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  1. 1. Crafoord d a y s 20-22 Sept 2004Programme РAbstracts РThe Crafoord lectures ΠThe Crafoord Prize in Polyarthritis 200 4
  2. 2. Cell Migration in Health and Disease Crafoord d a y s 20-22 Sept 2004Programme – Abstracts – The Crafoord lectures
  3. 3. CR A FOOR D SY MPOSIUM Cell Migration in Health and Disease Monday 20 September in Lund, Tuesday 21 September in Stockholm09.00 Opening of the Symposium Gunnar Öquist, Secretary General, Royal Swedish Academy of Sciences09.10 Integrin Cell Adhesion Molecules in Health and Disease page 12 Timothy A. Springer, Crafoord Laureate 200410.00 Coffee break10.30 Control of Leukocyte Traffic page 20 Sirpa Jalkanen, University of Turku, Finland11.15 Control of Interstitial Fluid Pressure and Acute Edema page 21 – Role of Integrins Kristofer Rubin, University of Uppsala, Uppsala12.00-13.00 Lunch13.00-14.00 Poster session14.00 Regulation of Human Neutrophil Apoptosis page 22 Tommy Andersson, Malmö University Hospital, Malmö14.45 Coffee break15.15 Cell Adhesion and Migration in Tumor Progression page 23 Richard O. Hynes, MIT, Cambridge, USA16.00-16.45 Towards an Understanding of Leukocyte page 6 Trafficking in Physiology and Disease Eugene C. Butcher, Crafoord Laureate 2004
  4. 4. CR A F O OR D L AU R E AT E S 20 0 4 Eugene C. Butcher Eugene C. Butcher, 54 years, Ph.D (medicine) at Washington University in St. Louis 1976; Professor of Pathology, Stanford University, California, USA. Timothy A. SpringerTimothy A. Springer, 56 years, PhD in biochemistry and molecular biology 1976; Latham Family Professor of Pathology, Harvard Medical School, Boston, Massachusetts, USA. 3
  5. 5. Inflammation and Immunity A short introduction to the 2004 Crafoord PrizeEugene Butcher and Timothy Springer have elucidated the function of cell adhesionmolecules that are expressed in white blood cells and direct their exodus across theblood vessel wall into tissue where they are crucial for the defence against disease. Cell adhesion molecules (CAMs) are cell surface proteins expressed by manycells including white blood cells. The CAMs are activated by specific signals fromperipheral tissues in response to injury or infection and their effector functions areimportant for processes of inflammation and immunity. Examples of CAMs are selectins and integrins. The selectins are glycoproteins,consisting of a single chain. They enable the blood cells to bind to activated en-dothelium in the blood vessel wall. As a consequence, the blood cell can ”roll” alongthe surface of the vessel and eventually adhere more firmly. The integrins consist oftwo sub-units – an alpha and a beta chain – and function as receptors on the whiteblood cells. The activity of the various molecules eventually allow the now flattenedblood cell to migrate between the cells in the vessel wall to the site of the diseaseprocess (Figure 1) Selectins Integrins Integrin Selectin Blood vessel Chemoattractants Diseased tissueFigure 1. Selectins in the cellmembranes of white blood cells allow them to adhere to the blood vesselwall in the area around an injurious process. Aided by the integrins they leave the blood vessel andmigrate to the damaged area where they act to eliminate the cause of injury. 4
  6. 6. Eugene Butcher has identified the selectins and their interaction with integrinsthat make them come to a halt, from high velocities along a stretch of some mil-limetres. Butcher has characterised the components of the process, including theligands and receptors forming the bonds, and has formulated the now classical mul-ti-step model to describe it. Timothy Springer demonstrated the crucial role of the CAMs in cellular immu-nity. The integrins can rapidly increase in number when a blood cell has identifiedan antigen, and improve the defence. Springer showed that the integrins constitutea molecular family all consisting of an alpha chain and a beta chain. These existin various configurations that can combine into a large number of defined variantswith different specificities against highly variable targets. In recent years Spring-er has worked to determine the structures at high resolution and to define thefunctional domains. Like Butcher, he has produced multi-step models to reconcilethe structure-function relationships. In recent years he has studied the molecularmechanisms that open and lock integrin in various conformations. If a disulphidebinding (sulphur-based) is introduced, an integrin can be locked in an open posi-tion and its function is lost. The flow of white blood cells in inflammation can thenbe controlled.New treatment strategies for inflammatory diseaseBoth Butcher and Springer have made successful attempts to apply their findings tothe treatment of medical illness. In some cases advanced trials are in progress. Butcher has shown that treatment with antibodies to the protein LFA-1 pre-vented or cured cerebral malaria in mice. The VCAM-1 protein plays an impor-tant part in the slow but progressive disease multiple sclerosis (MS), and Butcher’sgroup have showed how antibodies to this protein has stopped the development ofdisease in some trials. Springer’s findings have inspired numerous companies working on new curesagainst rheumatism, psoriasis, asthma, intestinal disorders, haematological disor-ders and AIDS. 5
