Granulocyte Colony—Stimulating Factor Multiplies Normal Blood ROS Generation at Less than 1 µg/l

DOI: 10.4236/opj.2018.81001   PDF   HTML   XML   669 Downloads   1,000 Views  

Abstract

Background: The neutrophils (PMN) are our main blood cells to combat fungi, bacteria, and fibrin. For normal function, an activated PMN generates a certain concentration of reactive oxygen species (ROS). If the generated blood ROS concentration is too low, then fungi, bacteria or fibrin might threaten the life of the patient, and it could be of great medical interest to stimulate PMN by physiologic drugs. Granulocyte-Colony Stimulating Factor (G-CSF) is a cell hormone that increases the cell number of PMN and that stimulates the individual PMN. The blood ROS generation assay (BRGA) is an innovative physiologic test to monitor the ROS generation of PMN in blood. Here the ROS generating action of G-CSF on normal PMN is quantified. Material and Methods: 40 μl 0 - 10.3 ng/ml (final conc.) G-CSF (in 5% human albumin) in black Brand? 781608 high quality polystyrene F-microwells was incubated in triplicate with 125 μl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 μl normal citrated blood. Immediately (BRGA) or after 60 min (BRGA-60-) 10 μl 5 mM luminol sodium salt in 0.9% NaCl and 10 μl 0 or 36 μg/ml zymosan A in 0.9% NaCl was added. The photons were counted within 0 - 318 min (37°C) in a photons-multiplying microtiter plate luminometer. At about 0.5 t-maxn (0.5 fold the time to normal maximum) the approx. SC200 of G-CSF was determined. Results and Discussion: The approx. SC200 of G-CSF on normal blood ROS generation was 0.2 μg/l (=20 IU/ml). In clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient. The BRGA helps to find out the correct stimulating G-CSF dosage for each individual. An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis. G-CSF plasma concentrations of 0.1 - 1 μg/l might favor singlet oxygen generation without immunosuppression or cell fragment-induced thrombin generation.

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Stief, T. (2018) Granulocyte Colony—Stimulating Factor Multiplies Normal Blood ROS Generation at Less than 1 µg/l. Optics and Photonics Journal, 8, 1-10. doi: 10.4236/opj.2018.81001.

1. Introduction

The main cells of innate immunology are the phagocytes (neutrophils = PMN, monocytes = MØ, dendritic cells = DC). Drugs that enhance the PMN function are of great clinical relevance in many diseases where PMN are needed against the disease [1] [2] . The present study aimed to analyze the drug granulocyte- colony stimulating factor (G-CSF) in the blood ROS generation assay (BRGA) [3] [4] , an innovative test for whole blood ROS generation working with luminol-enhanced photons emission primarily by diluted whole blood PMN [5] [6] [7] , stimulated by typical pathophysiological septic concentrations of the fungal compound zymosan A (ZyA; 1 - 2 µg/ml).

2. Material and Methods

40 µl 0 - 10.3 ng/ml (0 - 974 IU/ml) G-CSF (final conc.) (2nd International WHO Standard, human rDNA derived, protein expressed in E. coli; NIBSC, Potters Bar, UK; article nr. 09/136; 1000 ng G-CSF (containing less than 10 ng LPS [8] ), 10 mg arginine, 10 mg phenylalanine, 5 mg trehalose, 2 mg human albumin, 0.01% Tween 20® dissolved in 500 µl H2O followed by 500 µl 5% human albumin (CSL Behring, Marburg, Germany) in black high quality flat bottomed polystyrene microwells (Brand, Wertheim, Germany; article nr. 781608), diluted with 5% human albumin, were incubated in triplicate with 125 µl Hanks’ balanced salt solution (HBSS; modified without phenol red; SAFC Biosciences-Sigma, Deisenhofen, Germany; article nr. 55037C-1000 ML) and 10 µl freshest normal blood anticoagulated with 11 mM sodium citrate (within 30 min after withdrawal). Immediately (BRGA) or after 60 min (BRGA-60-) 10 µl 5 mM luminol sodium salt (Sigma, Deisenhofen, Germany) in 0.9% NaCl and 10 µl 0 or 36 µg/ml zymosan A (Sigma) in 0.9% NaCl were added. The photons were counted within 0 - 318 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5 s per well. The intra-assay coefficients of variation were less than 10%. At about 0.5 t-maxn (0.5 fold the time to normal maximum) the approx. SC200 of G-CSF was determined.

HBSS consisted of 185.4 mg/l CaCl2・2H2O, 200 mg/l MgSO4・7H2O, 400 mg/l KCl, 60 mg/l KH2PO4, 350 mg/l NaHCO3, 8000 mg/l NaCl, 90 mg/l Na2HPO4, 1000 mg/l glucose, pH 7.0 - 7.4. Expressed in molarity, the concentrations of the HBSS components are: 1.3 mM Ca2+, 0.8 mM Mg2+, 5.8 mM K+, 143 mM Na+, 144 mM Cl, 1.6 mM SO 4 2 , 0.4 mM H 2 PO 4 , 0.6 mM HPO 4 2 , 4.2 mM HCO 3 , 5.6 mM glucose.

