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NEWS&VIEWS: What's the particular physiological condition?

ROSEN: We focused on a family of receptors for a lipid that is produced by platelets and by a variety of tissue cells called sphingosine 1-phosphate. Sphingosine 1-phosphate acts on a family of receptors called the S1P receptors or the "edg receptors," originally defined as endothelial differentiation genes. They activate these receptors and regulate a range of physiological functions that include cardiovascular function and blood pressure. We showed that they control the recirculation of lymphocytes—which was never understood before.

We found that these sphingosine 1-phosphate lipids or synthetic chemical agonists of the S1P receptors are able to alter the trafficking of lymphocytes in a reversible way. These are very potent. They act on receptors in the low nanomolar and sub-nanomolar range. They lead to the misdirection of lymphocytes.

Lymphocytes circulate around the body in order to mediate immunity and mediate the bystander damage of tissues—whether it's a transplanted tissue as in a rejection or damage to normal tissue as in autoimmunity. This mechanism regulates the egress of lymphocytes from lymph nodes and, as we show in a paper that we've just submitted for review, the egress from thymus into the blood.

Lymphocytes normally come from two organs, the bone marrow and thymus, then enter the bloodstream. They circulate through the blood to secondary lymphoid organs—lymph nodes, Peyer's patch, the spleen. If they encounter antigen in the secondary lymphoid organ, they begin to proliferate and to undergo clonal expansion, producing various effector cells. These effector cells leave the lymphoid organs and return to the blood, having acquired the ability to enter the tissue spaces and remove what they recognize as non-self. This could be a transplanted organ, a viral or a bacterial antigen, or, in the case of autoimmune disease, normal tissue that has broken tolerance and has become recognized as non-self.

We've discovered a couple of key steps are regulated by these sphingosine 1-phosphate receptors. There is a biological toggle switch—an on-off switch—that is regulated as you activate these receptors. You essentially shut off a switch and, as you activate these receptors, the lymphocytes disappear in a reversible way from peripheral blood and you can protect an animal or a person from transplant rejection and from autoimmune-mediated tissue damage.

NEWS&VIEWS: At the same time, though, you are suppressing the immune system.

ROSEN: In fact, you are. One of the reasons we are particularly interested in this research topic is that it represents a mechanism of immunosuppression that has the potential to be significantly less dangerous than other mechanisms of immunosuppression.

Why do I say this? What are the other modalities of immunosuppression? Corticosteroids not only suppress lymphocytes, but also lead to significant reductions in function and number of myelomonocytic cells, neutrophils, and monocytes. So a patient's ability to withstand bacterial or fungal infection is compromised. In addition, corticosteroids cause significant changes in metabolism. They are diabetogenic and cause significant loss of bone mass, which can lead to osteoporosis and bone fractures.

Calcineurin inhibitors, like cyclosporin, are used for serious autoimmune disease and for treatment of transplant rejection. They have a number of mechanism-based toxicities including causing dose-dependent renal dysfunction and hypertension.

Rapamycin and the TOR kinase inhibitors cause significant alternations in blood lipids, some of which can cause acute heart attacks.

What we have here is a mechanism that can spare the use of these other drugs that are potentially very toxic. Secondly, it impairs lymphocyte recirculation, but doesn't impact on myelomonocytic cell function. So, it should not cause enhanced bacterial and fungal infections, which would be an advantage to patients. There are no metabolic consequences that one knows of associated with this mechanism. It should, therefore, not produce osteoporosis, pathologic fractures, or diabetes, nor should it promote renal dysfunction or blood pressure changes.

NEWS&VIEWS: What would you guess the potential side effects to be?

ROSEN: One could guess that potential side effects would be those associated with, for instance, the inability to clear a localized viral infection. There is data in the literature showing that the systemic response to a viremic delivery of antigen, in this case of LCMV in mice, is quantitatively normal, but distributed differently. In other words, you get T-cells, but the effector CD8 cells are largely restricted to the lymph nodes and don't get into the periphery. We see the same thing for CD4 effector cells in a paper that we've got coming out in the April 1 issue of Journal of Immunology. You can mount responses to systemic infection, but not necessarily in the right place.

One should always bear in mind that one would generally only use immunosuppressive strategy in somebody who was seriously ill. It's not something one would use for a disease that is self-limiting and non-disabling.

NEWS&VIEWS: What happens to the lymphocytes themselves? Are they sequestered in the lymph nodes, and, if so, what happens to them there—do they eventually go back into circulation?

ROSEN: Let's take it in two steps. What do we know? We know that cells will accumulate acutely in lymph nodes. Lymph nodes in the mouse for instance might increase in size by about 20 percent over the first two to three days. By 14 days, these nodes have returned to normal size and appear to maintain that normal size, so it doesn't seem to directly affect cell fate in that sort of timeframe. In the long-run, we don't know what happens to the fate of lymphocytes.

As you sequester cells in lymph nodes, you actually stop egress from the thymus. In fact, in a paper that we have currently sent out for review, we can actually stop export of cells from thymus by about 95% within two hours. We can switch this mechanism on and off and the cells arrest in the thymus.

Over time, the thymic cortex will thin and the medulla will become more cellular as more mature T cells are behind the barrier blocking their egress into blood. Do we understand the homeostatic mechanisms that come into play to regulate thymic size and the feedback loops? I would argue to you that we don't.

That becomes one of the future approaches that we would take. How do you use evoked responses to small molecules to unlock or understand homeostatic pathways that are very hard to discern in the steady state? By small molecules, I mean chemical probes, small organic molecular probes of protein function that allow one to measure and perturb the physiology in measurable ways—in other words, chemical biology. That is one of the general approaches that we like to take within the lab.


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