Impairment of G-CSF receptor on granulocytic progenitor cells causes neutropenia in protein malnutrition.

OBJECTIVE: It is well known that protein malnutrition (PM) states can affect hematopoiesis, leading to severe leukopenia and reduced number of granulocytes, which act as the first line of defense, and are important to the innate immune response. The aim of this study was to elucidate some of the mechanisms involved in the impairment of granulopoiesis in PM. METHODS: Male C57BL/6 mice were submitted to PM with a low-protein diet containing 2% protein. Control mice were fed a 12% protein-containing diet. Bone marrow histology and the percentage of granulocytic progenitors were evaluated after in vivo granulocyte-colony stimulating factor (G-CSF) stimulus. Cell proliferation, STAT3 signaling, and the expression of G-CSF receptor were evaluated in hematopoietic progenitor cells. RESULTS: Malnourished animals presented with leukopenia associated with reduced number of granulocytes and reduced percentage of granulocytic progenitors; however, no differences were observed in the regulatory granulopoietic cytokine G-CSF. Additionally, the malnourished group presented with impaired response to in vivo G-CSF stimulus compared with control animals. PM was implicated in decreased ability of c-Kit+ cells to differentiate into myeloid progenitor cells and downregulated STAT3 signaling. Furthermore, the malnourished group exhibited reduced expression of G-CSF receptor on granule-monocytic progenitors. This reduced expression was not completely reversible with G-CSF treatment. CONCLUSIONS: This study implies that PM promotes intrinsic alterations to hematopoietic precursors, which result in hematologic changes, mainly neutropenia, observed in peripheral blood in PM states.

The dark side of granulocyte-colony stimulating factor: a supportive therapy with potential to promote tumour progression.

Granulocyte-colony stimulating factor (G-CSF) is one of several cytokines that can expand and mobilize haematopoietic precursor cells from bone marrow. In particular, G-CSF mobilizes neutrophils when the host is challenged by infection or tissue damage. Severe neutropenia, or febrile neutropenia is a life-threatening event that can be mitigated by administration of G-CSF. Consequently, G-CSF has been used to support patients undergoing chemotherapy who would otherwise require dose reduction due to neutropenia. Over the past 10-15 years it has become increasingly apparent, in preclinical tumour growth and metastasis models, that G-CSF can support tumour progression by mobilization of tumour-associated neutrophils which consequently promote tumour dissemination and metastasis. With the increasing use of G-CSF in the clinic, it is pertinent to ask if there is any evidence of a similar promotion of tumour progression in patients. Here, we have reviewed the preclinical and clinical data on the potential contribution of G-CSF to tumour progression. We conclude that, whilst the evidence for a promotion of metastasis is strong in preclinical models and that limited data indicate that high serum G-CSF levels in patients are associated with poorer prognosis, no studies published so far have revealed evidence of increased tumour progression associated with supportive G-CSF use during chemotherapy in patients. Analysis of G-CSF receptor positive cohorts within supportive trials, as well as studies of the role of G-CSF blockade in appropriate tumours in the absence of chemotherapy could yield clinically translatable findings.

Hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy.

RATIONALE: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor function, and increased GM-CSF. OBJECTIVES: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. METHODS: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. MEASUREMENT AND MAIN RESULTS: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor ß chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. CONCLUSIONS: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.

G-CSF and GM-CSF concentrations and receptor expression in peripheral blood leukemic cells from patients with chronic myelogenous leukemia.

Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) are the principal cytokines in granulopoiesis and differentiation of granulocytic precursors. Their physiologic effects are mediated by binding to specific cell surface receptors (G-CSFr and GM-CSFr, respectively), which are widely expressed from immature bone marrow cells to mature peripheral granulocytes. The fact that concentrations of plasma G-CSF and GM-CSF and their receptors are changed in infectious diseases showing neutrophilia is known, but such changes in patients with chronic myelogenous leukemia (CML) have not been studied. Based on quantitative assays of plasma G-CSF and GM-CSF and their receptors on the peripheral granulocytes of CML patients and healthy controls, this study analyzes the differences between these groups in G-CSF and GM-CSF levels, as well as quantitative and qualitative changes in the receptors. Plasma levels of G-CSF and GM-CSF were measured in 47 patients in the chronic phase of CML and 25 healthy adults as normal controls. G-CSFr and GM-CSFr on peripheral granulocytes were analyzed by quantitative flow cytometry, and changes in G-CSF and GM-CSF receptor counts were also measured. Plasma concentrations of G-CSF and GM-CSF in CML patients were similar to normal controls (p>0.05). The quantity of G-CSFr on neutrophils was more highly expressed than on other cell types in both groups, and the amount of this receptor in patients with CML was less than in normal controls (p=0.001). GM-CSFr was expressed in higher concentrations on monocytes than neutrophils, and there was no difference in the amount of GM-CSFr on neutrophils. After incubation with excess G-CSF, the expressed amounts of G-CSFr on neutrophils and monocytes were decreased in both groups. However, G-CSFr on the monocytes was decreased in healthy controls (p=0.02) with no difference in patients with CML. The quantities of GM-CSFr expression on neutrophils and monocytes after incubation with excess GM-CSF were decreased in both groups. Granulocyte counts in peripheral blood of CML patients were not correlated with the plasma concentrations of G-CSF or GM-CSF, nor with the expression of G-CSFr or GM-CSFr on granulocytes. Granulopoiesis in patients with CML was not mediated by increased plasma CSF concentrations, and there was no difference in the amounts of G-CSFr or GM-CSFr expressed on the granulocytes. This suggests that a structural change may occur on monocytes of CML patients, since the binding capacity of G-CSFr to G-CSF on the monocytes is different from the normal controls.