  7. 7. THE CR A FOOR D LECTUR ES 2004 Towards an Understanding of Leukocyte Trafficking in Physiology and Disease Eugene C. Butcher, Stanford University, Stanford, California, USAYour Majesties, President of the Academy, members of the Crafoord family, mem-bers of the Academy, ladies and gentlemen, it is a great honor to be awarded theCrafoord Prize by the Royal Swedish Academy of Sciences, and I am pleased to besharing this prize today with my co-laureate, Timothy Springer. In accepting this honor, I thought I would share with you a personal perspectiveon the path that led to our studies of leukocyte trafficking and its role in the im-mune system. I wanted to be a scientist from an early age, and I was (and am)intrigued by everything from fundamental physics to the fundamental questions inbiology. My brother, who is here to share this day with me, knew he was to be anastronomer since the age of 9. I on the other hand was much less focused, and foundI couldn’t chose among the many fascinating mysteries of Nature until much later.By college, though, I had decided that biology was for me. I was attracted I thinkby the inherent wonder of life, it was and is still truly amazing to me that such acomplex and counterintuitive entity as a human can exist, with eyes, ears and brainhaving all evolved and developed in parallel, and all actually working, at least mostof the time. I went on to Medical School, largely to delay deciding what specificquestion to work on (or, putting it in a more positive light, to give me a better per-spective from which to chose an “Important Question”). The question I became fascinated with was how do cells recognize each other?And how do they know where to go? During processes such as embryogenesis, neu-rogenesis, and wound healing cells migrate and interact in highly specific and or-chestrated fashion. How do they do this? When I finished Medical School, the only tools available for defining moleculesinvolved in such events were immunologic, antisera were being used to inhibit cellfunctions, and to identify functional proteins so I decided I had to join an immuno-logy laboratory. When I went to train in pathology at Stanford University, however,the well-known immunology groups were all full. This turned out to be a stroke ofluck, however, because I ended up with the recently tenured Irv Weissman. Irv’s labwas an extraordinary environment for me, providing the combination of freedomand intellectual enthusiasm I needed. I was also very fortunate because, soon afterI arrived at Stanford, Judy Woodruff published a seminal paper in JEM, describing 6
  8. 8. (from my perspective) the most thrilling and accessible system ever devised forstudying cell-cell recognition. They showed that lymphocytes, rocked in the cold fora mere 15 minutes or so on frozen sections of rat lymph nodes, would recognize andbind with remarkable selectivity to specialized high endothelial venules (HEV), thesame vessels that Gowans and Marchesi had shown in the 1960’s recruit lymphocy-tes from the blood. A fantastically simple yet, as it turned out, powerful techniquefor studying the cellular and molecular mechanisms of leukocyte trafficking. One of the most exciting discoveries from our early studies was that lymphocytescan tell the difference between blood vessels in different organs in the body, sugges-ting a way that immune cells can be targeted to specific tissues (Figure 1a, b). It ap-peared that differences in blood vessels themselves could explain lymphocyte traf-ficking behaviours that had been described by pioneers in field, such as the abilityof some lymphocytes to selectively recirculate through the intestines or through theskin, or to home preferentially to the gut wall. Later studies revealed recognitionsystems for joint, BALT and brain and skin endothelium. Using Occam’s razor, weFigure 1A Figure 1B Tissue selective lymphocyte-endothelial recognition:Lymphocytes homing in a Lymphocytes Tissue selective between HEV in different can distinguish lymphocyte-endothelial recognition:high endothelial venule homing in a a clonal lymphoma cell line adheres to HEV in different Lymphocytes sites. Here Lymphocytes can distinguish between HEV(HEV) in vivo high endothelialin sections of sites. Here a clonal lymphoma cell line adheres to HEV venule lymph nodes but not Peyer’s patches (HEV) in vivo in sections of lymph nodes but not Peyer’s patches Lymphocytes HEV Lymphocytes HEV Endothelial cells Endothelial Lymphocytes cells Lymphocytes Peripheral lymph nodes Intestinal Peyer’s patch Peripheral lymph nodes Intestinal Peyer’s patch Lymphocytes α4β7 L-selectin CLA Homing receptors MAdCAM-1 PNAd (CHO) E-selectin Tissue selective vascular ligands (addressins) intestinal lymph node skin Endothelial cellsFigure 2. The ”lock-and-key” model 7
  9. 9. hypothesized a simple lock-and-key model of lymphocyte-endothelial recognition,in which lymphocytes express homing receptors that can target their migrationfrom the blood by interaction with tissue–selective endothelial “address signals” orvascular addressins (Figure 2).This model, although wrong, drove a search for the postulated homing receptors.We used the new monoclonal antibody technology to make an antibody to the pu-tative lymph node homing receptor: this molecule, the first identified lymphocyte-endothelial cell adhesion molecule, is now called the L-selectin, or CD62L. In sub-sequent studies, we identified tissue-selective HEV antigens, including a sulfatedcarbohydrate epitope defining the L-selectin ligands (“sulfoadhesins”) of the Perip-heral Node HEV Addressin, PNAd (which is also expressed by chronically inflamedvenules in many sites); and the mucosal addressin cell adhesion molecule MAd-CAM-1, which binds the lymphocyte intestinal homing receptor, an integrin calledα47. One antibody we made to human HEV, HECA452, was serendipitously found(by a particularly observant fellow) to cross react with a cutaneous T cell antigen(CLA) expressed on skin homing lymphocytes: this turned out to be an epitope as-sociated with E-selectin-binding carbohydrates. This E-selectin-CLA interaction isa hallmark of T cell recruitment in skin inflammation, for example in psoriasis. These tissue-selective lymphocyte and vascular adhesion molecules offer a wayto control inflammation locally or regionally. Immune responses require the re-cruitment of lymphocytes from the blood, and in the case of pathological immuneactivity, for example in arthritis, blocking lymphocyte recruitment can suppress theunwanted inflammation. Indeed, antibodies or small molecule inhibitors of severalof these lymphocyte adhesion pathways have made their way into