Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Photoconversion provides a trackable in vivo method for the quantitative analysis of leukocyte egress for a mechanistic insight into leukocyte residence and exit dynamics from multiple tissue sites in diseased states. Well, here we demonstrate the utility of leukocyte tracking from cutaneous tumors. The application of this assay to other tissues and diseases is limited only by the ability to administer light.

The main advantage of this technique is that to allows the quantification of endogenous, rather than transferred immune cell populations as a egress from inflamed tissues in vivo. To induce inflammation, after confirming a lack of response to toe pinch, use a micropipette to administer 20 microliters of dibutyl phthalate or DBP, to the ventral side of the ear pinna of an anesthetized Kaede transgenic mouse. After allowing the DBP to dry, pull the ear through a slit in a piece of aluminum foil and use double-sided tape to secure the ear flatly to the foil with the dorsal side facing upward.

Then, position that you're directly under a 405 nanometer light source and photoconvert for three minutes at 100 milliwatts of power. For vaccinia infection, use a micropipette to administer 5 times 10 to the 6th plaque forming units of vaccinia virus in 10 microliters of PBS on to the ear pinna of an anesthetized Kaede transgenic mouse and poke the pinna 25 times. 24 hours later, photoconvert the pinna as just demonstrated.

At the appropriate experimental time point, harvest the pinnae and the cervical and inguinal lymph nodes and use two pairs of tweezers to peel apart the ventral and dorsal side of each pinna. Then place the lymph nodes and the pinna pieces, with the inside of the ear facing down, into individual wells of a 24-well plate containing 1 milligram per milliliter of Collagenase D and 80 units per milliliter of Dnase diluted in HBSS containing calcium and magnesium. Use two 29 gauge needles to tease open each lymph node capsule and place the plate at 37 degrees Celsius for 30 minutes.

At the end of the incubation, press the digested pinna and lymph node tissues through separate 70 Micron nylon cell strainers to obtain single cell suspensions of each tissue. When the tumors have grown to the appropriate experimental size, shave any newly regrown fur around the tumor site and pull the tumor through a circular hole cut in a piece of aluminum foil. Position the tumor directly below the 405 nanometer light source and photoconvert for 5 minutes at 200 milliwatts of power.

24 hours after the photoconversion, harvest the tumors and brachial and inguinal lymph nodes from each animal and use scissors to mince the tumors within individual wells of a 24-well plate containing Collagenase D and DNase in HBSS. Place the lymph nodes into separate wells and tease open the capsules as just demonstrated. After incubating the tumors for one hour and the lymph nodes for a half hour at 37 degrees Celsius, press the digested tissues through separate 70 Micron strainers to obtain single cell suspensions of each tissue.

To analyze the single cell suspensions by flow cytometry, first collect the spleen or blood from a Kaede transgenic mouse and lyse the red blood cells with ammonium chloride potassium buffer. Split the cells into one unconverted Kaede FITC and one converted Kaede PE population and resuspend the single color Kaede PE cells in one milliliter of PBS in one well of a 24-well plate, then photoconvert the cells for 5 minutes under a 405 nanometer light source at 100 milliwatts of power. Next use the photoconverted Kaede PE cells and the unconverted Kaede FITC cells to set the photomultiplier voltages to 70 to 80%of the total range of each fluorescent channel.

Once the voltages for the Kaede proteins have been set, compensate for all of the other primary antibody stains using single fluorophore labeled compensation beads, according to the manufacturer's instructions. Then, run the samples on the flow cytometer according to standard flow cytometric analysis protocols. Single cell suspensions generated from the ear skin or cervical draining lymph nodes immediately following photoconversion exposure reveal a 78%conversion efficiency of all CD45 positive leukocytes in the skin, with no converted cells observed in the draining lymph nodes.

Quantification of the numbers of infiltrating CD45 positive leukocytes in photoconverted ears zero and 24 hours post-conversion, reveals that violet light exposure causes a statistically significant increase in leukocyte infiltration. This increase in CD45 positive leukocyte infiltration however, is small relative to the infiltration induced by a potent inflammatory stimulus such as vaccinia virus. Photoconversion also increases vascular permeability in the ear pinnae as measured by Miles assay, together indicating that effects of violet light on tissue inflammation should be considered when optimizing dose, exposure time and data interpretation.

Flow cytometric analysis of dissociated tumor cells from photoconverted animals reveals a significant conversion of intratumoral CD45 positive cells from Kaede FITC positive to Kaede PE positive, while draining lymph node cells remain unconverted. The photoconversion efficiency of CD45 positive leukocytes varied significantly between tumor types and sizes, with CD45 positive leukocytes within small melanoma tumors demonstrating the most efficient photoconversion. As the tumor volumes increase, the conversion efficiency decreases to around 50%Notably, melanin has a striking impact on photoconversion efficiency with a maximum photoconversion of CD45 positive cells observed within melanin-producing tumors of only about 30%in smaller tumors, that drops to 10%as the tumor volumes approach 400 cubic millimeters.

The examination of tumor draining lymph nodes 24 hours after photoconversion reveals the presence of Kaede red positive immune cells of various phenotypes, thus demonstrating leukocyte emigration from the photoconverted tumor. After its development, this technique paved the way for researchers in the field of Immunology to uncover the kinetics of steady state and inflamed leukocyte turnover in peripheral tissue. While attempting this procedure, it is important to ensure that only the tissue of interest is photoconverted, as inadvertent photoconversion of the surrounding tissue will compromise your results.

Individuals new to this method should optimize the exposure time for conversion of their tissue, as well as duration of the times before the tissue collection occurs based on the dynamics of leukocyte of interest.