Lymphovenous Canada: A report on the U.S. think tank "Conquering Lymphatic Disease - setting the research agenda"

I returned, in May 2001, from the very exciting Think Tank conference put together by the National Institutes of Health and the Lymphatic Research Foundation: Conquering Lymphatic Disease: setting the research agenda. This meeting was held in Bethesda, Maryland.

Although there is an international Lymphatic Congress of Lymphology held regularly, the Think Tank was an opportunity to get a different mix of scientific experts and thinkers from around the world together with a different agenda -- to do some brainstorming on how to promote and encourage more scientific research in this area. Many of the experts who attended didn't present their latest data. Instead, the focus was on discussion, interaction and collaboration at a very informal level.

The Think Tank was organized into four sessions:

1. genetics and development
2. physiology and pharmacology
3. lymphatic imaging
4. lymphangiogenesis/hemangiogenesis

As the two-and-a-half days unfolded, these groups met separately, and then we all got together and compared notes identifying, in many cases, what we don't know about the lymphatic system. It was interspersed with some presentations as well.

The strength of a lymphatic Think Tank is that it brings together people from a wide range of disciplines. The lymphatic system has the capacity to join disciplines together and make people talk to one another, and to consider new approaches to medical problems involving lymphatics.

Because the lymphatic system connects nearly all the organs and tissues in the body, it potentially is a target for therapy or intervention in any organ or tissue, with any disease. Clearly the lymphatics play a huge role in the inflammatory processes. Lymphatics and cancer is another important and relevant area. By knowing biological mechanisms, it opens the door to treating the lymphatic system as a whole

Professor Miles Johnston and myself represented Canada at the Think Tank. The international list of participants was impressive. One of the pioneer lymphologists, who has made substantial experimental contributions, was Dr. Joe Hall from the United Kingdom. It was a personal pleasure for me to have the opportunity to consult with him.

We were sorry that some of the experts in lymphatic filariasis were not there given the far-reaching research initiative being undertaken by the World Health Organization and over 25 partners. Contemporary thoughts that lymphatic filariasis is due, as much, to the host response to the organism as it is to the physical blockage will undoubtedly expand research interests in the area. Perhaps the next Think Tank set for 2002 will include this topic.

Getting back to the meeting...

Genetics and development:

The lymphatic system is finally cashing in on part of the action related to genetic research. There are at least two large family studies now -- one organized through the University of Pittsburgh, and the other one from Arizona -- that involve family trees and relationships that are quite extensive, which are defining where the genes are, on which chromosome, etc. As in all genetic studies, it's a long way from defining where the genes are, and being able to regulate these, and turn them on or off, or whatever, to become a treatment, but it's a starting place, and that part's very exciting.

The other part of genetics that's exciting is to a large extent pioneered by Dr. Aliltalo and colleagues from Helsinki. Using mice -- because they have a lot of similarities to humans -- they have been able to identify some of the genes that are involved in the lymphatic system development. They can knock these genes out, make a strain of mice that are missing these genes, and then study what the defects are in the lymphatic system.

Such studies show clearly that there seem to be markers of the lymphatic system. The cells lining the lymphatics are quite different from blood vessels. The development of the lymphatic system seems to be quite different from that of the blood circulatory system. This was suggested by the classical and quite old medical literature but now we are starting to see the genetics that proves it.

If one eliminates the genes that are fundamental to the development of the lymphatic system at the embryo stage, life is impossible. These animals get tremendous accumulations of fluid in the thoracic cavity around the heart, and the abdominal cavity, and they die about halfway through gestation. This has been informative in studying how the lymphatic system develops, and how it differs from the blood circulatory system.

These are called "knock-out mice" because they knock the genes out or put a nonsense gene in where the regular gene would be but there are "knock-in mice" too. Once you identify some of these genes, one can start splicing in genes that would be important in the development of the lymphatic system.

Some of the markers that are on the lining cells for the lymphatics. A pathologist can now stain these markers on the blood vascular lining cells to determine how they are different from the lymphatic cells. With dysplasias and other developmental abnormalities, in the very near future one should be able to diagnose whether the condition has a lymphatic origin, or a blood vascular origin. It may not be as simple as a blood test -- it might require a sample of tissue, a needle biopsy, or whatever -- but in time these tests will get easier and easier to do.

Once we can identify a process and understand something about it, more rational treatments always follow. I thought that these experimental developments were very encouraging.