animal models,and in some instances clinical trials, for autoimmune diseases including arthritis,diabetes, inflammatory bowel diseases, psoriasis, and asthma. As specific examples,antibodies to α47 are in clinical trials for colitis; and antibodies to sulfoadhesinare in development for several diseases of chronic inflammation. In spite of its successes, it soon became clear that this early lock-and-key hypo-thesis was simply wrong. The problem was that several lymphocyte homing recep-tors were also expressed by other white cells with very different homing properties,such as monocytes and neutrophils. Antibodies to L-selectin even inhibited neu-trophil recruitment into inflamed skin. Obviously, the real mechanism was morecomplex. By this time it was well established by Tim Springer and others that the integrinshe had identified, in particular the beta 2 integrin LFA-1, could participate in leu-kocyte binding to activated endothelium; and Alf Hamann had shown that LFA-1had an accessory role in lymphocyte homing as well. We found that activated neu-trophils rapidly shed L-selectin while upregulating beta 2 integrins, suggesting that 8
  10. 10. the molecules might act sequentially. Perhaps most telling were studies by KarlArfors, a pioneer in the field of microcirculation, who showed with John Harlan thatantibodies to beta 2 integrins prevented neutrophil sticking (stopping on the wallof inflamed vessels), but that the neutrophils still rolled along the endothelium. Wenaturally hypothesized that L-selectin might be responsible for this rolling and, ina collaboration with Ulrich von Andrian, then in Karls’ lab, we showed that rolling(and subsequent activation-dependent arrest) in a rabbit model were indeed inhi-bited by anti-L-selectin antibodies. This established sequential roles for L-selectinand integrins in a multi-step molecular process of neutrophil adhesion to vessels atsites of inflammation. At the same time (and unbeknownst to us), Mike Lawrence in Tim’s lab waspursuing elegant in vitro models that demonstrated that the vascular selectinscould mediate tethering and rolling of neutrophils on slides, and that this rolling,as in vivo, was essential for activation-dependent integrin-mediated arrest of cellsunder flow. In fact, Uli von Andrian first presented our results, and Tim theirs, atthe same meeting in Cold Spring Harbor in 1991. I stayed home from the meetingto walk the hills and write, because I had become incredibly excited by the ideathat this might be a general model for leukocyte-endothelial cell recognition andhoming---a model that could explain everything we’d learned about lymphocytehoming and its regulation in physiology and disease. One problem with generalizing the paradigm was that we had no idea what ac-tivating signals on endothelium could trigger lymphocyte integrins. We knew thatchemoattractant receptors, specialized receptors similar to those that we use fortaste, sight and smell, were responsible for activating neutrophil integrins for stick-ing and arrest, and we had evidence from our own studies with pertussis toxin thatsimilar receptors might be important for lymphocyte arrest on HEV as well. But nopotent chemoattractants for circulating lymphocytes were known. In another strokeof luck, I complained about this to a friend who just happened to know that Genen-tech (Tom Schall, building on his earlier work with Alan Krensky at Stanford) hadjust identified a novel lymphocyte chemoattractant related to the neutrophil activa-tor IL8, defining as it turns out what is now called the “chemokine family”. This wasenough for me to stick my neck out and propose that these might be the missinglymphocyte adhesion-triggers required for a general model. (Although it actuallytook years before we and others identified chemokines that trigger lymphocyte ar-rest on HEV, ultimately this large chemoattractant family has proven important tothe homing of every lymphocyte subset studied, and of monocytes, eosinophils, andindeed all white blood cell types). The hypothesis I put forth was that, for lymphocytes and other white blood cellsas well as neutrophils, endothelial interaction might be an active process involvingseveral sequential but equally important events, rolling, chemokine or chemoatt- 9
  11. 11. Figure 3. A general model of leukocyte recruitment as a multistep process 1-tethering and rolling 2-activation 3-arrest 4-diapedesis P-selectin-CHO Chemokines α4β7---MAdCAM-1 Chemokinesreceptor-ligand pairs *PSGL 1 E-selectin-CHO (N~30?) α4β1---VCAM-1 (N~30?)Interchangeable P-selectin-Sulfoadhesin Lipid mediators LFA-1--ICAM-1 Lipid mediators α4β7---MAdCAM-1 Formyl peptides Mac-1--?ICAMs Formyl peptides α4β1---VCAM-1 C5a Others? Chemerin? ?----VAP-1 Chemerin? Cathelicidin? CD44--HA Cathelicidin? Others? Specificity and diversity from combinatorial association of adhesion, activating and chemoattractant Table 1. Some examples to illustrate combinations of adhesion and activating factors in selective cell trafficking programs ractant-induced activation, activation arrest (chemokine or (activated rapid activation-dependent cell target site tethering slow rolling attractant) integrins) integrin-mediated arrest, naïve CD4 T cells lymph node L-selectin-PNAd L-selectin CCR7-CCL21 LFA-1-ICAM and potentially a fourth step CD8 T cells lymph node ? - VAP1 ± L-selectin-PNAd ?CCL21 ?LFA-1 HEV in which chemoattractants naïve CD4 T Peyers L-selectin- a4b7-MAdCAM CCR7-CCL21 a4b7/LFA-1 cells patch MAdCAM-1 CHO could also regulate migration naïve B cells Peyers L-selectin- a4b7-MAdCAM CXCL13,CXClL12, a4b7/LFA-1 patch MAdCAM-1 CHO CCL21 across the endothelial barrier skin homing skin CLA-E-selectin a4b1-VCAM-1 CCR4-CCL17 a4b1-VCAM1 into the surrounding tissues, memory CD4 cells CCR10-CCL27 LFA-1-ICAM1 completing the process. The small intestinal small a4b7-MAdCAM-1 CCR9-CCL25 ?LFA-1 memory T cells intestines idea was that, if several diffe- Memory CD4 T joints L-selectin-PNAd a4b1-VCAM1 ?- ?CCR7, ?CCR2, ICAM-1 rent receptors could be used cells (arthritis)_ ?P-selectin VAP1 ?others ?a4b1-VCAM1 IgA small a4b7-MAdCAM-1 CCR9-CCL25 ?LFA-1 interchangeably at each of plasmablasts intestines CCR10-CCL28 IgA lung a4b1-VCAM-1 CCR10-CCL28 LFA-1 these steps (which they can, plasmablasts see Figure 3 and Table 1), it IgG plasmablasts systemic (non selectins?, a4b1-VCAM-1 LFA-1? mucosal) would provide an efficient, Eosinophils various E-, P- or L-selectin a4b1/a4b7 eotaxins a4? And beta2 combinatorial mechanism integrins for generating diversity and Monocytes various E-, P- or L-selectin a4b1 MCP chemokines, a4? And beta2 specificity in homing. C5a, formyl peptides integrins This model explains how Neutrophils various E-, P- or L-selectin IL8, formyl beta 2 peptides, C5a, integrins several different homing leukotrienes 10
