Haemophilus influenza is a major cause of inflammation in various chronic lung diseases including COPD and pneumonia. This protocol describes methods for assessing the effect of haemophilus on lung inflammation. The advantages of this technique is it allows for a comprehensive assessment of innate and adaptive immune responses.
Incubate 450 microliters of peripheral blood with 50 microliters of an activated propidium iodide labeled H influenzae in a five milliliter tube for 20 minutes in a water bath at 37 degrees Celsius. Remove the sample from the water bath, add five microliters of DHR and vortex for 10 seconds. Then place it back in the water bath for another 10 minutes.
After removing the sample from the water bath, lyse the erythrocytes with five milliliters of 0.8%ammonium chloride solution and analyze the samples on a flow cytometer as described in the text. Divide the peripheral blood sample into aliquots for control and antigen stimulation. Add costimulatory antibodies to both samples.
Then add the non typable H influenzae to the antigen sample and incubate at 37 degrees Celsius and 5%carbon dioxide for one hour. Next, add the Golgi blocking agent brefeldin A to the samples and incubate them for another five hours. After lysing the erythrocytes as demonstrated previously, fix the leukocytes using 500 microliters of one to 2%para-formaldehyde for one hour.
Count the cells using a hemocytometer, then permeabilize 1 million cells with 100 microliters of 0.1%saponin for 15 minutes. Next, incubate the cells with appropriate fluorescent labeled antibodies. Wash the cells, then analyze them using a flow cytometer.
Determine the proportion of antigen responding cells by getting the relevant lymphocyte populations. Perform background staining on non stimulated cells for all the cytokines to be analyzed. Slice about 20 to 40 grams of the lobectomy sample into three to five cubic millimeter sections.
Place them inside a sterile 50 microliter chamber and mechanically fragment the tissue using an appropriate dis-aggregator. After tissue disaggregation, lyse the red blood cells as demonstrated and resuspend the cells in sterile RPMI. Then filter the cells through a 100 micrometer sterile nylon mesh and count the of viable cells using the trypan blue exclusion method.
For the infection assay, resuspend the lung cells in RPMI to a final concentration of 4 million cells per milliliter per tube. Then infect the cells at an MOI of 100 bacteria per cell. Loosen the cap half a rotation to allow gas transfer in the tubes.
Place the cells in the tube rotator and incubate them at 37 degrees Celsius while rotating at 12 RPM. One hour after stimulation, add grefeldin A to prevent the extracellular export of cytokines and incubate the cell suspensions for a further 16 to 22 hours with rotation. The following day, wash the cell suspension with 500 microliters of PBS containing 1%bovine serum albumin and 0.01%sodium azide.
Next, stain the cell suspension for specific human lymphocytes cell surface markers for one hour. Then wash the cells with PBS and fix and permeabilize them as demonstrated previously. Next, incubate the cells with intracellular cytokine staining antibodies for one hour.
Then wash the cells and resuspend them in 100 microliters of PBS before data acquisition on a flow cytometer. Measure proteolysis via the action of metalloproteinases, add fluorescein labeled gelatin substrate directly on the lung tissue section slides. Place the slides horizontally and incubate them in a light protected humidified chamber at 37 degrees Celsius for one hour.
To prepare negative controls, add one x reaction buffer without fluorescent gelatin to the sections for further analysis. Peripheral blood mononuclear cells were gated using forward and side scattered to define the phagocyte population. The phagocyte population was further defined by CD14 expression to label the monocytes.
ROS measurement was performed via oxidation of DHR 123 to produce fluorescence. The median fluorescence of the stimulated sample was compared to the control. Intracellular cytokine production by lymphocytes was measured using flow cytometry.
Cells were analyzed first for their expression of the leukocyte marker CD45 and then for CD three and CD four, CD eight. The CD three, CD four positive cells were assessed for intracellular cytokine production in control and stimulated samples. Protease activity was measured by NC two zymography in unfixed lung tissue sections.
Fluorescent staining indicated the presence of MMP activity, which was also co-localized with the expression of chromatin. Adding antibodies to the lung tissue samples requires concentration and staining of the cells will require optimization in preliminary experiments. The supernatant of the lung tissue samples can be further analyzed for other inflammatory mediators using techniques such as ELISA and These techniques can be used to assess the effect of potential therapies on lung inflammation.