Physiology and pharmacology:

The second topic area, physiology and pharmacology, was the section that I participated in. There were three short presentations. The person who preceded my presentation was Dr. Noel McHale from Belfast. Noel presented new data on the muscular "pumping" action of lymphagions in the lymphatic system. These tiny little lymphatic "hearts" pump lymph through the lymphatic vessels. When they contract, the smooth muscle in them contracts - a bit like blood vessel smooth muscles.

We have smooth muscle in our respiratory system, our gastrointestinal system and our reproductive system. We know that the smooth muscle is quite different than striated muscle in our biceps and it's also different than cardiac muscle. Lymphatic smooth muscle has characteristics that make it slightly different from all of these other types of smooth muscle. It's not like exercise muscle, it's not like vascular smooth muscle, it's not like cardiac muscle, it's a little bit different again, but it shares some common properties.

What McHale showed was that in order to propel lymph through the lymphatic system, one must have to have a pretty organized contraction system. One cannot have it just haphazardly contracting, or lymph will go every which way, or it will go nowhere. It must be co-ordinated, and one way that it seems to be co-ordinated, much like the heart, is with little pacemakers in each one of these little hearts or lymphangions. It has nerve tissue that Dr. McHale was able to stain and identify -- similar to our hearts -- which organizes the contractions and passes the message on. It operates in a totally separate rhythm, however, from the heart.

Once we can identify a structure that's controlling contraction, then we can start to target that structure with pharmacological products that make it work better, or inhibit it. I think that this notion will stimulate a very exciting next step.

We need to get a few more people to look at the contraction of the lymphatic vessels and go through all the compounds that are already out there that have been used with blood vessels for years and that might specifically affect the lymphatics. A screening process is required and to test these agents. The pharmaceutical companies would seem appropriate places to consider this screening process.

Would genetic research and the pharmacological developments primarily benefit people who are born with lymphatic disorders, or would it also benefit people with lymphatic conditions that develop later in life? That is a question that came up at various times in the meetings - whether primary and secondary lymphedemas should be considered together. The simple answer is yes, but no. In the final analysis, there will clearly be processes that apply to both.

If there is a genetic defect, at some point in time it may be possible to apply gene therapy and put in the right genes that treat that whole condition. There aren't too many people doing gene therapy related to the lymphatic system yet, but as we identify the genes, that may become feasible. Certainly the principle of gene therapy is a very attractive and an active area of research.

The regulation of lymphocyte circulation:

The area I spoke on at the think Tank relates to the experimental research that my laboratory is doing in the area of the regulation of lymphocyte circulation. Lymphocytes are cells that immunologists and clinicians tend to think are one of our white blood cells, which they are. But, they are called lymphocytes, with "cyte" being cell, and "lymph" being lymph. They get their name from being the cells in lymph.

There are a couple of organs in the body, the bone marrow and the thymus, that make these cells, but once they are released into the circulation, they go everywhere, just like motor vehicles go everywhere. Lymphocytes are continuously on the move. It is sometimes helpful to make the analogy between motor vehicle traffic and lymphocyte traffic. It turns out, however, that only 1 percent of lymphocytes are really in the blood at any one time. The rest move in other parts of our body. An intact lymphatic system is critical to maintain this lymphocyte traffic.

To study lymphocyte traffic, one must assess it as it functions to get a true picture of how it works. That's been difficult to do, because one needs a living organism, not post mortem or surgically removed samples. Once cells are fixed and removed for microscopic examination they are dead, and not moving. There is a simple analogy for this: if one has a helicopter flying over your city at peak hour, and one took a still photo of all your freeway systems and showed it to somebody who didn't know what a car is, they probably wouldn't know whether your freeway is a parking lot or whether the cars are all moving at 100 km an hour. Unless one takes a movie or uses a video camera to record the dynamics of traffic, you can't tell the difference between a traffic jam and the rapid flow of traffic.

We have been bombarded in medicine, for many years, with pathologists looking down the microscope at fixed, frozen, and stained tissues -- resulting in only the still picture. Until one gets people to see it or to look at this in different ways, the dynamics are not appreciated. One simply cannot assess the rate of movement nor the direction from still photos.

Back to our lymphocytes, the equivalent of all the lymphocytes in our blood move in and out of the blood system every few hours. Where do these cells go? The lymphocytes leave with the fluid that is destined to become lymph. They leave between the lining cells in the blood vessels, get into the interstitial tissue and fluid in any organ or tissue, and the lymphocytes are then picked up by the lymphatic system.