  12. 12. events could use a common receptor (e.g. L- selectin is used in lymphocyte, neu-trophil, eosinophil and monocyte recruitment: but specificity can be determined bythe use of different chemoattractants to trigger arrest). It also makes it easy to seehow new homing specificities can evolve simply by combining pre-existing receptorsin new ways. And it implies that leukocyte recruitment can be blocked for thera-peutic ends by inhibiting any of the 3 or 4 steps. This general multistep paradigm,with numerous variations and refinements, has been confirmed in many differentmodels of cell trafficking, by our lab and many others. From a desire to study a simple cell-cell recognition event, we had been led tothe discovery of a truly beautiful, combinatorial mechanism for targeting many andperhaps all cells of the immune system in their migration from the blood. Theremay be hundreds of different trafficking programs, allowing specific control of im-mune cell localization (and thus of immune responses) as a function of the tissuesite, the inflammatory state, the leukocyte type and its differentiation. Certainly,we now know combinations of adhesion and chemoattractant receptors that can ex-plain the localization of naïve lymphocytes and subsets of memory cells and dendri-tic cells to lymph nodes or to Peyer’s patches; of memory T cells for gut antigens tothe small intestines, and of cutaneous memory lymphocytes to skin; of IgA and IgGplasmablasts; of monocytes, eosinophils and more. Many of the molecules involvedare now being pursued as therapeutic targets to treat inflammatory diseases, inclu-ding multiple sclerosis, colitis, psoriasis, asthma and arthritis. In addition to therapeutics aimed at blocking homing and chemokine receptors,future efforts may take control of the expression of the receptors, redirecting theimmune response by altering cellular positioning and interactions. Redirection ofimmune cells may allow us to improve the efficiency of immune responses aftervaccination; or to shut down autoimmune responses. We may even be able to turnleukocytes into Trojan horses to deliver therapeutics to sites of our choosing in thebody. It has been with great pleasure that I have seen many of our observations servingas the basis, directly or indirectly, for efforts to develop novel therapies for inflam-matory diseases. My grandmother suffered greatly from rheumatoid arthritis, andalthough my passion will always be for the pure beauty of science, it is also my per-sonal hope to have contributed in some way to reducing the suffering and disabilityof this terrible disease. I thank the Crafoord family for their generous support ofsuch efforts. And I cannot end without extending my deep appreciation and indeb-tedness to the people who made it possible for me to be here, the many wonderfulmembers of my laboratory who have contributed their ideas and talents over theyears, my scientific colleagues and collaborators in academia and industry, and es-pecially my wife and family for supporting me with humor and some bemusementin my obsessive dedication to science. 11
  13. 13. Integrin Cell Adhesion Molecules in Health and Disease Timothy A. Springer, Dept. of Pathology, Harvard Medical School, Boston, USAYour Majesties, President of the Academy, members of the Crafoord family, mem-bers of the Academy, ladies and gentlemen, I am humbled to be invited to addressyou, and express my deepest gratitude to the Royal Swedish Academy of Sciencesfor the Crafoord Prize.I am now facing one of the greatest challenges of my life – explaining what I do to alay audience. I am not too good at doing this in words, so I am going to use movies,and even a modern dance, to bring the cells and molecules I work with to life. I am trained in biochemistry – the science of the molecules of life. And I alsowork in immunology, the study of how we develop immunity to bacteria and viruses,and fight infections. I have been lucky to work at Harvard Medical School and CBRBiomedical Research Institute, where opportunities abound to make connectionsbetween the molecules of life and the diagnosis and treatment of disease in pa-tients. I was fortunate to participate in the development of a new research field, celladhesion molecules of the immune system. The initial stage was discovering oridentifying molecules on the surface of white blood cells that allow them to bind,stick, or adhere, to use three different names, to other cells. While most of my col-leagues were looking for molecules that were unique to white cells or lymphocytes,and allowed them to recognize foreignness, I was looking for cell adhesion molecu-les that allowed them to recognize the context of the cell on which the foreignnesswas displayed. The cells that compose a multicellular organism must organize themselves intotissues and organs that work together for the good of the whole. This organizationand communication requires cell adhesion molecules that sense the context that acell finds itself in the body, and can control the movement of the cells between diffe-rent niches. While this is now accepted fact, it wasn’t at all accepted when I startedworking on it, and some people thought that physics and chemistry, without anymolecular specificity, might allow cells to come together and sort into organs, muchas oil or clay can separate out of water. However, we found that specific proteinswere required, and that when these were blocked, killer lymphocytes could not dotheir jobs in recognizing the foreignness that signifies an infected cell. Let us look at a movie of a white blood cell killing an infected cell. The white cellmoves around, first gingerly touching other cells, and when it recognizes foreign- 12
