3. Materials and Methods
3.6 In vitro stimulation of primary rat alveolar macrophages
3.6.1
Preparation and culturing of rat alveolar macrophages
Anaesthetised rats were exposed to the blast pressure wave as already described previously (3.4). The animals were sacrificed 10 minutes (min), 24 and 96 hours following this experimental induced blunt chest trauma, non-traumatized animals served as sham controls.
The alveolar macrophages were harvested by bronchoalveolar lavage (BAL). Therefore lungs were tracheotomized and five times rinsed with 10 ml cold NaCl: The latter three times while softly massaging the thoracic cage to increase the amount of the isolated BAL cells. Recovery of the NaCl was always > 75%. The lavage fluids were pooled and centrifuged (4°C) for 10 min at 270 x g, and the pellet was resuspended in 20 - 50 ml cold RPMI 1640 and centrifuged for another 5 min (190 x g, 4°C). The pellet was again resuspended in RPMI 1640 with phenol red, 10% heat-inactivated FCS and 1% penicillin/ streptomycin. BAL cells were cultured at an initial density of 5 x 105/ ml (except 3.6.3/ 3.11) on 96-well plates in 200 µl/ well RPMI 1640 (phenol red, 10% FCS, 1% penicillin/ streptomycin) for two hours at 37°C. The non-adherent
in vivo ex vivo
Materials and Methods
BAL cells were removed by washing the cells three times with fresh medium. The remaining adherent alveolar macrophages were cultured in 200 µl/ well RPMI 1640 (without phenol red, 10% FCS, 1% penicillin/ streptomycin) either non-stimulated, with endotoxin or whole, dead bacteria (3.6.2, 3.6.3). Depending on the isolated BAL-cell counts all experiments were run at least in duplicate.
3.6.2
Stimulation with dead, fluorescent bacteria
The experiments were started by incubating the alveolar macrophages with either fluorescence labelled Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) (Molecular Probes) in a final concentration of 250 µg/ ml. Both reagents were stored as concentrated stocks at 4°C and diluted with medium after sonicating (2 min) immediately before use. After three hours incubation at 37°C the cells and supernatant were either harvested and analysed (3.11) or subjected to different assays as described below (3.6.4, 3.6.5).
3.6.3
Stimulation with endotoxin
The cells were cultured at an initial density of 106 cells/ ml (100 µl/ well) (3.11) or 5 x 105/ ml (200 µl/ well) (3.6.5) in the presence of various concentrations of lipopolyssacharide (LPS) from salmonella abortus equi (Metalon) (0, 10, 100 ng/ ml, 1, 10, 100 µg/ ml) or lipoteichoic acid (LTA) from staphylococcus aureus (Konstanz) (0, 2, 10, 50, 100 µg/ ml). Both reagents were stored as concentrated stocks at 4°C and –20°C respectively and diluted with medium immediately before use. Restored endotoxin was sonified for 2 minutes before use. After three (3.6.5) or four (3.11) hours incubation at 37°C the supernatant was either harvested and analysed (3.11) or subjected to another assay as described below (3.6.5).
3.6.4
Fluorescence labelled E. coli/ S. aureus phagocytosis assay
This assay measures the ability of phagocytes to engulf fluorescence labelled E. coli/ S.
aureus particles. Prepared BAL cells were cultured in 96-well plates (5 x 105 cells/ ml, 200 µl/
well), allowed to adhere for 2 hours and stimulated with dead, labelled E. coli/ S. aureus (final concentration: 250 µg/ ml) as described in 3.6.5. After 3 hours incubation the phagocytosis was stopped by washing the cells three times with PBS (room temperature) to remove non-phagocytosed bacteria. The remaining PBS was removed by quickly turning the plate and gently pushing it on a lint-free paper towel. Then cells were lysed by addition of 100 µl/ well PBS + 0.1% Triton X-100. Fluorescence was determined at 530 nm excitation and 590 nm
Materials and Methods
emission wavelengths using a fluorescence microplate reader. Cells without bacteria were used to determine the background fluorescence. From every experiment a digital photograph was taken (Nikon Coolpix 995 through a special ocular with a Zeiss Axiovert 50).
Viability assays like Trypan blue or Sytox/ Hoechst staining at different steps during the assay procedure were used as an index for physiological culture conditions.
The used particle concentration of 250 µg/ ml as well as the incubation time of 3 hours is based on previous experiments, were the concentration signal relation and kinetic of particle phagocytosis was measured. Furthermore complete inhibition of ingestion at 0°C was a criteria for a quantitative phagocytic assay 158.