Now the lymphocytes are in lymph. This lymph filters through lymph nodes where the lymphocytes are able to interact with other cells to take part in and mount immune responses. The lymphocytes then leave the lymph node in the lymph and are eventually returned to the blood. Thus a local immune response (for example from a vaccination site) becomes amplified and disseminated by these lymphocytes in lymph. Some of the lymphocytes become antibody-forming cells, some produce other effector molecules which gives us immunity, and some lymphocytes become the purveyors of immunological memory. This memory is the fundamental aspect of immunity and immunization. Lymph cells are extremely important!

In order to study the dynamics of lymphocytes, we use the lymphatic system to collect a few million lymphocytes and label them using coloured dyes. Then we put the labelled lymphocytes back into the laboratory animal we are using to track where they go by sampling blood, lymph nodes, spleen, pre-nodal lymph that's going into the lymph nodes and post-nodal lymph that's coming out of the lymph nodes.

We sample in all these places and count the number of labelled cells that we see, and then map out how long it takes them to go from A to B, and whether they all make it there, or whether they like to go to some areas (tissues) better than others. We find that some lymphocytes like to go to the gastrointestinal tract better than to the skin. Some like to go to the skin better than the gastrointestinal tract. Some refer to this as the "homing" of lymphocytes.

When I started in immunology, all lymphocytes were thought to be the same, and then scientists determined that there were T lymphocytes and B lymphocytes. T lymphocytes come from the thymus (which is located in the chest in the neck region), and the B lymphocytes were the ones that eventually made antibody. Now we have probably fifty or more different kinds of lymphocytes that we can identify, and they all have different functions and many have different migratory properties.

Many of these lymphocytes are produced by the thymus before birth. If the thymus does not produce these cells before birth, then a person is born immunity deficient. Once the T lymphocytes have initially been produced, however they continue to replicate themselves throughout your life. As already mentioned, the lymphocytes are continuously leaving the blood, going through the tissues into lymph, into the lymph nodes and back through the large, collecting lymphatic -- the thoracic duct -- the largest lymphatic vessel in the body.

Back to our lymphocytes -- they are trafficking around, like little cars. They follow thoroughfares such as blood, but they also go through all organs and tissues, the skin, and wherever. They move into the pre-nodal lymphatics, leading up to the lymph node, being carried there with plasma proteins. They traffic around and around: blood to lymph, blood to lymph, blood to lymph, many times. We call that lymphocyte recirculation.

One of the standard roles or functions of lymphocytes is to recognize our own components from something that's foreign. They have receptors on their surface that are able to do that very well, and say, "This structure here is me, and that structure is not me, so I'm going to react against it." Whether that reaction is a transplanted organ, a virus, or bacteria or some kind or cancer. We call this "immune surveillance" and the ability of the lymphocytes to recirculate between blood and lymph makes them ideally suited for this surveillance role.

In the case of lymphedema it is clear that the traffic of lymphocytes in the tissues is suppressed, perhaps because the lymph flow is suppressed. So there is less of this immune surveillance, and typically, those areas are prone to infection more than other areas. Dr. Waldemar Olszewski told the Think Tank participants that the number of lymphocytes in the lymph of humans with lymphedema was greatly reduced.

Lymphocytes are carried passively by the flow of lymph, but they are also actively migrating cells that can swim well, if you like. So both methods of transport are going on, and it's conceivable that one could manipulate one part of the process, and make them swim better, even though the river is still moving slowly.

To track these cells one needs to identify, collect, stain or otherwise label the cells, put them back and then track them. Only 1 percent of our lymphocytes are in the blood, but that's still a pretty big number. In a human, it's about 10 billion lymphocytes.

These days cell tracking is done with coloured dyes and an instrument called a flow cytometer. A flow cytometer expels cells out of a nozzle in a very small flow stream. A laser intersects the stream and measures a variety of cell parameters, feeding this data to a computer which records these cells at hundreds per second.

Now, instead of using a microscope and examining a couple thousand cells, and taking all day to do it, we look at ten thousand or a hundred thousand cells in every sample, so we can measure these events much more effectively. We can also separate out different cells as they flow. The technology now allows us to do experiments that a few years ago were impossible.

Experimental science flourishes on quantitative methods, which give us reproducible and objective criteria to test our ideas. Most recently, Bill Ristevski in our laboratory discovered a method to label all of the white cells in the blood without first removing the blood to label outside the body. We look forward to being able to study this traffic in more depth in the immediate future and hope to persuade clinical colleagues to begin to think about new applications to a variety of diseases in patients.