3. Results

In albumin samples, the BRGA maximum of 2389 RLU/s was reached after 124 min. In NaCl samples, the maximum of 1694 RLU/s was reached after 137 min. At 318 min, the blood ROS generation was 51% or 37% of the maximum, respectively (Figure 1).

Figure 1. Blood ROS generation in presence of albumin or 0.9% NaCl in BRGA. 40 µl 5% human albumin (Figure 1(a)) or 0.9% NaCl for control (Figure 1(b)) in black Brand® 781608 high quality polystyrene F-microwells were incubated in triplicate with 125 µl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood. 10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 36 µg/ml zymosan A in 0.9% NaCl were added. The photons were counted within 0 - 318 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo). In albumin samples the maximum of 2389 RLU/s was reached after 124 min, in NaCl samples the maximum of 1694 RLU/s was reached after 137 min. At 318 min the blood ROS generation was 51% or 37% of the maximum, respectively. The experiment was repeated twice, the standard deviations were <10%.

In albumin samples, the BRGA-60-maximum of 6502 RLU/s was reached after 84 min. In NaCl samples, the maximum of 6254 RLU/s was reached after 84 min, too. At 264 min, the blood ROS generation was 43% or 22% of the maximum, respectively (Figure 2). This means that a protein-poor environment facilitates the down-regulation of the ROS generation.

In the BRGA, the approx. SC200 was 0.2 ng/ml G-CSF (=20 IU/ml) (Figure 3). In the BRGA-60-, there appeared an approx. IC50 of 2 ng/ml G-CSF. Higher conc. of G-CSF again stimulated the ROS generation (Figure 4). This means that

Figure 2. Blood ROS generation in presence of albumin or 0.9% NaCl in BRGA-60-. 40 µl 5% human albumin (Figure 2(a)) or 0.9% NaCl for control (Figure 2(b)) in black Brand® 781608 high quality polystyrene F-microwells were incubated in triplicate with 125 µl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood. After 60 min 10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 36 µg/ml zymosan A in 0.9% NaCl were added. The photons were counted within 0 - 264 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo). In albumin samples the maximum of 6502 RLU/s was reached after 84 min, in NaCl samples the maximum of 6254 RLU/s was reached after 84 min, too. At 264 min the blood ROS generation was 43% or 22% of the maximum, respectively. The experiment was repeated twice, the standard deviations were < 10%.

Figure 3. Approx. SC200 of G-CSF in BRGA. 40 µl 0 - 10.3 ng/ml (final conc.) G-CSF (in 5% human albumin) in black Brand® 781608 high quality polystyrene F-microwells were incubated in triplicate with 125 µl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood. 10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 0 or 36 µg/ml zymosan A in 0.9% NaCl were added. The photons were counted at 44 min (37˚C); approx. SC200 = 0, 2 ng/ml = 20 IU/ml.

Figure 4. Approx. IC50 of G-CSF in BRGA-60-. 40 µl 0 - 10.3 ng/ml (final conc.) G-CSF (in 5% human albumin) in black Brand® 781608 high quality polystyrene F-microwells were incubated for 60 min (37˚C) in triplicate with 125 µl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood. 10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 0 or 36 µg/ml zymosan A in 0.9% NaCl were added. The photons were counted at 42 min (37˚C). There appeared an approx. IC50 of 2 ng/ml G-CSF. Higher conc. of G-CSF again stimulated the ROS generation.

Figure 5. Biogenesis of ROS/photons by activated neutrophils.

within the first incubation time of one hour (37˚C) in the BRGA-60-, G-CSF seems to be inactivated to some extent.

4. Discussion

By contrast, in the BRGA, very low concentrations of G-CSF stimulate blood ROS generation. This could be of pharmacologic interest: in clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient. The BRGA helps to find out the correct stimulating G-CSF dosage for each individual. An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis [9] - [17] .

The normal plasma concentration of G-CSF is about 25 ± 20 pg/ml, and in acute infections, the G-CSF concentration can increase up to about 100 fold [18] [19] [20] ; upon subcutaneous injection of 300 µg filgrastim, the G-CSF plasma concentration has increased about 1000 fold (blood half-life about 4 h), activating on neutrophils the CD11b/CD18 expression and the respiratory burst, on monocytes/dendritic cells the generation of immune suppressive interleukin-10, on endothelial cells the release of von Willebrand factor and F8, on hepatocytes the release of fibrinogen [21] . There could be an enhanced generation of thrombin/systemically circulating micro-thrombi [14] [22] . Thus, respective blood hemostasis, a G-CSF dosage of about 300 µg seems to be “too much of a good thing”. The present work indicates that a G-CSF plasma concentration around 1 ng/ml (injection of about 3 µg G-CSF, i.e. 100 fold less than currently used) might favour the physiologic singlet oxygen generation (Figure 5) against pathogens without pathologic thrombin generation or immune suppressive side effects [23] - [47] . The BRGA is a powerful tool to compare new analogues of G-CSF (e.g. the E. coli product filgrastim or the CHO product lenograstim). Dose-finding studies are highly indicated to establish the range of beneficial G-CSF concentrations for each individual patient.

Conflicts of Interest

The authors declare no conflicts of interest.

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