Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony- stimulating factor

Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low-affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG-CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G- CSF injections, but was reinduced during the next treatment cycle with rhG-CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64).

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Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony- stimulating factor

Blood ◽

10.1182/blood.v78.4.885.885 ◽

1991 ◽

Vol 78 (4) ◽

pp. 885-889 ◽

Cited By ~ 134

Author(s):

R Repp ◽

T Valerius ◽

A Sendler ◽

M Gramatzki ◽

H Iro ◽

...

Keyword(s):

Fc Receptors ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

High Affinity ◽

Healthy Donors ◽

High Affinity Receptor ◽

Affinity Receptor ◽

Stimulating Factor

Abstract Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low-affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG-CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G- CSF injections, but was reinduced during the next treatment cycle with rhG-CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64).

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Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy

Blood ◽

10.1182/blood.v82.3.931.931 ◽

1993 ◽

Vol 82 (3) ◽

pp. 931-939 ◽

Cited By ~ 106

Author(s):

T Valerius ◽

R Repp ◽

TP de Wit ◽

S Berthold ◽

E Platzer ◽

...

Keyword(s):

Antibody Therapy ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

High Affinity ◽

Healthy Donors ◽

Polymerase Chain Reaction Technology ◽

High Affinity Receptor ◽

Factor Therapy ◽

Affinity Receptor ◽

Stimulating Factor

Abstract Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes-- Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy.

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Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy

Blood ◽

10.1182/blood.v82.3.931.bloodjournal823931 ◽

1993 ◽

Vol 82 (3) ◽

pp. 931-939 ◽

Cited By ~ 3

Author(s):

T Valerius ◽

R Repp ◽

TP de Wit ◽

S Berthold ◽

E Platzer ◽

...

Keyword(s):

Antibody Therapy ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

High Affinity ◽

Healthy Donors ◽

Polymerase Chain Reaction Technology ◽

High Affinity Receptor ◽

Factor Therapy ◽

Affinity Receptor ◽

Stimulating Factor

Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes-- Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy.

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Neutrophil Activation and Hemostatic Changes in Healthy Donors Receiving Granulocyte Colony-Stimulating Factor

Blood ◽

10.1182/blood.v93.8.2506.408k31_2506_2514 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2506-2514 ◽

Cited By ~ 2

Author(s):

A. Falanga ◽

M. Marchetti ◽

V. Evangelista ◽

S. Manarini ◽

E. Oldani ◽

...

Keyword(s):

Activation Parameters ◽

Antigen Expression ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Elastase Activity ◽

Healthy Donors ◽

Stimulating Factor ◽

Plasma Markers

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 μg/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF–treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.

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Neutrophil Activation and Hemostatic Changes in Healthy Donors Receiving Granulocyte Colony-Stimulating Factor

Blood ◽

10.1182/blood.v93.8.2506 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2506-2514 ◽

Cited By ~ 102

Author(s):

A. Falanga ◽

M. Marchetti ◽

V. Evangelista ◽

S. Manarini ◽

E. Oldani ◽

...

Keyword(s):

Activation Parameters ◽

Antigen Expression ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Elastase Activity ◽

Healthy Donors ◽

Stimulating Factor ◽

Plasma Markers

Abstract Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 μg/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF–treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.

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A role for the carboxyl terminus of human granulocyte-macrophage colony-stimulating factor in the binding of ligand to the alpha-subunit of the high affinity receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37496-3 ◽

1994 ◽

Vol 269 (8) ◽

pp. 5548-5553

Author(s):

G.F. Seelig ◽

W.W. Prosise ◽

J.E. Scheffler

Keyword(s):

Alpha Subunit ◽

Carboxyl Terminus ◽

Colony Stimulating Factor ◽

High Affinity ◽

High Affinity Receptor ◽

Macrophage Colony Stimulating Factor ◽

Affinity Receptor ◽

Macrophage Colony ◽

Stimulating Factor

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In vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor on neutrophil functions.

Journal of the Japan Society of Blood Transfusion ◽

10.3925/jjtc1958.35.647 ◽

1989 ◽

Vol 35 (6) ◽

pp. 647-652

Author(s):

Akimichi Ohsaka ◽

Seiichi Kitagawa ◽

Akira Yuo ◽

Takashi Obata ◽

Youichi Amemiya ◽

...

Keyword(s):

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

In Vivo Effects ◽

Stimulating Factor

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In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody

Blood ◽

10.1182/blood.v86.3.1124.1124 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1124-1130 ◽

Cited By ~ 24

Author(s):

J Michon ◽

S Moutel ◽

J Barbet ◽

JL Romet-Lemonne ◽

YM Deo ◽

...

Keyword(s):

Binding Site ◽

Cancer Patients ◽

Bispecific Antibody ◽

Neuroblastoma Cells ◽

Bispecific Antibodies ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Stimulating Factor

Abstract Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.

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Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils

Blood ◽

10.1182/blood.v84.11.3885.bloodjournal84113885 ◽

1994 ◽

Vol 84 (11) ◽

pp. 3885-3894 ◽

Cited By ~ 129

Author(s):

M de Haas ◽

JM Kerst ◽

CE van der Schoot ◽

J Calafat ◽

CE Hack ◽

...

Keyword(s):

Healthy Volunteers ◽

Plasma Levels ◽

Subcutaneous Administration ◽

Secretory Vesicles ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Specific Granules ◽

Stimulating Factor

In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1- antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.

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