Modulating effects of fumonisin B1 and ochratoxin A on leukocytes and messenger cytokines of the human immune system.

Fumonisin B1 and ochratoxin A are mycotoxins of importance to public health and agro-economics. Although much is known about their cellular toxicity and carcinogenesis in animals, there are no reports of adverse effects on immune cells (leukocytes) or on the immune modulation of the molecular messengers (cytokines) in humans. This study was designed, therefore, to determine and compare the morphological effects of fumonisin B1 and ochratoxin A on lymphocytes and neutrophils harvested from the circulation of healthy volunteer subjects and patients with oesophageal and breast carcinomas. Both fumonisin B1 and ochratoxin A reduced the number of viable lymphocytes and neutrophils harvested from the circulation of volunteer subjects carcinoma patients in a dose-dependent manner. Leukocyte secretion of cytokines on exposure to the mycotoxins was evaluated by immunocytochemical methods. Expression of granulocyte-colony stimulating factor (G-CSF), tumour necrosis factor (TNF-alpha) and chemokine (CX3CR1) receptors were determined on the circulating leukocytes and the immunolabelling visualized by brightfield-and electron-microscopy. Cytokine levels were determined in the circulation of healthy volunteer subjects and in patients with oesophageal and breast carcinomas since they reflect the status of the immune system in humans. The findings of this study on immunocytes (leukocytes) and the immune molecular messengers (cytokines) suggest that fumonisin B1 and ochratoxin A have an immuno-suppressive effect in humans, in particular patients with cancer by impairing immune surveillance.

Quantities of receptor molecules for colony stimulating factors on leukocytes in measles.

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.

Quantities of Receptor Molecules for Colony Stimulating Factors on Leukocytes in Measles

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery.

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies.

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells.

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.

Clinical significance of granulocyte colony-stimulating factor (G-CSF) receptor expression in acute myeloid leukemia.

We examined granulocyte colony-stimulating factor (G-CSF) receptor (GR) expression on leukemic cells from 44 adults with newly-diagnosed acute myeloid leukemia (AML). GR expression was higher in female patients. G-CSF was administered to AML patients after initial induction therapy without significant acceleration of leukemia, irrespective of GR expression level. G-CSF administration after initial chemotherapy did not adversely influence clinical outcome of GR-positive patients. However, at first relapse, leukemia regrowth was accelerated in 3 of 15 GR-positive patients who received G-CSF after re-induction. It remains to be determined whether leukemia acceleration due to G-CSF contributes to re-induction failure and if G-CSF therapy is a significant risk in relapsed, GR-positive AML patients.

Leukemic cells with 11q23 translocations express granulocyte colony-stimulating factor (G-CSF) receptor and their proliferation is stimulated with G-CSF.

We report a 20-month-old boy with acute lymphoblastic leukemia with the 11q23 translocation whose blasts markedly increased in peripheral blood after intravenous granulocyte colony-stimulating factor (G-CSF) administration, but disappeared after stopping G-CSF. The in vitro study showed that the leukemic cells separated from this patient expressed G-CSF receptor (G-CSFR) and an addition of G-CSF stimulated their proliferation by 3H-thymidine incorporation assay (stimulation index, 4.9). To clarify whether or not leukemic cells with 11q23 translocations generally express G-CSFR and show proliferative response to G-CSF, we performed the similar in vitro experiments using eight leukemic cell lines with 11q23 translocations. We found that all cell lines examined expressed G-CSFR (20-98%) and proliferation of seven leukemic cell lines was significantly enhanced in response to G-CSF (stimulation index >1.5 in five cell lines), suggesting a possible participation of the G-CSF/G-CSFR interaction in the process of growth regulation of leukemic cells with 11q23 translocations.

Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils.

We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism.