| Background: Neutrophils, or polymorphonuclear cells (PMNs), are the most significantinflammatory cells in the organism. After pathogenic bacteria entering into an organism,PMNs will assemble to the place of pathogenic bacteria and then devour, thereforedigesting and degrading pathogenic bacteria, as well as giving full scope to naturalimmunity against pathogen. From the past research, after Mycobacterium tuberculosis(Mtb) infecting the organism, the immunity of resisting tuberculosis is mainly cellimmunity. Furthermore, Mtb enters into macrophage through complement receptor andinterrupts macrophage’s activity and the mature of lysosome to bring into long-termsurviving and latent infection. However, in the current date, PMNs will devour Mtbfirstly in the early stage of acute TB (pulmonary tuberculosis). Besides, PMNs is themost leading macrophage in the infection of active TB patients, and there is not onlysimple phagocytosis in the biologic influence. Hence, to obtain full-scalecomprehension of immunity protection mechanism about organism resisting infection,there are important academic meaning and clinical application value on theinvestigation of PMNs killing Mtb’s activity and natural immunity function to resist TBinfection.Objective: To investigate the optimal concentration of FDA and BCECF-AM inlabelling of Mtb and to modify the method for killing of Mtb by PMNs of humanperipheral blood using flow cytometry. To observe the effects of several chemicalregents and temperature on PMNs killing of Mtb and also to observe the effects ofcytokines such as Th1type (IL-2and IFN-γ), Th2type (IL-4), common type (GM-CSF)and Th17cell type (IL-17) on human PMNs killing of Mtb.Methods:1. Mtb were labelled with different concentrations of fluorescent FDA and2’,7’-DEAD-5&6-BCECF-AM in PBS (pH7.2), the positive rate and meanfluorescence intensity (MFI) of Mtb were measured by flow cytometry.2. FDA labelled Mtb and BCECF-AM labelled Mtb were incubated for30minutes inthe0℃ice-bath,37℃,65℃and95℃water bath, thereafter washing, and usingflowcytometry to test lablle rates and MFI. 3. FDA labelled Mtb and BCECF-AM labelled Mtb were incubated with differentconcentration of ethanol, carbinol and PFA reagent for30minutes in the roomtemperature, washing, and using flowcytometry to test lablle rates and MFI..4. The whole blood were incubated with FDA-Mtb and BCECF-Mtb for7minutes in37℃, thereafter washing and lysing erythrocytes, followed adding auto serum, incubatedfor0～60min in37℃. The killing rates of Mtb by PMNs were measured byflowcytometry.5. Percoll isolated PMNs were incubated with FDA-Mtb and BCECF-Mtb for7min in37℃, thereafter washing and adding auto serum, incubate for0～60min in37℃. Thekilling rates of Mtb by PMNs were measured by flowcytometry.6. Pretreatment of whole blood with Th1type (IL-2and IFN-γ), Th2type (IL-4),common type (GM-CSF) and Th17cell type (IL-17) for1h before incubation withFDA-Mtb for7min in37℃, thereafter washing and lysing erythrocytes, followedadding auto serum, incubated for30min in37℃. The killing rates of Mtb by PMNswere measured by flowcytometry.Results:1. The lablle rates in PBS in different concentration of FDA (0.025μM～2.0μM) andBCECF-AM (0.025μM～2.0μM) increase from85%to99%as well as from33%to99%respectively. They both have significant difference (p <0.01); MFI increases from45to1506as well as from25to2202respectively, indicating significant difference (p<0.01).2. Mtb lablled with FDA and BCECF-AM, after being in0℃ice-bath,37℃,65℃,95℃water bath in30min, FDA-Mtb’s lablle rates decreases from99%in0℃to62%in65℃. Besides, it will decline with the rise of temperature. When the temperature is95℃,the marked ratio is almost zero (0.21%), indicating significant difference (p <0.01). MFIdeclines from2015in0℃to87in95℃， indicating significant difference (p <0.01) aswell; BCECF-Mtb’s lablle rates decreases from99%in0℃to95℃, the lablle rates isalmost zero (0.18%), indicating significant difference (p <0.01). MFI declines from2344in0℃to93in95℃, indicating significant difference (p <0.01) as well.3. Mtb lablled with FDA and BCECF-AM, after acting with different general chemicalreagents (ethanol, methanol, PFA) in30min, FDA-Mtb’s MFI in the ethanol decreasesfrom opponent’s1598to655, in the methanol declines to118, in the PFA declines to 185, all indicating significant difference (p <0.01); BCECF-Mtb’s MFI in the ethanoldecreases from opponent’s1318to708, in the methanol declines to187, in the PFAdeclines to648, all indicating significant difference (p <0.01) as well.4. Mtb lablled with FDA, act with full blood in37℃in0min,10min,30min and60min. The percentages of PMNs killing Mtb are14.95,20.59,31.50,44.58respectively;Acting with separating PMNs in37℃in0min,10min,30min and60min, thepercentages of PMNs killing Mtb are11.37,12.02,27.20,58.44. Both the percentagesof PMNs killing Mtb indicate an increasing trend (p <0.01) in0～60min activity of37℃; Similarly, Mtb lablled with BCECF-MA, act with full blood in37℃in0min,10min,30min and60min. The percentages of PMNs killing Mtb are18.83,19.64,34.65,40.17respectively; After acting with separating PMNs in37℃in0min,10min,30minand60min, the percentages of PMNs killing Mtb are15.94,18.74,29.46,38.44. Boththe percentages of PMNs killing Mtb indicate an increasing trend (p <0.01) in0～60min activity of37℃.5. After using different concentration of IL-2(40~320U/ml) to deal with PMNs inadvance,1hour later, it is apparent to show compared with its opponent, killingpercentage of PMNs in30min has increased in40U/ml. As the increase ofconcentration, the killing ratio increases (p <0.01). For instance, in320U/ml the killingrate is up to48%. After using different concentration of IFN-γ (200~1600U/ml) to dealwith PMNs in advance,1hour later, as the increase of concentration, the killing rateincreases (p <0.01). For instance, in200U/ml the killing ratio is28%. In1600U/ml thekilling ratio is up to56%.6. After using different concentration of IL-4(50~400U/ml) to deal with PMNs inadvance,1hour later, it is apparent to show compared with its opponent, killingpercentage of PMNs in30min has increased to23%in50U/ml. As the decrease ofconcentration, the killing rate decreases (p <0.05). For instance, in400U/ml the killingratio is down to12%. After using different concentration of GM-CSF (50~400U/ml) todeal with PMNs in advance,1hour later, there is no statistics significance about killingpercentage of PMNs in30min compared with its opponent.7. After using different concentration of IL-17(0.8~6.4ng/ml) to deal with PMNs inadvance,1hour later, compared with its opponent, killing percentage of PMNs in30min has increased in0.8ng/ml. As the increase of concentration, the killing ratedecreases (p <0.05). For instance, the killing rate increases to56%in6.4ng/ml. Conclusion:1. Flowcytometry can acutely detect different concentration of FDA and BCECF-MAaffecting Mtb marked ratio and MFI. Moreover, two fluorescents can mark Mtb stably.2. IN different temperature, different concentration of ethanol, methanol and PFA, Mtb’slivability and the lablle rates of flowcytometry test have positive correlation with MFI.3. Flowcytometry can able to detect the killing rate to FDA-Mtb and BCECF-Mtb byhuman peripheral blood PMNs and full blood separating PMNs. Besides, the killing ratecan enhance as the extension of time.4. Th1type IL-2, IFN-γ and Th17type IL-17can help to enhance the function of PMNskilling Mtb. Otherwise, Th2type IL-4reduce the function of PMNs killing Mtb, whichshows these types have the adjustment function in PMNs resisting TB immunity. |
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