still largely unknown. However, there is accumulating evidence suggesting that proteolytic disruption of the CXCR4/SDF-1 signaling pathway may represent an important mechanism regulating mobilization of neutrophils in response to G-CSF [14 15 16 17 ]. It is thought that SDF-1/CXCR4 signaling represents a key retention signal for neutrophils/hematopoietic progenitors in the bone marrow and that desensitization of CXCR4 may be an important mechanism in inducing cell mobilization from the bone marrow in response to G-CSF treatment. In line with this hypothesis, we observed an increased sensitivity of bone marrow-derived neutrophils from GRK6−/− animals to migrate toward SDF-1. In naive as well as in G-CSF-treated animals, we demonstrated enhanced SDF-1-induced chemotaxis of GRK6−/− neutrophils as compared with WT cells. Based on these data, we propose that impaired desensitization of CXCR4 in the absence of GRK6 causes the observed, impaired neutrophil mobilization in GRK6−/− mice. Increased sensitivity of the CXCR4/SDF-1 signaling pathway might promote increased retention of neutrophils in the bone marrow, resulting in little or no increase in circulating neutrophils in response to G-CSF.
Our present results, showing that desensitization of the calcium response to stimulation with SDF-1 is impaired in GRK6−/− neutrophils, and the study by Fong et al. [26 ], showing increased SDF-1-induced GTPase activity in GRK6−/− lymphocytes, are consistent with the hypothesis that GRK6 is required for desensitization of CXCR4, presumably via agonist-induced phosphorylation of the receptor. It is important to realize, however, that CXCR4 has not yet been identified as a substrate for phosphorylation by GRK6.
Surprisingly, in contrast to neutrophils, chemotaxis of T and B cells toward SDF-1 is inhibited in the absence of GRK6 [26 ], whereas we show here that the chemotactic response of neutrophils toward SDF-1 is increased. We cannot exclude that the difference in chemotactic activity between PMN and T/B cells reflects differences in cell type-specific characteristics of the CXCR4 or the way these distinct cells respond to CXCR4 activation. However, it seems more likely that the differences in SDF-1-induced chemotaxis between PMN and T/B cells are a result of differences in cell type-specific mechanisms of migration that are affected by GRK6. Indeed, it has been suggested that T cells and PMN use different mechanisms to migrate; T lymphocytes increase locomotion by reducing the duration of migration breaks, and PMN migrate by reducing the frequency of migration breaks [35 ]. In this respect, it is of interest that we also have data showing increased migration of GRK6−/− PMN toward the chemoattractant leukotriene B4 [50 ].
Retention of neutrophils in the bone marrow is controlled by adhesive interactions involving vascular cell adhesion molecule 1 (VCAM-1), which is expressed by stromal cells, and the integrin very late antigen 4 (VLA-4), which is expressed on neutrophils [36 37 38 39 ]. Inhibition of the interaction between VCAM-1 and VLA-4 by infusion of anti-VCAM-1 or anti-VLA-4 was shown to be sufficient to promote mobilization in vivo [37 , 40 41 42 ]. It is interesting that SDF-1 was observed to stimulate VLA-4-dependent engraftment of immature progenitor cells in nonobese diabetic/severe combined immunodeficiency mice [43 ] and to promote VLA-4-mediated adhesion of bone marrow cells to fibronectin, VCAM-1, and bone marrow stroma [44 ], suggesting that CXCR4 signaling is involved in regulation of integrin function. Altered modulation of VLA-4-mediated adhesion of neutrophils as a result of altered CXCR4 signaling could thus represent an underlying mechanism for the observed, impaired mobilization of neutrophils in GRK6−/− mice.
Release of neutrophils from the bone marrow in response to G-CSF has been associated with proteolytic degradation of SDF-1 by serine proteases [17 ], leading to disruption of CXCR4/SDF-1 interactions [45 46 47 ]. In addition, these serine proteases were shown to inactivate the VCAM-1 [48 , 49 ]. It can therefore not be excluded that the impaired neutrophil mobilization in response to G-CSF is the consequence of impaired proteolytic degradation of SDF-1 or VCAM-1. However, the possible role of GRK6 in regulation of protease activity is unknown.
In conclusion, we demonstrate that the increase in circulating PMN in response to peg-G-CSF is significantly impaired in the absence of GRK6, suggesting a crucial role for this kinase in regulation of neutrophil mobilization via regulation of responsiveness of GPCRs, which are controlled by GRK6, such as CXCR4.
Received July 10, 2003; revised August 12, 2003; accepted October 21, 2003.
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modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells Exp. Hematol. 29,345-355[CrossRef][Medline]This article has been cited by other articles:
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