Macrophages are the garbage trucks of the immune system. Their name, in Greek, means literally "big eaters." Indeed, a single macrophage cell can engulf and destroy a microbe bigger than itself.
"When you have an infection," observed molecular cell biologist Michel Desjardins at the University of Montreal, "there are two things your immune system has to do: first, engulf and kill the pathogenic agent, and clear the infection as rapidly as possible. This is what we call innate immunity. It's the role of the macrophage, which is a white blood cell of the immune system, growing at the site of infection. It's the first line of defense against infection.
"Second, the immune system also has to make sure that some of these antigenic microbes will be cut up into small pieces, by the process of antigen presentation. That way, some of the fragments can be presented to T cells and B cells, which start building an antibody response against these foreign infectious particles. This is the adaptive immunity. So if your body sees the infectious agent again, your B cells will remember, and generate antibodies very rapidly."
Besides his faculty position as associate professor of cell biology at the university, Desjardins is director of proteomics at Caprion Pharmaceuticals Inc. in Montreal. He is senior author of a paper in the journal Cell, dated July 12, 2002. Its title: "Endoplasmic-reticulum-mediated phagocytosis is a mechanism of entry into macrophages."
"All textbooks, and all experts in the field," Desjardins told BioWorld Today, "think that phagocytosis is the internalization or engulfment of microbes, for example, in macrophages. The ER - endoplasmic reticulum," he explained, "is a membrane organelle that is inside the cell. There are a lot of these membranes there, and they are involved in the biosynthesis of proteins. When we synthesize proteins, they will start to be synthesized on ribosomes, and then get translocated inside the ER lumen. There they will be modified, glycosylated and exported to the cell surface or to other organelles inside the cell."
ER Has Unsuspected Affair With Microbe
"So it's not an organelle that's supposedly involved in phagocytosis whatsoever," Desjardins observed. "But when we were looking at the proteomics," he added, "we realized to our surprise that there was a lot of protein from the ER. And no protein indicating from other organelles. We were able to demonstrate that the ER, when a microbe is sitting at the cell surface, binds to a receptor at the surface of a macrophage, for example. We realize now that the ER, or part of it, is migrating inside the cells, directly under where the invading microbe is attached. And the ER will then fuse with the plasma membrane, opening itself to the surface. So now we have a continuity between the ER and the cell surface, right underneath the microbe.
"The microbe can slide inside this open membrane, which will then close, showing that the major part of the phagosome is made by ER and not by the plasma membrane. This by itself is a total surprise in the field, because it is not known that the ER can fuse with the plasma membrane. It's believed to be an organelle that has other roles to play.
"And then," Desjardins continued, "it challenges the well-established concept that phagocytosis is a process that involves the plasma membrane. This is already another important aspect of what we found, because we were using proteomics. Instead of looking at one protein at a time, we were screening hundreds of proteins. So this has very important implications in terms of immunity - between macrophage and microbes. We found that this process, using the ER during phagocytosis, was in fact taking place in macrophages."
Desjardins described the key role of proteomics in his paper's findings: "First of all, for us and I think for the biotech people who have heard about proteomics, it was such a hype thing for a while, but was not delivering so much of what people were expecting, really because proteomics is a young science - not as easy as genomics. It's a science that will take more time to deliver. But in our proteomics analysis, we wanted to understand more how host cells are taking care of microbes in infections. So we launched a proteomics effort, trying to identify all the proteins that are present on the phagosome, which is the organelle formed by the invagination of the plasma membrane to engulf the particles. Still we had a lot of protein from the ER.
"We knew that the way by which pathogens are entering the cells is also followed by inert latex particles. So instead of using a microorganism to start with, we induced the formation of phagosomes by feeding macrophages with very small latex beads, the size of a microbe, about 1 micron. So macrophages internalize these latex beads into phagosomes, and we were able to form them in macrophages."
Proteomics Promises Therapeutic Payoffs
"Then we used Caprion's CellCarta fractionation approach, which it applies to all of its proteomic efforts, even dealing with cancer and metabolic diseases. With this approach, instead of looking at the whole cells, we're looking at different organelles, so that we gain more information about the location of proteins, but also to enrich and purify proteins that are present only in low-copy numbers. It's a method of amplification to a certain degree, that we can use for proteomics.
"We had the macrophage and we isolated the phagosome organelles from the rest of the cell by a simple centrifugation on a sucrose gradient. Then we identified several hundred proteins by mass spec, which allowed us to build the phagosome proteome. This was the basic proteomic approach that we used. After that we were able to identify close to 500 proteins that are associated with this compartment, the phagosome, which is important for clearance of infectious disease.
"This knowledge," Desjardins foresees, "will likely transform into the ability to find drug targets that we can then stimulate or inhibit in the host cell to help clear infectious agents. When we know this, then we'll be able to use both antibiotics to attack the microbe and drugs designed to stimulate the response of the host. So far, this is almost impossible, because the knowledge is not yet there. But we now realize that proteomics is changing this, and generating lot of potential targets. When we know the players involved, then there will be a better way to develop drugs or therapeutic approaches."