Characterization And Significance Of Neutrophil Responses In Rat Blood During A Prolonged Bacterial Stimulation Period

Neutrophils are a group of important phagocytes and act as the vital first defense in the innate immune system against bacterial infection in the body. It is generally considered that the intracellular killing processes are the mechanisms for the bactericidal function of neutrophils, which are related to reactive oxygen species (ROS) produced by NADPH oxidase during the respiratory burst and proteases released from cytoplasmic granules into phagosomes after the neutrophils encounter bacteria or their products. The major killing process completes in about 30 minutes. These proposed neutrophil bactericidal mechanisms are largely based on the results obtained from studies using purified neutrophils. Experimental observation has mainly focused on the events occurring during the respiratory burst, or shortly after, in response to the stimulation of neutrophils by bacteria or their products. As a result, there is currently lack of knowledge with regard to the events happening in a period longer than 30 minutes after mixing of neutrophils with bacteria or their products.This study investigated changes in the biological properties of rat peripheral blood neutrophils during a prolonged period of bacterial stimulation (up to 300 min). Oxygen consumption assay and chemiluminescence detection approach were used to study biochemical responses of neutrophils after bacterial stimulation. Viable bacteria in testing samples were quantitatively measured by bacterial culture method. Optical microscopic method was employed for determination of phagocytosis of bacteria. After incubation of Pseudomonas aeruginosa with fresh rat blood in vitro, two distinct peaks of production of reactive oxygen species were observed, that is, Peak I and Peak II. The Peak I period started shortly after addition of the bacteria to the blood sample, characterized as a short duration (less than 30 min) and small peak value with significant oxygen consumption. The Peak II period started at about 60 min after bacterial addition, with a prolonged duration (up to about 150 min), a peak value much higher that that of Peak I and no observable oxygen consumption. During the entire reaction period (about 300 min), the permeability of plasma membrane did not change significantly in neutrophils that were undergoing chemiluminescent reactions, since no extracellular chemiluminescent reaction could be detected using horse radish peroxidase. Apparently, the Peak I and Peak II had distinct biological properties, representing two different functional states of the neutrophils.Bacterial killing studies demonstrated that rat blood had significant bactericidal effect, which was directly related to neutrophils but not plasma components. The bactericidal process by the blood could be divided into two distinct periods. The first was a rapid bacterial inhibitory/killing (RBIK) period, which only covered a short period (less than 10 min), corresponding to the first part of the Peak I or earlier period. The second was a delayed bacterial inhibitory/killing (DBIK) period, which followed the rapid period and lasted to the end of Peak II. During the RBIK period, the rate of bacterial inactivation could be as high as about 90%. Most of the effect took place within 3 min after the reaction started. However, during this period, phagocytosis of bacteria by neutrophils was insignificant, since only less than 5% neutrophils phagocyosed bacteria. Apparently, the marked inactivation of bacteria in this period was related to extracellular bactericidal mechanisms of neutrophils, not intracellular ones. During the DBIK period, the rate of phagocytosis of bacteria by neutrophils increased with time. After Peak II (200-250 minutes after the start of the reaction), the rate of phagocytosis of bacteria by neutrophils could reach 30% to 60%. As the reaction time lapsed during this period, the increase in the rate of the number of phagocytosing neutrophils and the average number of phagocytosed bacteria per cell were inversely proportional to the decrease in the number of viable bacteria in the system. Therefore, the intracellular inhibitory/killing mechanisms of neutrophils could play an important role in inactivation of bacteria in this period.Based on the results of this study, we propose that there are at least two major functional classes of neutrophils in peripheral blood, i.e early-responsive neutrophils (ERN) and delayed responsive neutrophils (DRN). ERN reacts quickly to stimulus with significant oxygen consumption. DRN exhibits a delayed response to stimulus without oxygen consumption. We also propose that extracellular mechanisms of neutrophils can play a vital role in bacteria inactivation during infection. The major function of phagocytosis and other related intracellularly-confined mechanisms of neutrophils are to clear foreign substances from the body.The major reason for the apparent discrepancy between our results and others with regard to neutrophil killing mechanism in infection is discussed.