  14. 14. ness, it grabs hold tightly, and delivers a lethal potion that kills the cell. It can thendetach, and go on and kill other cells. With the light microscope, we cannot see anyof the molecules on the surface of the white blood cell, but the dynamic movementsof its surrounding surface membrane suggests that many protein molecules on themembrane must undergo choreographed movements to enable adhesion to the tar-get cell. There must be dynamic regulation, so that the adhesion molecules canalternately hold onto one cell, then let go, and then hold on to yet another cell. In the first stage of our work, we identified the molecules that were required forkilling, because blocking these molecules with antibody probes prevented the func-tion of killing. We called these lymphocyte function-associated molecules 1, 2, and3 (LFA-1, LFA-2, and LFA-3). Later we identified intercellular adhesion molecules,or ICAMs. In a later stage of our work, we showed that these molecules really func-tioned as adhesion molecules, and not in some other step such as killing, and werespecific receptors and counter-receptors for one another. In other words, LFA-1 ona white cell bound to ICAM on an infected cell. Similarly, LFA-2 or CD2 on a whitecell bound to LFA-3 on an infected cell (Figure 1). At the time, it was quite a reve-lation that adhesion molecules, as well as receptors for foreignness, were requiredfor protective immune responses. Remarkably, blocking either of the two adhesionpathways was almost as effective as blocking foreignness receptors in preventingimmune responses.Figure 1. Molecular Basis of White Cell (T Lymphocyte) Interactions (circa 1983) LFA-1 ICAM-1Killer or Helper ICAM-2White Cell Foreign or LFA-2 LFA-3 Antigen-Interaction White Cell AgR/ Presenting CD3 HLAClass Cell CD8An ensemble of surface molecules, both antigen receptors and adhesion molecules, are required forefficient white cell recognition of foreignness. MAb to each can individually inhibit responses. We proposed that these pathways must also be utilized in immune responses thatgo awry and cause harm, such as in the autoimmune diseases rheumatoid arthritisand psoriasis. We reasoned that blocking the action of these molecules could be aneffective treatment for autoimmune diseases. Indeed, this vision has now been rea-lized. In early 2003, the FDA approved an LFA-3 decoy called Amevive (alefacept)developed by Biogen for moderate-to-severe plaque psoriasis. Amevive works bylooking like LFA-3, and blocking LFA-2 (Figure 2). The generic name for this drug 13
  15. 15. Figure 2. Molecular Basis of White Cell (T Lymphocyte) Anti-adhesive Therapeutics (2003) LFA-1 ICAM-1Killer or Helper ICAM-2White Cell Foreign or LFA-2 LFA-3 Fc LFA-3 Antigen-Interaction White Cell AgR/ Presenting CD3 HLAClass Cell CD8 Fusion protein containing LFA-3extracellular domain and Fc portion of IgG1 (Amevive; Alefacept = LFA-cept). Found effective in phase III clinical trials of moderate to severe plaque psoriasis. FDA approved in Jan2003, currently being sold by Biogen.is derived from our name LFA-3, i.e. al-ef-a-cept = LFA + (inter)cept. Further-more, yet another drug developed by Genentech was approved in late 2003, also formoderate-to-severe plaque psoriasis. This drug is an antibody to LFA-1 (Figure 3).These drugs have reversed psoriasis and dramatically improved the quality of lifefor many who have suffered the debilitating effects of this disease, and with redu-ced side effects compared to previous therapies. Extending treatment to other ty-pes of autoimmune diseases with these drugs is currently being explored. AlthoughI did not participate in drug development, both drugs were directly derived from mybasic research discoveries, and therefore 2003 was a very exciting year for me. There is a conundrum that I glossed over. If white cells recognize contextualadhesion molecules on other cells, as well as foreignness, don’t they waste most ofFigure 3. Molecular Basis of White Cell (T Lymphocyte) Anti-adhesive Therapeutics (2003) LFA-1 Ab ICAM-1Killer or Helper ICAM-2White Cell Foreign or LFA-2 Fc LFA-3 Antigen-Interaction White Cell AgR/ Presenting CD3 HLAClass Cell CD8 Antibody to LFA-1 found effective in phase III clinical trials for psoriasis (Raptiva, Efalizumab). FDA approved in Oct 2003, currently being sold by Genentech. 14
  16. 16. their time on the wrong cells? At a cocktail party, wouldn’t they spend most of theirtime with uninteresting cells with no foreignness, and never meet the foreignersthey are supposed to recognize and kill? The answer to this problem, we found,is that the adhesion molecule LFA-1 binds very weakly or not at all in its restingstate, but if a foreignness receptor gets activated, this activates signals inside thecell. These signals tickle the feet of the integrin inside the cell, and this gets com-municated to the legs of the integrin, which extend and cause the binding site in itshead to change shape and bind ICAM tightly (Figure 4). In turn, when LFA-1 bindsICAM, this sends further signals into the cell that tell it is in the right context forkilling, and essentially say “full speed ahead.” In my lab and others, further molecules were discovered that bore a family re-semblance to LFA-1, and these proteins are now called the integrin family. Theyare present on cells in all tissues in all multicellular animals, ranging from spongesthat arose over a billion years ago, to us. Integrins are essential for development ofan egg cell into a multicellular organism, and a wide range of processes throughoutlife including cell migration and wound healing.Figure 4. Activation of integrins White cell Intracellular signals Signaling by integrins, Integrin activation, outside-in inside-out Integrin shape change Foreignness recognition Activation signal recognition Binding to ligand (ICAM) ICAM Interacting cell 15
