In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

The protocol described here measures lung macrophage efferocytosis after exposure to a criteria air pollutant ozone. These data indicate that ozone exposure decreases alveolar macrophage efferocytosis and thus could be a mechanism for why elevated levels of ozone increase the incidence of lung diseases. The main advantage of this technique is that it allows an in vivo assessment of alveolar macrophage efferocytosis without any ex vivo manipulations of the macrophages that may alter their phenotype or function.

This technique has revealed a novel mechanism by which the lung cannot repair itself after air pollution exposure. Additionally, this technique may reveal novel pathways to targets that alter efferocytosis in the lung. This method can be applied to any model of lung injury or inflammation.

However, the user will need to optimize when it is best to assess efferocytosis following lung injury. Performing the oropharyngeal aspiration can be technically challenging. I would advise working with your veterinary staff to make sure you have optimized your technique to perform this procedure.

Begin by culturing Jurkat T cells in basal cell culture medium supplemented according to the manuscript directions at 37 degrees Celsius and 5%carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days. Prepare apoptotic cells by growing them to 90%confluency which takes three to four days after passaging.

Grow cells in five T75 flasks to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters and transfer them to a 50 milliliter conical tube. Count the cells using Trypan Blue staining and a hemocytometer.

Pellet the cells by centrifugation at 271 times g for five minutes and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100 by 20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.

Prepare approximately nine culture dishes. Set one dish aside as an unexposed control and expose the remaining dishes to UV.Set the UV crosslinker to the correct energy level by pressing the energy button and entering 600 with the number pad. Irradiate all dishes with the cells except for the control making sure to remove the top cover of the tissue culture dish during UV exposure.

Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50 milliliter conical tube and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant and resuspend the cells in 24 milliliters of sterile PBS.

Centrifuge the tube again and remove the supernatant. Prepare the cells for dosing mice by resuspending them in PBS at the appropriate concentration. Once the mouse is properly anesthetized, place it in a semi-recumbent supine position.

Use surgical string tied between pegs on a slanted acrylic sheet board to suspend the mouse by the maxillary incisors. Use a pair of blunt non-ridged forceps to lightly grab and pull the mouse tongue and instill the apoptotic cells into the oral cavity with a P200 pipette. Dosing is successful when mice make a crackling noise one to two seconds after receiving the dose.

With a gloved finger, gently cover the nose of the mouse until it inhales while the tongue is retracted. Keep the nose covered until no liquid is visible in the oral cavity and the mouse has taken two or more inhalations. Remove the mouse from the inoculation board and return it to the cage to allow it to recover from anesthesia making sure to place the mouse on its back to prevent debris from blocking the nares.

After all of the mice have awoken from anesthesia, wait 90 minutes to allow alveolar macrophages to engulf influx of apoptotic cells. To collect lavage fluid, prepare the euthanized mouse according to the manuscript directions. Slowly push PBS into the lungs allowing them to inflate, then pull the same volume back out making sure the PBS is not exiting the nostrils.

Collect the pooled lavage from each mouse in a 15 milliliter tube. Centrifuge the bronchoalveolar lavage or BAL at 610 times g for six minutes at four degrees Celsius, collect the supernatant into a 1.5 milliliter tube, and freeze it at minus 80 degrees Celsius. The pellet represents the cells from the bronchoalveolar space.

Add one milliliter of ACK red blood cell lysis buffer to the pellet to remove the residual red blood cells. Vortex and lyse for one minute on ice, then add four milliliters of PBS to stop the lysis reaction. Centrifuge the cells at 610 times g for six minutes at four degrees Celsius and aspirate the supernatant with a vacuum aspirator.

Then, resuspend the cells in one milliliter of PBS with 10%FBS and count the cells on a hemocytometer without Trypan Blue. Centrifuge 120 microliters of each sample onto slides at 12 times g for three minutes using medium acceleration and a cytocentrifuge. Leave the slides to dry overnight.

The next day, stain the slides with hematoxylin and eosin in order to calculate both efferocytic and differential cell counts. Then, view slides under a Brightfield setting on a biological microscope using a 20X or 40X objective. Calculate the efferocytic index based on the ratio of alveolar macrophages that phagocytosed apoptotic Jurkat T cells to alveolar macrophages without apoptotic cell uptake making sure to count at least 200 cells from each slide.

This protocol was used to investigate the effect of ozone exposure on pulmonary inflammation. Ozone exposed mice displayed an increase in macrophages and neutrophils in the air space compared to the filtered air control group and had an increase in BAL protein, a marker of alveolar epithelial dysfunction. Prior to dosing the mice, apoptosis in Jurkat T cells was confirmed with flow cytometry.

The 60 milli joule UV exposure level and four-hour incubation time resulted in repetitive results of approximately 75%apoptotic cells. Efferocytic macrophages were identified as macrophages that had engulfed a Jurkat T cell compared to regular alveolar macrophages. There was a significant decrease in the efferocytic index of the ozone exposed group compared to the filtered air controls which indicates that ozone induced pulmonary inflammation is associated with decreased clearance of apoptotic cells.

Attention to detail and writing down any observed differences is critical during this procedure. It is also recommended to blind the samples for analysis and have two different people count the macrophages. After isolation, the BAL cells can be used for mRNA analysis, stained for additional markers by immunofluorescence, and/or run on a flow cytometer to distinguish phenotypic changes.