The present study demonstrated that treatment with rhG-CSF following chemotherapy increased the number of G-CSF receptors per cell as well as the number of neutrophils in cancer patients. The rhG-CSF clearance closely correlated with both the number of G-CSF receptors per cell and that of circulating neutrophils. Results of previous studies indicated that hematopoietic cytokines exhibit common regulatory mechanisms, and a similar relationship between cytokine clearance and the number of target cells has been described elsewhere for rhG-CSF, recombinant human macrophage CSF, recombinant human granulocyte-macrophage CSF, and rhEPO (9
). In addition, upregulation of receptor mRNA by the ligand has also been reported for G-CSF and interleukin 2 (12
). However, to our knowledge, no in vivo study has previously reported the relationship between G-CSF receptor and neutrophils or rhG-CSF clearance.
rhG-CSF is eliminated through saturable and unsaturable mechanisms. In a rat model, a high dose of rhG-CSF decreases clearance to a plateau level, and clearance of a high dose of rhG-CSF is abrogated by nephrectomy (15
). In humans, a high intravenous dose of rhG-CSF (≥10 μg/kg) also decreases clearance to a plateau level (6
). Clearance of rhG-CSF administered subcutaneously is inversely correlated with circulating neutrophil counts (13
), and clearance of rhG-CSF administered intravenously decreases in patients with renal failure (1
). These findings indicate that saturable and unsaturable clearance mechanisms of rhG-CSF mainly involve the neutrophils and kidneys, respectively. In this study, since creatinine clearance did not change during rhG-CSF therapy, the kidney was probably not involved in the observed change in rhG-CSF clearance.
At the time of chemotherapy-induced neutropenia, the number of G-CSF receptors per cell decreased to almost half the level found in healthy individuals. G-CSF receptors are normally present on myeloid progenitor cells to peripheral neutrophils (2
). The number of receptors per cell increases with differentiation, and neutrophils in bone marrow have fewer receptors than do peripheral neutrophils (1
). On the other hand, chemotherapy has been shown to inhibit the functions of peripheral neutrophils, but to our knowledge, no study of G-CSF receptors has been reported. There are two possible explanations for the reduced number of G-CSF receptors per cell observed in the present study: (i) increased release of neutrophils from bone marrow and (ii) direct inhibition by chemotherapy.
Treatment with rhG-CSF increased the percentage of G-CSF receptor-positive neutrophils to 73.0%. In a series of preliminary studies, when the neutrophil count spontaneously returned to the prechemotherapy level without rhG-CSF therapy, the percentage of G-CSF receptor-positive neutrophils remained low (51.5% ± 11.8%; n
= 4). Other investigators have shown that rhG-CSF enhances the expression of G-CSF receptor mRNA in human neutrophils in vitro (17
), supporting our results in vivo. Steinman and Tweardy (12
) reported that the upregulation of murine G-CSF receptor mRNA by rhG-CSF is rapid and due to transcriptional activation without the synthesis of new protein. rhG-CSF increased not only the number of circulating neutrophils but also the density of neutrophil G-CSF receptors, which accelerated the increase in the total number of G-CSF receptors. We also showed that rhG-CSF clearance closely correlated with the number of G-CSF receptors per neutrophil. Accordingly, rhG-CSF is thought to be eliminated through G-CSF receptors on neutrophils rather than by nonspecific endocytosis by neutrophils.
G-CSF receptors are also expressed on platelets, monocytes, endothelial cells, and certain cancer cell lines (2
). Soluble G-CSF receptors, anti-G-CSF antibodies, proteases, and G-CSF receptor antagonists such as complement component C5a are present in peripheral blood (2
). Accordingly, these receptors and substances may modify G-CSF clearance. However, platelet and monocyte counts did not change significantly in this study, and the density of G-CSF receptors on neutrophils accounted for as much as 91% of rhG-CSF clearance. In rats treated with cyclophosphamide, rhG-CSF enhanced the expression of G-CSF receptor mRNA on bone marrow cells but not that of soluble G-CSF receptors when serum rhG-CSF levels dropped (7
). Although rhG-CSF therapy induces circulating anti-G-CSF antibodies, the antibodies do not inhibit cytokine function (5
). To our knowledge, no study has previously reported the in vivo role of circulating proteases, receptor antagonists, or G-CSF receptors in cancer cell lines. These findings suggest that G-CSF receptors on cells other than neutrophils and circulating G-CSF-related substances are unlikely to contribute to changes in rhG-CSF clearance following chemotherapy.
In conclusion, rhG-CSF increased the density of G-CSF receptors on neutrophils, which closely correlated with rhG-CSF clearance. The pharmacokinetics of rhG-CSF are probably modulated in a complex fashion, and further studies are necessary to determine the optimal usage of rhG-CSF.