  17. 17. The importance of integrins on white blood cells was underscored when we foundpatients with an inherited deficiency of integrins. These patients have recurringbacterial infections, and often die in infancy. Their white cells cannot leave thebloodstream to fight infections. Along with Eugene Butcher, we found that a multi-step process is required for white cells to leave the circulation and emigrate intosites of infection. In the first step, cells roll along the vessel wall. Then activatingmolecules bind to the white cells. Finally, integrins get activated and mediate firmadhesion to the vessel wall, and provide traction for emigration out of the vesselinto tissue. Remarkably, we were able to reproduce these steps with purified adhe-sion molecules in artificial flow vessels in the lab. Initially, the lymphocytes roll ona special adhesion molecule called a selectin. When activation signals are added,they signal the lymphocytes to make their integrins sticky. When this happens, theintegrins rapidly bind ligands like ICAM on the vessel wall and cause the lympho-cytes to stick firmly (Figure 5). In the next step, the cells migrate out of the vessel,and use the integrins like feet to walk out of the blood vessel and then around inthe tissue until they find the infectious agents. The activity of integrins is coordi-nated to aid the walking process. The integrins become stickiest in the directionthat the cell senses activation stimuli. We have probes that show the integrins getselectively turned on in membrane projections that are newly formed in the direc-tion in which the cell is attracted by activating stimuli , so that new adhesions tothe surface on which the cell is walking can form in the direction in which the cellis moving.Figure 5. Reconstitution of Rolling, Activation, and Firm Adhesion in Vitro G Protein-Coupled Receptor Inactive Integrin Active Integrin Carbohydrate Ligand Step 2. Addition of chemoattractantactivator to perfusate solution P- Selectin ICAM-1 Step 1. Rolling Step 3. Firm Adhesion 16
  18. 18. Recently, we and other labs have looked at integrins under the electron micros-cope and by X-ray diffraction in crystals. We have learned that integrins must un-dergo unusually large changes in structure in order to pass signals back and forthacross the cell membrane. There are two kinds of integrins, one of which containsan extra “I domain,” but overall they undergo very similar shape changes (Figure6). We have determined the structure of the sticky part of LFA-1, its I domain, andshown how it changes shape when it becomes sticky for ICAM-1. In even more recent work, we have studied an integrin on platelets. This integrinbecomes activated during bleeding so that it binds fibrin, and allows the platelets toaggregate and plug up holes in blood vessels. However, improper activation of thisintegrin can also cause thrombosis in heart attacks and stroke. Therefore, drugshave been developed that bind to this integrin for prevention and treatment of co-ronary artery thrombosis. We have determined structures that show exactly howthese drugs bind, and also reveal the highly active conformation of integrin. Withother work, this reveals how integrins work as protein machines in transmittinginformation across membranes. They have a head that binds ligands and two longlegs that cross the membrane. In the resting shape, the legs are highly bent. Whenthe integrin becomes active, a signal is transmitted between the ligand binding siteand one of the upper legs. When the binding site changes shape to bind tightly toligand, this transmits a signal that causes one of the upper legs to separate mar-kedly from the other at the knees. This causes the integrin to extend and stand upFigure 6. Integrin Shape Change In Signaling across the Cell Membrane Extended, Extended, medium-sticky highly-sticky integrin integrin Head Bent, non-sticky integrin Upper leg Lower leg Outside Membrane Inside cell 17
  19. 19. on the cell surface. This positions the active head far above the surface in a goodposition to bind ligand (Figure 6). Separation of the legs is transmitted into the cellto signal further activation. Integrins are actually not fixed in one of these shapes,but can dynamically equilibrate between them, as will be better brought out next.In summary, we understand much about how signals are transmitted between theintegrin head and legs outside the cell, and also through the membrane, since theintegrin feet inside the cell separate when the legs do. To bring integrins to life, I have collaborated on a modern dance in which twointegrins are the stars. We also see two activation signals, and a ligand, to whichthe integrins will stick when they get activated. Imagine that these integrins are onthe membrane of two different cells in your bloodstream. They are continually onpatrol as they circulate in your blood. Integrins change shape, up and down, evenwhen they are at rest and are not yet active. Ligands are always eager to bind, butnot integrins. Integrins require activation before they want to stick. The ligandwants to adhere, but the integrins are not sufficiently aroused. Suddenly activation signals arrive on the scene, meaning something bad is hap-pening offstage, like a blood vessel rupture or an infection. These signals travelthrough the blood and alert the cells. Now, the activation signals enter the cells, and excite the integrins to becomeinterested in binding ligand. The integrins change into a different, highly activeshape that is specialized for ligand binding. The integrins now want to, and do bind ligand, but binding is