Modifizierte durchflusszytometrische Methode zum empfindlicheren Nachweis der Produktion von reaktiven Sauerstoffspezies neutrophiler Granulozyten in vitro

An improved technique is described providing a more sensitive qualitative as well as a more reliable and informative quantitative evaluation of the generation of reactive oxygen species (ROS) by neutrophils in vitro. A major drawback of presently practised flow cytometric ROS-measurements is the variable loss of considerable amounts of PMN during their activation on plastic. These variable cell losses provide unreliable results because it is unknown to which extend the residually measurable cells really represent the initial population to be tested. In order to largely avoid such cell losses our new technique monitors the reactivity of PMN in 96 well microtiter plates on an isotonic Percoll cushion of 100 µL per well (Percoll-test) instead of placing the cell directly on plastic. For evaluation of the Percoll-test most of the parameters investigated were achieved contemporarily by identical cell preparations and conditions – usually within the same microtiter plate – however placing the cells directly on plastic. Thus, the only differences in these comparative tests were the contact of PMN to either plastic or Percoll. Both types of ROS-tests included triplicates of 3 x 105 purified PMN in 50 µL Dulbecco`s modified Eagle`s medium (DMEM) alone, with the addition of Dihydrorhodamine 123 (DHR), a green fluorescing indicator (FL1) of intracellular ROS-production, in a final concentration of 0,75 µg/mL with or without 1 to 10.000 nmol/L of phorbol-12-myristat-13-acetate (PMA), a receptor independent activator of cells. All tests were incubated for 45 min. at 37°C in 5% CO2 in air and stopped by placing them on wet ice (4°C). Prior to flowcytometric evaluation the addition of a defined amount of red fluorescing (FL4) CyTM5-labelled mononuclear reference cells permitted quantitation of the harvested PMN. Addition of the infra red fluorescing (FL4) dye To-Pro-3 allowed evaluation of cell viability. These comparative investigations were mainly performed with PMN purified from the peripheral blood of various healthy cows. They provided a number of surprising results: About 50% of the PMN incubated on plastic without any addition were lost for evaluation within 45 min. of incubation at 37°C. Stimulation of these cells by 10 to 10.000 nmol/L of PMA increased PMN losses to more than 80 and 95% of the input. Due to the fact that the residually harvested cells comprised an increasing number of “dead” (To-Pro-3 positive) cells (9% without and up to 30 % with PMA) the remarkable cell losses might be caused by cell death in addition to strong adherence to plastic. An additional loss factor is most likely the polystyrene tubes usually employed for flow cytometry (FACS tubes). Simple storage of none activated PMN for 40 min. at 4°C in these tubes can cause a considerable cell loss. This can be reduced or avoided by using polypropylene tubes instead plus measuring the cells in the flow cytometer immediately after harvest from the well into the tube. Identical treatment of PMN on a 100 µL Percoll-cushion per well instead on plastic reduced cell losses to a maximum of 5% of the input when incubated with or without up to 100 nmol/L of PMA. More than 100 nmol/L of PMA increased cell losses but hardly above 20%. Moreover the cell viability of the stock PMN preparation (< 2% “dead” cells) will not change during 45 min. of incubation with and without up to 10.000 nmol/L of PMA. The reduction of PMN losses achieved by the Percoll-cushion could by no means be reached by any of the 5 different brands of commercial microtiter plates tested. Also the functional differences were remarkable between plastic or Percoll incubated PMN: Plain incubation of PMN on plastic without any addition resulted in an increase in cell size plus an induction of ROS-production (by means of DHR fluorescence) by factors between 14 and 27 as compared to the stock suspension of PMN. In contrast to plastic Percoll did neither increase cell morphology nor did it induce ROS-production by more than 2. These results indicate an unpredictable costimulatory influence of the plastic surface on PMN. This view is supported by the results achieved in the presence of PMA as cell activator: On plastic a minimum concentration of 100 nmol/L of PMA was required to induce a clearly detectable ROS-production and only 1.000 or 10.000 nmol/L of PMA were able to achieve a plateau level of ROS. On Percoll, however, the same PMN responded 10 to 100 times more sensitive to PMA: With 10 nmol/L of PMA a strong ROS-production could be reliably documented and 100 nmol/L of PMA was enough to generate the plateau level of ROS which was frequently clearly above the plateau on plastic. In addition to improved sensitivity the range of reactivity was considerable wider on Percoll as compared to plastic: PMN stimulated with 10 nmol/L of PMA diplayed a ROS-production which was 373 – 634 times that of the same cells under identical conditions but without PMA. And with 100 nmol/L of PMA even factors of 914 – 1.080 were achieved. These were remarkable higher than those on plastic and with higher concentration of PMA: 1.000 to 10.000 nmol/L of PMA resulted only in factors of 45 – 63. As 30 to 100 nmol/L of PMA are sufficient to induce maximal ROS-production in PMN on Percoll these concentrations stay below those were cell losses on Percoll might exceed 5%. Thus, ROS studies on Percoll can be performed without any PMN loss greater than 5 % for most of the species tested. This new technique has been shown to be basically applicable to cattle as well as to dogs, humans and horses. It also made possible to document that 0,1 up to 100 autologous platelets have no detectable influence on the PMA induced ROS-production of PMN. In contrast, 50 % of autologous MNC reduce the ROS-production of PMN due to absorption of PMA and perhaps also by means of regulatory influence.