Quantifying Bordetella-induced Neutrophil Infiltration Using a Myeloperoxidase Assay Sandra M. Fuentes, Research Scholar Faculty Mentor: Dr. Eric Harvill The Pennsylvania State University Abstract Bacteria from the genus Bordetella are gram negative cocobacilli. Several members spread via contaminated aerosols and colonize the respiratory tract of mammals. Two important species from the genus are B. bronchiseptica and B. pertussis. Phagocytosis by neutrophils contributes to the clearance of these bacteria. Neutrophils produce an enzyme called myeloperoxidase (MPO) that can be used as an indicator of the relative number of neutrophils present. In this study, an MPO activity assay was used to detect neutrophil infiltration into the lungs of mice infected with B. bronchiseptica or B. pertussis. The lower limit of detection of the MPO activity assay was between 22,000 and 44,000 neutrophils. The assay was able to detect the myeloperoxidase activity of samples homogenized in HEPES or PBS. Perfusion of the lung was critical for accurate quantification of neutrophils present in the lung. The results of this study suggest that this MPO assay can be used to quantitate neutrophil infiltration into the lungs of mice infected with Bordetella. Introduction Bacteria from the genus Bordetella are gram negative coccobacilli. Closely related members of this genus spread via contaminated aerosols and colonize the respiratory tract of mammals. Two very similar species of Bordetella are Bordetella bronchiseptica and Bordetella pertussis. B. bronchiseptica, infects a wide range of mammals, but it very rarely infects humans, while B. pertussis, is strictly a human pathogen and is responsible for whooping cough, a disease that most commonly affects children younger than one year old. Even though there are vaccines available for whooping cough there is still a high incidence of the disease in children. Immunity to Bordetella has been well studied. Studies done with mice deficient in B cells showed that these cells are necessary for the clearance of both B. pertussis and B. bronchiseptica (1). It is also known that serum antibodies can rapidly clear B. bronchiseptica but not B. pertussis (1). Another type of cell from the immune system that contributes to the clearance of Bordetella from the lung is the neutrophil. Neutrophils are a type of leukocyte that are attracted to the site of infection to phagocytose the bacteria. Neutrophils adhere to bacteria by an Fc receptor that recognizes the constant chain of the immunoglobulins that have opsonized or covered the bacteria. A study performed by Lenz, et al (2) using a technique that distinguishes extracellular bacteria from phagocytosed bacteria showed that there is a low survival rate among phagocytosed B. pertussis. Only 1-2% of the bacteria remain viable after phagocytosis by neutrophils.
A variety of virulence factors from B. pertussis and B. bronchiseptica are known to affect neutrophil function after bacterial infection. The adenylate cyclase toxins (Cya) of these two bacteria are very similar and it has been suggested by different studies that the Cya inhibits neutrophil function and consequently phagocytosis (3, 4, 5). Another virulence factor that affects the phagocytic activity of neutrophils is filamentous hemagglutinin (FHA). Antibodies produced against FHA during the course of infection interfere with phagocytosis by neutrophils (5). The role of pertussis toxin, present only in B. pertussis, in neutrophil function is not yet known. The number of neutrophils in the lung is not the same during an infection with B. bronchiseptica as it is with B. pertussis. This difference may be due to a variety of factors, such as differences in complement activity, differences in the role of tumor necrosis factor alpha (TNF) or the activity of pertussis toxin. The roles of all of these components of the immune response to the bordetellae are currently being studied in the lab. Previous studies performed in this lab have shown that complement deficient mice have a reduced number of neutrophils. Also a study by Sadiko et at has shown that, activation of NFκB, a factor that contributes to the production of TNF, produces an increase in the number of neutrophils infiltrating the lung (6). In another experiment with mice infected with a pertussis toxin mutant the amount of neutrophils in the lung was measured and the results obtained suggested that pertussis toxin inhibits neutrophil infiltration in the lungs. Finally, histology data from complement receptor type three (CR3) knockout mice shows a greater degree of pathology, consisting of neutrophil and macrophage infiltrates, after infection with Bordetella. In all of these experiments the number of neutrophils was measured using differential cell count or histology. A more sensitive assay can give a more accurate number of neutrophils in the lung. For the purpose of producing a more accurate number we used another method for neutrophil quantification. Neutrophils produce an enzyme called myeloperoxidase that can be used to identify the amount of neutrophils infiltrating a tissue after inflammation. For this study, we used an assay that allowed us to detect the presence and relative quantities of myeloperoxidase present in the lungs of mice infected with B. bronchiseptica or B. pertussis. A variety of studies have used this assay to measure the decrease or increase in the amount of neutrophils during inflammation (7, 8). For this study on neutrophil infiltration of the lungs of mice during B. pertussis or B. bronchiseptica infection, we measured myeloperoxidase activity using a modification of the protocol developed by Schneider et al (9). Methodology A. Infection of the mice: Wild type and a variety of genetically modified strains of mice were infected with wild type strains of B. bronchiseptica (RB50) or B. pertussis (BP536). Mice sedated with isoflurane were inoculated by pipetting 50 µl of PBS that contained 5x10 5 CFU to the external nares of the mouse. B. Extraction, homogenization and preparation of lung samples a. Samples pre-prepared for cytokine analysis:
