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   . The present study aimed to analyze the drug granulocyte- colony stimulating factor (G-CSF) in the blood ROS generation assay (BRGA)   , an innovative test for whole blood ROS generation working with luminol-enhanced photons emission primarily by diluted whole blood PMN    , 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  ), 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 , 0.4 mM , 0.6 mM , 4.2 mM , 5.6 mM glucose.
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.
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  -  .
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    ; 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  . There could be an enhanced generation of thrombin/systemically circulating micro-thrombi   . 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  -  . 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.
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