In the future we might be able to replace cells where people have certain lymphocytes missing, or where they are not being manufactured by the body. For example, some lymphocytes have receptors for HIV, the virus, and the virus infects such cells and kills many of them. But there's a controversy in the HIV literature whether all these cells are killed because they can't find them in the blood. The alternative is that they might be hiding in other parts of the body such as lymph nodes, the lymphatic system, the tissues, or in the spleen. A blood test, currently used to determine whether the HIV virus exists, doesn't show whether the virus is in other organs or tissues.

I think both cell death and cell redistribution is going on. Lymphocytes interact with other cells, and cause inflammatory processes to either get worse or better in various organs and tissues. One informative way to measure this is to collect and analyze the lymph directly from all these organs and tissues.

There is a relationship between the traffic of lymphocytes and inflammatory-type conditions. If we consider rheumatoid arthritis, tuberculosis, Crohn's Disease, or multiple sclerosis (MS), one of the features of chronic inflammatory diseases, and auto-immune diseases, is an infiltration of lymphocytes. Where there are typically and normally only a few lymphocytes zipping through, in these diseases, these other organs start to resemble lymph nodes.

In these auto-immune conditions, the lymphocytes look like they're accumulating in certain sites. However, the living processes are extremely dynamic. The lymphocytes are entering in increased numbers and they are also coming out in increased numbers or trafficking. If one takes a static photograph of it, it looks like a parking lot. However, when measured in real life, there is as much lymphocyte traffic as there is in a lymph node. Recall the analogy of the traffic jam versus the peak hour traffic.

There's a whole school of thought that thinks there are changes in the antigens in our own joints that make it look like an infection, are very similar to ones that are normally in the gastrointestinal tract. People get flare-ups of a type of arthritis for example, after having a gastrointestinal infection or virus. It's called antigenic mimicry, and there are several interesting reports where people who didn't really have joint disease, or it was very mild, suddenly get these viral infections, and later they get significant joint lesions. People talk about the two areas being controlled by similar molecules.

Clearly research in this area can have an impact, first of all in identifying the problems in terms of the production of different types of lymphocytes, which may lead to an impaired immune system, and secondly, for diagnostic purposes. If one could understand and regulate lymphocyte traffic, the possibility to regulate pathological and immunological processes may become more feasible.

Imaging:

One of the highlights for me at this Think Tank was the interaction and dialogue between the scientists involved in imaging techniques with scientists more concerned with biological phenomena.

When people think of imaging the lymphatic system, one obvious application is in cancer, where one of the routes of metastasis is through the lymphatic system, and the regional node, sometimes called the sentinel node.

As an immunologist, we want to have that lymph node as happy as it could be to counteract the cancer, but what is often done is exactly the opposite. If there is cancer in the regional lymph node, it is taken out. It's a dilemma: the immune system is clearly protective but should one not remove as much cancerous tissues as possible? There is no clear answer. The best rationale may differ with different cancers and circumstances.

The obvious application of imaging is with these regional nodes so that one could say to the surgeon, in breast cancer for example, whether there is cancer in there, or not, so that appropriate steps can be taken. Whether that step is radiation without removing the nodes, or chemotherapy, or nothing can be determined on the basis of the imaging. The surgeon might not have to do any scalpel work at all. It could potentially lead to the elimination of biopsies or lymph node dissection in this area altogether.

In order to get contrast agents into the lymphatic system for imaging, there are two obvious routes: one is to inject it into the local drainage area, say under the skin, the other is to administer the agent intravenously.

With the first approach if one wanted to look at the node behind the knee, one would inject the contrast media between the toes, typically somewhere in the lower leg, and it would travel up through the lymphatic system. The node would fill and one would take the picture to see the node. Again, at the Think Tank, Drs. Charles and Marlys Witte from Tucson, showed data on the use of lymphscintigraphy to visualize the lymphatic vessels and lymph nodes. They use very small amounts of a radioactive tracer for gamma camera images.

A new intravenous approach has just come onto the market using a contrast agent that is commonly used for magnetic resonance images (MRI's). This agent is put in the blood through a simple injection. By the next day some of this agent has moved from the blood into the lymphatic system. The cells in the nodes gobble up this contrast media allowing the nodes to show up on the imaging screen.

Older diagnostic imaging tools weren't designed to target the lymphatic system. They were commonly designed to visualize the blood vascular system. The interesting thing is that now investigators and companies are specifically targeting the lymphatic system.

With increasing awareness of the lymphatic system, techniques and agents should be developed which do not compromise lymphatic function. One of the standard dyes used in the past for visualizing lymphatics in animals or in humans, inhibited the lymphatic pumping. With the new compounds and systems, they go through pretty rigorous testing prior to release onto the market. It is significant that the lymphatic system is now a target for such testing.

