TESTING THE HYPOTHESIS
A number of different experiments were performed to determine how most leukocytes traveled across the blood brain barrier and what molecules were involved.
EXPERIMENT 1: LEUKOCYTE MIGRATION THROUGH THE BLOOD BRAIN BARRIER
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Methods
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In order to see how leukocytes traveled across the blood brain barrier, they were made to do just that on the cell model of the BBB.
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Peripheral blood mononuclear cells (PBMCs), a type of leukocyte, were used in this experiment as they travel faster than any other leukocyte and are therefore easily observable. Blood was drawn from human volunteers and PBMCs within were separated out by centrifugation, a technique that uses high speeds and gravity to separate substances by their densities.
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The cell models were divided into two groups. One group received molecules that encourage leukocyte migration, TNF and CCL2. These molecules have also been speculated to increase the amount of leukocytes that barreled right through the cell (transcellularly). Observing whether this is true could pinpoint exactly how and why transcellular migration occurs, if it does at all.
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The other group of cells received nothing else and acted as a control to observe how leukocytes migrate without any other factors in play.
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The PBMCs (leukocytes) were added to the cell model they were supposed to travel across and warmed for one hour inside a CO2 incubator so that migration could take place. One hour is the perfect amount of time for many leukocytes to travel through the cells, but only far enough that it would be easy to tell whether they traveled through the gaps or through the cell itself.
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Why CO2? Carbon dioxide plays a major role in homeostasis and pH regulation within our bodies- it keeps everything balanced (along with water). Without a CO2 incubator, the cells may be drastically altered in terms of pH, rendering them unusable.
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The cells were then placed in a solution, ice-cold para formaldehyde, which would freeze them where they were at the moment of migration so that it could easily be observed how exactly they were getting through the cells. After staining the cells with dye to visualize the gaps in between, the cells were observed under a microscope to see if the leukocytes moved through the gaps of the endothelial cells or straight through the cells themselves.
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Results
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In order to view their method of transport, leukocyte migration had be halted before it completely finished so that it was easy to see where leukocytes had crossed the cells; half of leukocytes hadn’t traveled through yet. However, even then, it was clear which was the primary method of travel.
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The figure to the left highlights the drastic difference between paracellular migration and transcellular migration not only in the control cells (in which this might be expected), but also in the ones carefully constructed to simulate the blood brain barrier. The white bars represent para cellular transport. Clearly, many more leukocytes preferred this method of transport rather than the other one (represented by the black bars).
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What are the small lines extending from the top of each bar? No one experiment could predict what happens to each and every leukocyte in the blood brain barrier; it's impossible. The lines (called error bars) indicate how uncertain experimenters are that their results represent what would actually happen in the blood brain barrier. Smaller lines means less uncertainty. In this case, it's fine to use these results to make conclusions about the actual blood brain barrier as the error bars are relatively short.
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Over 98% of leukocytes migrated paracellularly across the endothelium. As stated before, this means that the majority of leukocytes travelled through the gaps in between endothelial cells and somehow bypassed the tight junctions that guarded them.
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The pluses and minuses lining the bottom of the figure show which molecules were added to the model; if you take your finger and move left from a plus or minus, you will reach the molecule that was added (plus) or not added (minus).
When CCL2 (again, a molecule that encourages leukocytes to migrate) was added, transcellular migration straight through the cell went up to 15%, slightly higher than in the model without CCL2.
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Under both normal conditions and specialized conditions meant to encourage travel straight through the cell, transcellular migration rarely occurred.
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The majority of leukocytes travel paracellularly through the blood brain barrier.
EXPERIMENT 2: AFFECT OF SIGNALLING MOLECULES PECAM-1 and CD99
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PECAM-1 and CD99 are both signaling molecules that have been shown to be involved in encouraging leukocytes to travel across the endothelium cells of the blood brain barrier. Pinpointing their affects in this model helps solve a piece of the puzzle that is leukocyte migration. The purpose of this miniature experiment is not to see how leukocytes travel (we already figured that out with experiment 1), but to see what affects how much they travel in general.
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Methods
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Peripheral blood mononuclear cells (PBMCs), a type of leukocyte, were again used to simulate leukocyte migration in this experiment; see experiment 1 for why they were used and how they were prepared.
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The PBMCs were then separated into two groups. One group was given something which would block their respective signalling molecules, anti-PECAM-1 or anti-CD99. Blocking the signalling molecules would isolate the affect they had on leukocyte migration; if more leukocytes migrated when these molecules weren't blocked, it would indicate that PECAM-1 and CD99 played a clear role in encouraging leukocyte migration.
The other group of cells was given a non-blocking control where the signalling molecules were free to act as they normally did in the blood brain barrier.
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The altered PBMCs were then placed onto the endothelial cell model and stored for 1 hour at 37 degrees Celsius in a CO2 incubator so that the leukocytes could migrate through the cell model (see experiment 1 for why a CO2 incubator was used).
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Both groups were then viewed under a microscope to assess how many of the PBMCs (leukocytes) had migrated when the signalling molecules were present and how many had migrated when the molecules were blocked.
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Results
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The figure to the right highlights the drastic difference in amount of leukocyte migration when signalling molecules were blocked vs. when they weren't.
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When PECAM-1 and CD99 weren’t blocked, most leukocytes (90%, to be exact) migrated through the endothelial cells, which is normal for leukocyte travel in the blood brain barrier.
When they were blocked, however, leukocyte migration decreased to 15%-20%.
This is 70% less than when the signalling molecules were allowed to perform their job.
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PECAM-1 and CD99 clearly play an important role in leukocyte migration through the blood brain barrier.
EXPERIMENT 3: TIGHT JUNCTIONS DURING LEUKOCYTE MIGRATION
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Tight junctions are the gatekeepers of endothelial cells. Experiment 1 showed that leukocytes primarily travel in between the gaps of endothelial cells, somehow bypassing the tight junctions that guard them. This experiment looks into a possible explanation.
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Methods
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Peripheral blood mononuclear cells (PBMCs), a type of leukocyte, were again used to simulate leukocyte migration in this experiment; see experiment 1 for why they were used and how they were prepared.
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The PBMCs were placed on the cell model of the blood brain barrier and allowed to migrate through it for 10 minutes. In other experiments, the leukocytes given an hour to migrate. The purpose of this experiment, however, was to capture an image of the leukocytes during travel to see what was happening to the tight junctions. A shorter migration time would mean that the leukocytes wouldn't have finished traveling through the blood brain barrier, which in turn means you can pinpoint exactly what is happening to tight junctions during the process.
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Immediately after the 10 minutes of migration were up, the cell models were placed in ice-cold para formaldehyde which freezes them in place for easy observation. The model was then stained with a fluorescent dye that labelled claudin-5, one of the proteins that make up tight junctions. Labeling this protein would allow for easy observation under the microscope of the junctions and how they behave during migration.
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Results
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When tight junctions were stained and observed during migration, something strange occurred.
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The tight junctions disappeared.
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The figure to the left shows endothelial cells of the model during leukocyte migration, just as the leukocyte slips between the gap in the cells.
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The tight junction (indicated by the yellow arrow) which would normally seal the gap was markedly absent.
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This result shows that tight junctions behave similarly to another protein that seals endothelial cells elsewhere: VE-Cadherin, which moves out of the way of incoming molecules trying to cross it and reforms itself immediately after.
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Tight junctions that normally guard endothelial cells from incoming molecules are plastic, and move out of the way to allow leukocytes to cross them.
