What Makes a Cell Stay

By Jason Socrates Bardi

As the mysteries of the immune system fall one by one under the piercing eye of modern science, scientists find answers—but also further questions.

A case in point is the question of certain immune cells passing through the tissues of the body known as the lymph nodes. For years, scientists have known that lymphocytic cells such as T cells can become sequestered in the lymph nodes instead of passing through them. But what makes a cell stay there was not known until recently.

At least one factor that keeps T cells in lymph nodes had been firmly established—chemical "agonists" that turn on a particular type of receptor protein known as the S1P receptor. However, a detailed description of how it worked was lacking.

Now, a team of researchers from The Scripps Research Institute and The University of California, Irvine has combined chemical biology with an advanced technique known as two-photon imaging to observe the egress or exit of cells from the medullae of lymph nodes in living tissue, and they have come up with an explanation for why the cells stay.

By applying antagonists (deactivating the S1P receptor) and agonists (activating the S1P receptor) and looking at the effect of these chemicals on the cells' egress, they have found evidence that the signaling of the S1P receptors opens and closes what are known as endothelial gates through which the lymphocytes pass.

In combining these chemical probes with the advanced imaging technique of two-photon imaging, says Scripps Research Professor Hugh Rosen, "we were able to get mechanistic insights that you couldn't get by any other means." Rosen was one of two lead authors on the study along with Professor Michael D. Cahalan at UC Irvine.

The S1P Receptors

S1P, or sphingosine 1-phosphate, is a molecule produced by platelets—those flat, circulating, cytoplasmic fragments in the blood that are essential for clotting—and by a variety of tissue cells. S1P is produced by endothelial cells, for instance, at sites of inflammation where there are inflammatory cytokines like tumor necrosis factor-alpha.

A few years ago, Rosen and his colleagues showed that S1P receptors can control the recirculation of lymphocytes. Furthermore, they found that either S1P lipids or synthetic chemical agonists of the S1P receptors are able to alter the trafficking of lymphocytes in a reversible way.

These very potent synthetic chemicals bind to S1P receptors in the low nanomolar and sub-nanomolar range. These small molecules are also immunosuppressive, disrupting antigen responses by misdirecting peripheral T cells to the wrong lymph nodes by inhibiting recirculation. They also regulate the release of new, mature T cells from the thymus. Future therapies could potentially harness this effect to prevent the rejection of organ transplants or the effects of autoimmune diseases.

Rosen and his colleagues, Christopher Alfonso and collaborating Scripps Research principal investigator Michael McHeyzer-Williams, have shown that when S1P agonists interact with thymus, they cause the T cells to lose a surface receptor, called CD69, on their surface, promoting their maturation in the medulla. In addition, a biological switch is activated that shuts off emigration of mature T cells from thymus, preventing T cells from reaching the periphery  (PNAS 2003, Eur. J. Immunol. 2006).

"What we have," says Rosen, "is a biological toggle switch—an on-off switch—that is regulated as you activate these receptors. As you activate these receptors, the lymphocytes disappear in a reversible way from peripheral blood, protecting an animal or a person from transplant rejection and from autoimmune-mediated tissue damage."

In the last few years, Rosen and his laboratory have focused on the study of the mechanism under various conditions in vivo, and this is what led to the collaboration with Cahalan and his colleagues.

Competing Theories

Scientists had at least two theories explaining why chemicals that activate S1P receptors cause T cells to halt their exit from the lymph nodes. Because of the action of the S1P receptor in controlling the fate of T cells in the periphery, many scientists thought that the same action accounted for the arrest of cells in the lymph nodes: T cells would express S1P receptors on their surface, and when the S1P receptors were activated, this would signal other molecules to halt them.

Complicating the problem, however, was the fact that the endothelial cells lining the spaces inside the lymphatic tissues also express S1P receptors. As such, another theory posited that endothelial cells within the thymus were responsible for the halting the cells.

To see if they could find support for this alternative theory, members of the Scripps Research and UC Irvine team studied the S1P receptor by combining the chemical biology approaches that Rosen has pioneered over the last few years with the technique of two-photon imaging, the use of which Cahalan is an expert in.

The technique is basically a way of imaging tissue through fluorescence by subjecting it to two streams of low-energy infrared photons simultaneously. These photons can penetrate up to one millimeter into tissue. Because these photons are low energy, they do not damage the tissue, enabling researchers to look at the workings of living cells.

For instance, the technique allows the live image processing of T cell arrest in the lymph nodes of rodents. Video sampling of individual images allows the creation of real-time videos of cellular movement and interactions, such as the halt of the lymphocytic cells in the lymph nodes.

Using a selective S1P1 receptor agonist, Rosen, Cahalan, and their colleagues observed that when the S1P receptors were activated, the T cells were log-jammed at the "stromal gates" of the lymph nodes, but did not lose their motility elsewhere in the node. Then, by using an antagonist that was created by Scripps Research Professor Chi-Huey Wong and his laboratory, the investigators observed the opening of the stromal gates and the resolution of the log jam.

The most reasonable explanation for this, says Rosen, is that the selective agonist works on the endothelial S1P receptors rather than the T cell S1P receptors. Hence, the S1P receptors on the endothelial cells are controlling the lymphocyte arrest, halting the flow of cells through the lymph node the way that a faucet twisted shut would stop the flow of water through a tap.

The article, "Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses" by Sindy H. Wei, Hugh Rosen, Melanie P. Matheu, M. Germana Sanna, Sheng-Kai Wang, Euijung Jo, Chi-Huey Wong, Ian Parker, and Michael D. Cahalan was published by the journal Nature Immunology 6, 1228 - 1235 (2005), with an accompanying Nature "News & Views." To access the article online, see: http://dx.doi.org/10.1038/ni1269.

This research was supported by the National Institutes of Health.

Send comments to: mikaono[at]scripps.edu

"What we have is a biological toggle switch-an on-off switch-that is regulated as you activate these receptors," says Professor Hugh Rosen. Photo by Kevin Fung.