Some of the samples used for the MPO activity assay were also used for cytokine analysis. These lung samples were extracted from the mouse without being perfused and were homogenized in 1ml of phosphate buffer saline (PBS). The samples were centrifuged at 13,000 x g for 3 minutes, and the supernatant was then used for the MPO activity assay. b. Samples prepared for the MPO activity assay exclusively: The lungs of mice were perfused with 3 mls of 1X PBS. The lungs were then prepared following the protocol of Schneider et al (9) with some modifications. The lungs were homogenized in 50 mm HEPES ph 8.0 and half of the homogenate was used for total and differential cell count. The rest of the homogenate was stored at -80ºC from 1hour to overnight to lyse the cells. After thawing, the sample was centrifuged for 30 minutes at 10,000 x g at 4ºC. The supernatant was then used for the MPO activity assay. c. Blood samples: The blood samples were obtained by either ocular bleeding or cardiac puncture. The sample was then layered on a Histopaque gradient and centrifuged for 30 minutes at 3,000 rpm. The layer of white blood cells was extracted and used for the MPO activity assay and differential cell count. C. Myeloperoxidase activity assay: The protocol for MPO detection was modified from Schneider, et al (9) Each sample was diluted serially, from a 1:2 dilution to a 1:64 dilution, with 10mM citrate buffer ph 5.0. For an assay with only one sample, 75 µl of each dilution was added to different wells of a 96 well plate. Substrate solution (75 µl) was added to each well. This substrate solution consisted of 3 mm Tetramethylbenzidine (TMB), 120 µm resorcinol and 2.2 mm H 2 O 2 in deionized water. The reaction was stopped after two minutes with 150 µl of a cold stop solution that consisted of 4N H 2 SO 4. For the background OD for each dilution the same reagents were added to a different well, but the stop solution was added before the substrate. The OD was read at 430 nm. Myeloperoxidase activity was determined by subtracting the value of the background from the value of the dilution. All the reagents were kept in the refrigerator until used and the substrate solution had to be made fresh before every assay. The background reactions were assayed right after the sample reaction to avoid the precipitate that forms when the acid stays in the well for too long. D. Cell Count: The part of the sample that was taken for the cell count was digested with a digestion buffer made up in a cell culture media containing 10% fetal calf serum, 1% Penicillin-Streptomycin solution (10,000 µg/ml), 1 mg/ml collagenase and 10 µg/ml DNase (10) for 30 minutes. Then the sample was layered on a Histopaque gradient for 30 minutes at 3,000 x g. The layer of white blood cells was removed. Part of this sample of white blood cells was used for total cell count and part was used for differential cell count. For the total cell count the white blood cells were diluted 1:10 in PBS and counted in a hemacytometer. For the differential cell count, the white blood cells were diluted 1:10 or 1:100, as necessary, in 1% BSA
in PBS and then centrifuged at 300 x g for 5 minutes on to glass slides using cytospin cartridges. The cells were stained with a modified Wright/Giemsa stain. Results Construction of a standard curve with blood samples. To relate an OD reading from the MPO activity assay with the amount of neutrophils present in the sample we used the blood from a mouse infected with B. pertussis to perform the MPO activity assay and standardize to a differential cell count as described in Materials and Methods. The results (Fig. 1) show that there is a rapid decrease in myeloperoxidase activity in the first 3 OD readings which correspond to the 1:2, 1:4 and 1:8 dilutions. After these values there is very little detection of MPO activity. These results indicate that the assay loses sensitivity when the number of neutrophils in the sample is between 22,000 and 44,000 neutrophils. To be able to correlate an OD reading to a certain number of neutrophils the OD values of the dilutions that had over 44,000 neutrophils were used to construct a standard curve. This standard curve, shown in Figure 2, shows a linear relationship between myeloperoxidase activity and the number of neutrophils present in the sample. The results of this assay show that we can use the OD reading activity to obtain the number of neutrophils that are present in the lung. Analysis of MPO activity in a lung sample. To determine if MPO activity could be detected in lung homogenate the lung of a mouse infected with B. bronchiseptica, extracted and perfused on day three post inoculation was used. The lung was extracted and homogenized in HEPES and stored at -80ºC overnight. The next day the lung homogenate was centrifuged and the resulting supernatant was diluted and assayed for MPO activity. Except for the last three dilutions the OD reading of the samples was significantly above background (Table 1). The results from this assay show that myeloperoxidase activity can be detected within lung tissue homogenate. Analysis of lung sample homogenized in PBS. The lung of a TNF knockout mouse on day 28 post inoculation with B. pertussis was used to determine if the phosphate saline buffer used to homogenize the samples stored for cytokine analysis interfered with the assay. The lung was extracted without perfusion and homogenized in PBS. The lung homogenate was then centrifuged and the supernatant was stored at -80ºC for later analysis. The MPO activity of the lungs of two wild type mice, on day three post inoculation with B. brochiseptica, homogenized in HEPES, as described above, was used as a positive control. The OD values of the lung homogenized in PBS were significantly above background at the highest dilutions. Results from this experiment show that PBS does not interfere with the assay. Analysis of perfused and unperfused lungs. To determine whether perfusion made a difference in the value of MPO activity an assay was performed to compare MPO detection in a perfused and an unperfused lung. The lungs of two wild type mice were used. One of the lungs was perfused before extraction to remove blood inside the tissue. Both lungs were rinsed in PBS to remove peripheral blood. The results (Fig. 3) show that there is four-fold higher level of myeloperoxidase activity in the unperfused lung. Since