reversible. Whenonly one integrin binds a ligand at a time, it is easy for the ligand to get away. Now two integrins bind the same ligand, and stick to it more avidly. Now it isvery hard for the ligand to get away, and the integrins can cause the blood to clot,or leukocytes to fight infection. Binding of multiple integrins causes further activation of the cell. Because theintegrins can bind ligand, they tell the cell it is doing something important, andurge it on to do even more, including to divide and make more cells to finish thejob. Thus integrins enable cells to integrate events outside the cell with events insidethe cell, and for cells to communicate with their nearest neighbors. Basic researchon adhesion molecules has resulted in the development of new drugs to treat au-toimmune disease and heart attacks, and many more drugs directed to integrinsare under development. The research I have described was a team effort involvingmany PhD students, postdoctoral fellows, and collaborators. I am sorry that I donot have time to name them all now, but I am most pleased that some of them arehere today to share in this honor. 18
  20. 20. A BST R ACTS 19
  21. 21. Control of Leukocyte Traffic Sirpa Jalkanen, MediCity, University of Turku, Turku, FinlandLymphocytes continuously recirculate between the blood and tissues in search oftheir cognate antigens. These cells, together with polymorphonuclear granulocytes,also rapidly accumulate at sites of inflammation. The physiological and inflamma-tion-induced leukocyte trafficking processes have several features in common. Inboth cases the blood-borne leukocyte initially makes transient tethering contactswith the endothelial lining of blood vessels. These interactions result in reductionof leukocyte velocity and in characteristic rolling behavior of the cell. The rollingleukocyte can then become activated. Only the activated leukocyte has the abilityto stably adhere to the endothelial cell and seek for the interendothelial junctionsthrough which it can penetrate the vessel wall and thus enter the tissue. There-after, the cell percolates through the tissue stroma, and in case of lymphocytes,it finally leaves the tissue via efferent lymphatics and is carried back to systemiccirculation. Leukocyte emigration into tissue is the hallmark of inflammation. It is the pro-totype response to a wide variety of noxious stimuli as diverse as acute and chronicinfection, autoimmune disorders, ischemia-reperfusion injury, graft rejection anddefence reactions against malignant cells. These conditions include a wide rangeof clinically important disorders as exemplified by bowel infections, arthritis, dia-betes, heart infarction, kidney rejection and cancer development just to mentiona few. Analogously, malignant cells need to migrate to distant sites of the body toform metastases. They seem to use similar or comparable mechanisms as leukocy-tes do to travel into different tissues. Within these disease entities, adhesion molecules can be utilized in developingdiagnostic aids and therapeutical agents for many purposes.The concept of anti-adhesive therapies has proven to be valid in few efforts performed to date. Howe-ver, finding an optimal target for drug development is a huge challenge. We haveidentified and characterised novel molecules responsible for leukocyte migrationinto sites of inflammation. Moreover, we have elucidated mechanisms that normallymphocytes use when exiting the tissues and cancer cells use for metastasis forma-tion. These results can be benefited in the development of new types of drugs forthe treatment of harmful inflammations and cancer. 20
  22. 22. Control of Interstitial Fluid Pressure and Acute Edema – Role of Integrins Kristofer Rubin, Dept. of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, SwedenThe current concept in interstitial physiology holds that the interstitium acts as apassive fluid reservoir. During the last decade data have been presented suggestingthat this model should be revised. It has become evident that connective tissue cellsactively control the interstitial fluid pressure (IFP) and thereby fluid flux over theblood vessel wall. A rapid lowering of IFP plays a fundamental role in the develop-ment of edema in burns and in the initial swelling during inflammatory reactions.Integrin-mediated contacts provide a common pathway by which cells can controlIFP. Present data suggest that connective tissue cells exert this control by a processrelated to fibroblast-mediated compaction of three-dimensional collagen lattices invitro. Inflammatory swelling can be modulated both by exogenous and endogenoussubstances, thereby suggesting that connective tissue cells can serve as targets forpharmacological intervention aimed to control edema. Furthermore, these newconcepts in interstitial physiology and means to regulate IFP may be of importancefor drug delivery into carcinoma, where a pathologically elevated IFP seems to limitthe uptake of therapeutic drugs. 21
  23. 23. Regulation of Human Neutrophil Apoptosis Tommy Andersson, Malmö University Hospital, Malmö, SwedenThe human neutrophil is the most abundant granulocyte and the major type ofcell involved in an acute inflammatory response. Neutrophils are armed with vari-ous systems of enzymes, that can find and kill pathogens, but unfortunately, these”weapons” cannot distinguish between the host tissues and the ”invaders.” The-refore, an extensive neutrophil reaction leads to continuous release of toxic me-tabolites, which causes successive self-destruction of host tissues and possibly alsoorgan failure. Such a series of destructive events has been implicated in diseasessuch as rheumatoid arthritis, myocardial infarction/reperfusion injury, atheroge-nesis, asthma, cystic fibrosis, emphysema, and vasculitis. Resolution of an acuteinflammatory process depends on termination of neutrophil emigration from bloodvessels and clearance of extravasated neutrophils and their metabolic products.Outside the blood