3.6.5
Assay for superoxide production
The production of superoxide after stimulation with either endotoxin or whole, dead bacteria was performed by assaying the superoxide sensitive reduction of the water soluble and cell impermeable tetrazolium salt WST-1. The advantage of this system is the higher sensitivity and lower background in comparison to the method using the reduction of ferrycytochrome C
159. Isolated BAL cells were seeded in 96-well plates as described in 3.6.1. After 2 hours adhesion the non-adhered BAL cells were removed by washing. Then 200 µl of 500 µM WST-1 in Hanks balanced saline solution (HBSS) were added to each well.
In the experiments with whole, dead bacteria (see 3.6.2) to half of the wells either 250 µg/ ml E. coli or S. aureus were added respectively. If endotoxin was used for stimulation to half of the wells 0.1 µg/ ml LPS or 100 µg/ ml LTA was added. Therefore, three different groups (non-stimulated, stimulated with endotoxin or bacteria) can be defined. To one well of each of these groups 1000 U/ ml superoxide dismutase (SOD) was added. Then the plates were incubated at 37°C for 3 hours.
After 3 hours, the extinction of the single wells was read by a plate reader at a wavelength of λ = 450 nm. The concentration of superoxide and the rate of its production was calculated considering the following facts:
The number of moles n is defined by the product of volume V of the sample (V =100⋅106l) and its molar concentration C: n=V ⋅C
According to Lambert/ Beer, the molecular concentration C is calculated by dividing ∆E (change in WST-1 extinction caused by its reduction by superoxide) through the product of WST-1`s molar extinction coefficient ε (37000 M-1 cm-1) and the diameter d of the sample (d = 0.3cm):
d V E
n ⋅
⋅ ∆
= ε
Materials and Methods
The difference in extinction ∆E was calculated for each group by forming the difference between extinction En of each single well of a group and the average extinction ESOD for the appropriate wells containing SOD: SOD
n E
E E= −
∆
Since superoxide – in contrast to cytochrome C – is reduced by two electrons, 2 moles of superoxide are needed to reduce 1 mol of WST-1, the formula has to be multiplied by the factor 2, resulting in the final equation:
d
The rate of production of superoxide R is the quotient of the number of molecules produced during a certain time t, which was 180 minutes in these experiments:
After inserting the known constants, the average rate for one group can be expressed as:
∑
∆ ⋅⋅
= 1 200.2
n E
R [pmol superoxide/ minute].
3.6.6
Preparation of washed and haemolysed erythrocytes and haemolysat
Peripheral venous blood was drawn from rats. To prevent coagulation Liquemin in NaCl (1 : 10) was added. The blood was diluted 10-fold in ice-cold NaCl and centrifuged for 5 minutes at 270 x g and 4°C. The supernatant was carefully removed and the washing procedure was repeated twice. Then a 10-fold volume of RPMI 1640 (without phenol red) was added and the erythrocyte cell count was determined using the Neubauer counting chamber. After another 5 minutes centrifugation step at 270 x g and 4°C, cell pellet was diluted in RPMI 1640 (without phenol red) to a final concentration of 8 x 109 erythrocytes/ ml corresponding approximately to whole blood erythrocyte counts. The experiments were performed with 1.25 % washed erythrocytes. To determine the impureness with leukocytes the leukocyte counts were assessed (3.9). To get haemolysed erythrocytes the washed erythrocytes were frozen in liquid nitrogen and then thawed at RT. This procedure was repeated three times. The haemolysat was obtained after centrifugation for 10 minutes at 10600 x g and 4°C.3.6.7
Lysis of erythrocytes
BAL fluid was centrifuged for 10 minutes at 270 x g and 4°C and the pellet was resuspended in medium (1:20) containing 0.17 M ammonium chloride to lyse the erythrocytes and incubated for 5 minutes at RT. To stop the lysis a five-fold excess of fresh medium was added, pellet was resuspended and centrifuged again for 5 minutes at 190 x g and 4°C.
Materials and Methods 3.6.8
Experimental design of the in vitro experiments
Anaesthetised rats were exposed to blast wave thorax trauma (3.5 cm distance from the nozzle). Animals were sacrificed 10 min, 24 and 96 hours after trauma, non-traumatized animals served as sham controls (3.4.4). The alveolar macrophages were harvested by BAL (3.6.1) and cultured either non-stimulated, with endotoxin (LPS or LTA) (3.6.3) or whole, dead bacteria (E. coli or S. aureus) (3.6.2) for four and three hours, respectively.
Supernatants were either harvested for TNF measurement (3.11) or subjected to a phagocytosis (3.6.4) and superoxide (3.6.5) production assay respectively.
In some experiments either whole blood, plasma, washed erythrocytes, haemolysed erythrocytes or haemolysat (3.6.6) was added together with the bacterial stimulus.
In addition total and differential BAL cell counts (3.9) were determined.
Figure 3.6 summarizes schematic the experimental design of the in vitro experiments.
Figure 3.3: Timescale of the in vitro experiments.