Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

During an infection, activated lymphocytes release inflammatory mediators — cytokines — to elicit an immune response.

To quantify inflammatory mediators in an infected tissue, begin with a cell suspension containing human lung-tissue-derived immune cells.

Incubate the cells with Haemophilus influenzae — a respiratory pathogenic bacterium. The bacteria's surface antigens interact with pattern recognition receptors on phagocytic immune cells. This initiates bacterium internalization, processing into smaller peptides, and the presentation of the peptide by the major histocompatibility complex molecule.

The antigen-MHC-II molecule on phagocytic cells binds to T lymphocytes through the T cell receptors, along with co-stimulatory receptor interactions, resulting in T lymphocyte activation and the production of pro-inflammatory cytokines.

Treat the cells with Golgi blockers, impairing cytokine release — causing their accumulation inside the cells. Wash the cells with a blocking buffer to block non-specific binding sites.

Stain the cell suspension with a mix of fluorophore-labeled antibodies that bind specifically to human T lymphocyte-surface markers, enabling lymphocytes' labeling.

Fix the cells, and permeabilize them with a pore-forming surfactant. Incubate the permeabilized cells with fluorophore-labeled anti-cytokine antibodies, which enter the cells and interact with specific cytokines.

Using flow cytometry, measure the cytokine fluorescence signal within labeled lymphocytes, correlating with inflammatory mediator production due to bacterial infection. 

Slice about 20 to 40 grams of the lobectomy sample into 3 to 5-cubic-millimeter sections. Place them inside a sterile 50-microliter chamber, and mechanically fragment the tissue using an appropriate disaggregator. 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 number 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 Brefeldin 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, stay in the cell suspension for specific human lymphocyte 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.