vessels, neutrophils spontaneously undergo apoptosis, and aretherefore removed by phagocytic cells at the site of inflammation. Neutrophil apo-ptosis can be modulated by several factors in the local environment, such as the Fasligand (FasL), but the mechanisms involved are poorly understood. In this presentation, I describe and elucidate intracellular signalling mechanismsthat are involved in regulation of spontaneous and Fas-induced apoptosis in humanneutrophils. Using two different methods it was possible to detect constitutive ac-tivity of p38 mitogen-activated protein kinase (p38) in newly isolated neutrophils.The p38 survival signal was transiently lost during both spontaneous and Fas-in-duced apoptosis, an event that favoured induction of the apoptotic process. Duringthe transient loss of p38 activity there was a temporary Fas-induced increase inphosphatidylinositol 3-kinase (PI3K) activity, which also had a pro-apoptotic im-pact on the neutrophils. In addition, my experiments showed that the active formof p38 associates with caspase 8 and caspase 3, an interaction that led to p38-indu-ced phosphorylation of serine-362 and serine-150 on these caspases. These bioche-mical modifications impair the activities, and possibly also the stability, of caspase8 and 3 and thereby weaken the capacity of these enzymes to induce apoptosis.Finally, I will demonstrate that the short-lived decrease in the phosphorylationlevels of p38 and caspase 3 is regulated by protein phosphatase type 2A (PP2A). Byexerting that effect, PP2A increases the activity of caspase 3 and thereby enablesthe Fas-induced apoptotic response in human neutrophils. 22
  24. 24. Cell Adhesion and Migration in Tumor Progression Richard Hynes, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge. MA USAInvasion and metastasis (collectively malignancy) are the processes that make can-cer a dangerous disease. We understand much less about them than we do aboutthe initiation and development of primary tumors, yet malignancy is what kills.So, there is a pressing need to understand the molecular and cellular changes thatcontribute to invasion and metastasis. These processes involve loss of positionalcontrols, which are intrinsically more complex than the loss of growth controls in-volved in primary tumor growth. Invasion requires both loss of adhesion for “homebase” and acquisition of invasive and migratory properties, which themselves re-quire acquisition of novel adhesive interactions. Good examples exist in human tu-mors both of loss of adhesion (e.g., cadherins in colon and stomach carcinomas) andgain of adhesion (e.g., integrins in malignant melanomas and many carcinomas).Changes in cell adhesion probably also contribute to the arrest and extravasationof tumor cells from the vasculature. Despite these anecdotal cases, we do not havea good picture of the changes in cell adhesion that contribute to the many stepsrequired for a successful metastasis. In part this is because the processes are com-plex; in part it is because we have until very recently lacked a sufficiently completepicture of the molecules involved in cell adhesion. The situation has changed radi-cally in recent years and we now have the possibility to attempt a detailed inventoryof the changes in cell adhesion associated with the multiple steps of invasion andmetastasis. The coordinated action of adhesion molecules and cytokines during leukocytetraffic is one of the best understood cellular adhesion processes and offers a modelfor mechanisms used by tumor cells during their metastatic spread. It turns outthat some tumor cells do indeed exploit molecular players familiar from the leu-kocyte adhesion cascade in metastatic spread and that host adhesion moleculescontribute as well, both to metastatic spread and in the response of the innateimmune system to tumor growth. This can be demonstrated using mice lackingvarious cell adhesion receptors. Screens of metastatic cells for alterations in geneexpression also reveal important alterations in adhesion and chemokine receptorsand also many alterations in molecules controlling cell migration. Analyses of thissort offer promising opportunities both for understanding tumor progression andmalignancy and for developing therapeutic approaches. 23
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  26. 26. T H E R O Y A L S W E D I S H A C A D E M Y O F S C I E N C E S isan independent, non-governmental organisation founded in1739. The major aims are to promote research in mathematicsand the natural sciences. The Academy participates in and promotes internationalscientific cooperation through its seven scientific institutes bypublishing scientific journals, by distributing scientific infor-mation and by promoting contacts between scientists andsociety. Prizes and grants are awarded annually from fundsheld in trust by the Academy. The Nobel Prizes in Physics andChemistry have been awarded by the Academy since 1901 andthe Prize in Economic Sciences in memory of Alfred Nobelsince 1968. The Academy has about 350 Swedish members of whom167 must be under 65. There are also 167 foreign members.Members belong to one of the Academy’s ten classes. The head of the Academy is the President, assisted by threevice-presidents – all elected to these honorary positions for acertain period. Together with the Secretary General, who is afulltime employee, they form a Presidium. Academy work is carried on in the classes and in perma-nent or ad hoc committees. The Academy has a secretariat ofabout 30 employees, headed by the Secretary General. The Academy has bilateral agreements on exchange ofscientists with academies in other countries, and representsSweden in the International Council for Science (ICSU). TheAcademy also administers the Swedish national committees,which handle contacts with ISCU’s international scientificunions.P.O. Box 50005, SE-104 05 Stockholm, SwedenPhone: +46 8 673 95 00, Fax: +46 8 15 56 70E-mail: info@kva.se, Web site: www.kva.se ISSN 0283-2747