Angiogenesis:

The term lymphangiogenesis is quite different than blood vascular angiogenesis, but the traditional textbooks don't make these distinctions. Is it straightforward to imagine blood vessels growing, but of course lymphatic vessels grow very effectively, too.

One of the unknowns that investigators are targeting is how lymphatic vessels can be grown outside the body and what agents cause lymphatic cells to divide and proliferate. This is part of the lymphangiogenesis process. Again, these are recent developments, starting from the very basic work of growing these cells.

In situations where there is poor lymph drainage obviously one needs lymphatic vessels, and, they must be functional. If they are not present, for whatever reason, because of burns, or because of congenital missing genes in somebody's makeup, one needs to get the vessels there.

There are potentially a few ways to do this. One could inject into, say, a burned area, compounds that cause the few lymphatic vessels that remain undamaged to proliferate. Once one screens these, in culture, and test them in animal studies, then one can start to say, "Okay, this makes lymphatic vessels grow much more effectively than just letting them take their own course. Let's try it locally." We need to search for such agents.

There seems to be some thought that when lymphatic vessels are irradiated, they cannot regenerate in the same way lymphatic vessels normally regenerate after they are severed. This needs to be investigated more, particularly in relation to radiation therapy in cancer, lymph node removal and regeneration.

This is quite a complex area to test scientifically. One complicating factor is that cancers are all quite different and react differently. To study them systematically in animals, inbred animals could be helpful. These are typically rats and mice, with tiny lymphatic vessels, which cannot be easily accessed for experiments. In the larger animals, the tumour models are lacking. It is difficult to do human studies in this area for ethical reasons.

Next steps:

In terms of this conference, there will be some follow up. The Foundation has talked about ways to move the research agenda along over the next few years. The second meeting is suggested for 2002.

The National Institutes of Health are restructuring, just as we are here with the Canadian Institutes for Health Research. Research is done in compartments, or departments, if you like. Review panels such as immunology, transplantation, pathology and genetics exist but lymphatic research panels do not exist.

Scientists are very concerned that what they've worked on traditionally isn't going to have a name anymore, then their interest, which might have been a big area, may fall through the cracks, just like lymphatic studies have sometimes fallen through the cracks.

There's hope from the NIH Think Tank that there will be something with a lymphatic title to it that will keep this momentum going. One possible development would be a request for applications in the area of lymphatic research. We all do better if we have a name to categorize our studies. I think Wendy Chaite and Marlys Witte deserve tremendous credit for conceiving and organizing this first Think Tank.

The future?

One of the problems is that people don't look at diseases of the lymphatic system as a major killer disease, like heart and stroke and cancer, although it plays a role in all of these conditions. Lymphatic studies need to look flashy and have some kind of sex appeal if we want to attract new researchers and young people into this field of study. We need to look modern, use contemporary technologies, and fit in with current advancements. Although I have alluded to the ways in which money and resources direct research agendas, scientific curiosity remains a very powerful incentive.

It's gratifying for me to see former students, such as Dr. Allen Young, who was at this Think Tank. After spending five years in lymphatic and immunological research in Switzerland, he is now moving to Boston where he will continue to do studies in his area of expertise -- detailed analysis of the cells in lymph. He's now a world expert in that field. Dr. Miles Johnston and his group have some very exciting and novel data relating to connections between the central nervous system and the lymphatic system. These are not only intellectually fascinating but, in time, will be relevant to patients' welfare.

Science needs communication, interaction and it needs money. For the young investigators, it has to not only stimulate their interest and curiosity but it should be a pleasant experience where they have fun doing it. Money is part of it -- but the money needs to be available for meetings as well as research to allow these young people to talk about what they are doing. They get very excited about their work and they need the opportunity to pass that excitement on to their colleagues, their students and to patients and families with lymphatic disorders. Partnerships are clearly effective!

I thank Cathy McPherson for asking the appropriate questions and putting some semblance of order into my ramblings and often-incoherent responses.

Reference

Lymphocyte recirculation in development and disease. Seminars in Immunology, Volume 11-2, 1999. Editors: J. Hay and A. J. Young. Academic Press.

Professor Jack Hay works in the Department of Immunology at the University of Toronto. The Lymphatic Research Foundation, which organized and sponsored the think tank, may be contacted through their web site at: www.lymphaticresearch.org

If you have concerns about your condition, please consult your doctor or a health care specialist in this area.