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IMMUNOLOGY Remote-control Immunity Up Close Mechanism Shown for Recruitment of Monocytes to Inflamed Lymph Nodes at Distance from Wound Neither Galen nor Aristotle would have mistaken the modest lymph node, at times no larger than a lentil, for the center of intelligence or the soul. Yet its importance is demonstrated by the 25 million medieval Europeans killed by Yersinia pestis, cause of bubonic plague; and in our vulnerability to anthrax, a more topical example. Ulrich von Andrian has discovered a "remote control" mechanism that cells at a wound use to alert the nearest lymph node and initiate an immune response. Photo by Pam Murray "The lymph node sieves out microbes, and it is the staging area for our fight against infection in the periphery," said Ulrich von Andrian, HMS associate professor of pathology at the Center for Blood Research. Of primary interest is the monocyte--the target of many Y. pestis virulence genes--that homes to the node at the first sign of trouble. This leukocyte spawns not only macrophages, which gobble up bacteria, but also dendritic cells. The latter chop up antigens and serve them to naive T cells, spurring proliferation. Wounds send out chemokines, chemical SOS signals, to attract leukocytes to the site of infection. But what brings monocytes to the relatively distant lymph node? In a study published in the November Journal of Experimental Medicine, von Andrian and coworkers show that similar SOS signals generated at the wound work from afar, by "remote control." In their model, chemoattractants travel from the wound to the lymph node, where they stick to the tacky endothelium and flag down monocytes racing through the circulation. "It's really a question of trafficking," said von Andrian, describing the movement of T cells and other leukocytes in the body. Their byways are the lymphatic and vascular systems. When the skin is broken--from a paper cut or fleabite--lymph vessels in the area, which are the tributaries that carry interstitial fluid back to the blood, are unprotected. This is how microbes sneak into the body. In defense, a gauntlet of strategically placed lymph nodes, jam-packed with naive T cells, is ready for the monocytes to arrive and prepare for a counterattack. Researchers can induce this dynamic by injecting a powerful mix of adjuvant and foreign protein under the skin. Following this procedure, Roger Palframan, a postdoc in the lab, showed that monocytes home to the inflamed lymph node--a result von Andrian said is tucked away and nearly forgotten in a classic electron microscopy study published in 1964 by Marchesi and Gowans. Their micrographs revealed mysterious monocyte-like cells associated with microvessels called high endothelial venules (HEV), and they were present only during inflammation. It seemed clear from Palframan's work that the Marchesian mystery cells, identified in the new study by specific cell markers, were indeed monocytes. Marchesi's micrographs provoked many questions: how did the monocytes get there? And what unknown signals were responsible for the monocyte-endothelial cell interaction? Almost four decades later researchers still did not have all the answers. Applying the Brakes A general model has emerged describing how lymphocytes and neutrophils home to their targets. Normally, these cells zip through the blood at high speed. But when the immune system is challenged, life becomes--in more ways than one--gripping. Specific receptors hook onto counter-receptors on the endothelium, slowing the cell's forward motion to an adhesive roll. But the cell cannot stop unless it gets a second, chemokine-dependent activation signal. Little is known about the molecules involved in monocyte-braking in vivo. Von Andrian and coworkers suspected that one candidate might be the chemokine monocyte chemotactic protein, MCP-1. Studies had shown that it recruits monocytes to other peripheral sites of inflammation. In order to observe the migration of monocytes in vivo while manipulating chemokine levels, von Andrian teamed up with Barrett Rollins, HMS associate professor of medicine at Dana-Farber. They produced local inflammation in an MCP-1-deficient knockout mouse, and they found that unlike in the wild-type mouse, there was no significant accumulation of monocytes in the inflamed lymph node. By injecting MCP-1 into the granuloma, the researchers were able to restore wild-type monocyte behavior. Thus, MCP-1 was not only essential for monocyte homing in vivo; it could also travel to the lymph node from a distant source. Further experiments shed light on the source. The researchers observed a gradient of MCP-1 from the skin to the draining node. The only direct anatomic connection between these two regions is, suggestively, the afferent lymphatics. The final proof came from experiments by co-author Antal Rot that showed radioactive MCP-1 injected into mouse skin accumulated in high endothelial venules, "clearly showing that MCP-1 not only goes into the node but that it gets selectively concentrated in the HEV," said von Andrian. Collaring Immune Cells In order to determine the route the monocytes use to enter the lymph node, the researchers tracked the cells in vivo. Co-author Dan Littman of NYU provided a mouse strain with a green fluorescent protein (GFP) gene knocked into a locus specifically expressed in monocytes. After injecting peripheral blood cells from these mice into a second wild-type mouse, the fluorescent subpopulation could be monitored in much the same way that radiocollared animals are tracked in nature. There was one problem: not only are monocytes a relatively rare species--representing just 1 to 3 percent of mouse leukocytes--but the researchers would identify only a tiny fraction of the total population: the newcomers that homed to the node during the experimental period. It would be a bit like collaring all the bears in Yellowstone and identifying those who ransack the trash at the Old Faithful Lodge--within the first hour. In a tour-de-force experiment, Palframan injected the blood cells from a large number of GFP-positive donors into a wild-type mouse, waited four hours, and then measured monocyte migration. He found specific monocyte recruitment to inflamed, but not normal, lymph nodes. Furthermore, he proved the cells' mode and mechanism of entry: in this short time frame, the only route fast enough was the blood, and monocytes could no longer take that route when the animals were treated with an antibody that neutralizes MCP-1. Moving Pictures Against the window in von Andrian's office leans a meticulous 19th century pen-and-ink drawing of a frog's webbed foot. He takes it into his hands when he talks about the circulation. Copied from a rare book, the drawing illustrates the discovery of blood cells rolling along the endothelium--as seen through a microscope, the light shining through the diaphanous web. It is the first documented instance of microscopy in a living vertebrate. A century and a half later, von Andrian uses the same method of intravital microscopy to directly observe the living lymph node in an anesthetized mouse--with a few technological modifications. Using this technique, he can record the live motion of adhesive interactions between a single leukocyte and the endothelial wall. Is it aesthetics that makes these films so compelling? Or the knowledge, while watching, that this is the most realistic glimpse of how the immune system operates? The image is grainy. What inspires is seeing the action in real time, on location. The chemokines are a complex family. Since there are about 50 of these molecules already identified, von Andrian has his work cut out for him. But, if he has his say, it is just this kind of technology that will help scientists out of the trafficking morass and onto solid ground. --Anne Mahon