the only difference between these two samples was the perfusion step, the increased MPO activity in the unperfused lung is due to the neutrophils in the blood. These results show that perfusion of the lung is necessary to obtain an accurate reading of MPO activity from the neutrophils that were present in the tissue. Analysis of infected and uninfected lungs. To determine whether the MPO assay that we have modified is able to detect differences in neutrophil numbers between infected and uninfected mice, the lungs of three uninfected and three mice on day 1 post inoculation with B. bronchiseptica were analyzed for MPO activity. The lungs were perfused and homogenized in HEPES. The homogenate was centrifuged and the supernatant was used for the assay. The results shown in figure 4 are the average of the OD values of the three infected and the three uninfected mice. The average MPO activity of the infected lung is almost 6 times higher than the average of the uninfected lung. However, the standard deviation of the average OD of the infected mice was very broad. This is because one of the three mice inoculated with B. bronchiseptica had OD values similar to the uninfected mice. It is possible that this mouse was not properly inoculated, which would explain the very low OD reading and the broad error bars. The results of this assay indicate that it may be possible to detect differences between infected and uninfected lungs with this MPO assay. Discussion Inflammatory reactions, such as those caused by the infection of bacteria, are usually followed by the infiltration of neutrophils to the site of inflammation. Neutrophils contain in their granules the enzyme myeloperoxidase, that catalyses the oxidation reactions of H 2 O 2. In these oxidation reactions halide ions are transformed to hypohalous acids and used for the killing of the bacteria. Since this enzyme is specific to neutrophils it is often used as an indicator of neutrophil infiltration into tissues. Myeloperoxidase can be detected in a colorimetric assay that uses H 2 0 2 and the chromogen TMB. The advantage of using this assay is that it allows for the objective quantification of the number of neutrophils in blood samples and tissues. In this study we modified the MPO activity assay protocol for rats developed by Schneider, et al (8) to analyze the lungs of mice infected with Bordetella. With this assay, myeloperoxidase activity can be detected in samples that have over 44,000 neutrophils/75µl. The assay loses sensitivity when the number of neutrophils is lower. With the standard curve it is possible to correlate the reading of MPO activity with the number of neutrophils in the lung. The MPO assay performed on lung tissue homogenate showed that this modified assay works for tissue as well as for blood. In the study performed by Schneider et al (9) they used a clear supernatant instead of the red supernatant that resulted after the first centrifugation of the homogenate because they believed that hemoglobin interfered with the assay. However, visual observation from our study suggests that even though it takes longer to start, the reaction is much stronger in the red supernatant. As shown with the lung homogenized in PBS, this buffer does not interfere with the assay and can be used to homogenize the lung instead of HEPES buffer. However, to obtain an accurate reading of the myeloperoxidase activity of the neutrophils that
infiltrated the lung, it is critical for the lung to be perfused, since the MPO of the neutrophils in the blood will also be detected. We conclude that this assay can be used to detect myeloperoxidase activity in the lungs of mice infected with Bordetella. It can be used to answer questions that address the differences in neutrophil infiltration and the importance of neutrophils in the clearance of infection.
Tables and Graphs Figure 1: Standard curve of the number of neutrophils in the blood of a mouse infected with B. pertussis 0.4 0.35 OD - Background (430nm) 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50000 100000 150000 200000 Number of Neutrophils Figure 2: Standard curve of the number of neutrophils in the blood of a mouse infected with B. pertussis OD - Background (430nm) 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50000 100000 150000 200000 Number of neutrophils
Table 1: Detection of MPO activity in a lung from a wild type mouse on day three post inoculation with B. bronchiseptica Dilution Myeloperoxidase activity (OD 430nm Background) 1:4 1.025 1:8.780 1:16.257 1:32.125 1:64.094 1:128.039 1:256.021 Table 2: Detection of MPO Activity in lung homogenized in PBS Myeloperoxidase activity (OD 430nm Background) Dilutions Balb/c RB50 day 3 (5) (HEPES) Balb/c RB50 day 3 (6) (HEPES) TNF 536 day 28 (PBS) 1:4.414.185.914 1:8.132.043.78 1:16.031.025.267 1:32.038.003.144 1:64.02 -.005.08 1:128.015.004.068 1:256.006.005.027
F igure 3: M P O assay of perfused vs. unperfused lung sam ples 2 Myeloperoxidase activity (OD 430nm) 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Perfused lung Unperfused lung 0 1 Figure 4: MPO activity assay of uninfected vs. infected wild type mice 1.4 Myeloperoxidase activity (OD 430nm) 1.2 1 0.8 0.6 0.4 0.2 Uninfected Infected 0 1
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