Science
Membranes: key players in immune response Destruction of foreign cells depends on recognition sites on their membranes; chemical ways are evolving to study properties of the membranes Jeffrey L. Fox C&EN, Washington The body's immune system, charged with the task of recognizing and destroying invader cells, is a chemist's nightmare. Its varied components, including proteins, glycoproteins, cascading pathways, nu merous cell types, and obscure factors, make up a seemingly medieval compen dium of incantations and special dia lects. Yet, a few courageous apostate chem ists have joined earlier converts in a de termined effort to translate immunology into a vernacular tongue, to make chem ical sense out of biological cant. A prime target of those efforts—indeed, a prime target of the immune system when it re sists an invader—is the membranes of cells. One of the principal acts of the immune
Antibody molecules play pivotal role in immune system
Άη IgG-type antibody molecule is a glycoprotein of 150,000 molecular weight, containing two light and two heavy chains joined by disulfide bridges. It binds two antigen molecules through sites on its two Fab branches, and can bind immune effector cells through its Fc stem.
system is to poke holes in membranes of cells that invade the body. That concept, simple as it sounds, has not been simple in the making. Nor is it fully understood how the immune system assures itself that a particular membrane is a target worthy of attack, rather than an innocuous membrane best left alone. Scrutinizing target cells and their membranes is, in many ways, impractical. The chemistry of such cells is intricate but not uniform. Thus, chemists have chosen to abide by the wisdom exercised by anyone starting serious target practice: Make the target simple. Dr. Harden M. McConnell and his many collaborators at Stanford Univer sity have put that wisdom into practice by devising chemically simple targets for the immune system to attack. Their targets consist of membrane vesicles, which are spheres of a single phospholipid bilayer, and of liposomes, which are multilayered phospholipid spheres. The composition of such targets can be controlled. Thus, for example, the phospholipid mixture may be varied; spin-label probes may be added; those molecules or others may be used as the recognition sites (the antigens) for other components of the immune system; and the targets may be made and loaded at will with appropriately chosen markers. Other research groups have favored another simple target membrane that is several steps closer to biological reality, that of red blood cells. Such cells lack nuclei, making them far simpler than most other living cells. Moreover, red blood cells can be made into "ghosts" by opening and then resealing their mem branes. The process permits the loading of markers, such as radioactive or dyetagged molecules, and empties the cells of many molecules that might interfere with measurements. Molecules of the redblood-cell membrane, like those on arti ficial lipid vesicles, can serve as antigens to the body's immune system. In devising these membrane systems as targets for the immune system, there's been a corollary and parallel benefit: Special methods for studying the physical and chemical properties of the targets' membranes have been devised. The sci entists active in this research assert that it's not enough to make membranes to specification. One must also describe them in ample detail. "The aim of all this," McConnell says, "is to make as simple as possible the search for the crucial factors in recogni tion. The initial act of recognition is the most challenging one to me." That entails
McConnell: search for crucial factors
boiling down the recognition process to its bare chemical bones. For example, McConnell and colleagues have studied the complement-mediated immune pathway during the past several years. Complement is the name given to a series of proteins in the blood that can cause target invader cells to disintegrate when triggered by an appropriate immune signal. That signal, in part, involves the binding of other components of the im mune system, namely antibodies, to those target cells. "We raise antibodies by injecting a rabbit with a particular spin-label probe affixed to a carrier molecule," McConnell explains. "Meanwhile, that labeled probe is incorporated into lipid vesicles." The antibodies from the rabbit can "recog nize" the spin-label probe, called the an tigen in this role by immunologists. The complement components are still inactive at this stage. Thus, the spin-label probe residing in the membrane of the vesicle —an artificial target cell—becomes the sitting duck for the next phase of the ex periment. Adding the antibody sets off the com plement cascade. Ultimately, it pokes holes in the membrane of the vesicle. Loss of membrane integrity in the vesicle is the equivalent of killing a living cell. But what are the initial steps? The antibody plays a pivotal role. It must bind the antigen (the spin-label probe) before anything else happens. But to leave things at that misses a crucial point, McConnell says. The antibody must bind antigen that is in the membrane. Thus, if antigen Jan. 22, 1979 C&EN
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A laser beam directed through a grid and into a microscope bleaches dye molecules in a membrane, superimposing a striped pattern. Its rate of spreading is a direct measure of molecular lateral diffusion, according to McConnell and Smith
were free in solution instead of being embedded in the vesicle membrane, the complement cascade would not begin and the membrane would remain intact. Not only is the presence of the antigen in the vesicle membrane essential, but the physical state of that membrane influences the course of subsequent complement reactions taking place in solution. For instance, if the lipid composition of the membrane is altered so that it is more solidlike than fluid, complement activation is slowed down. "To us, that's a fascinating result," McConnell says. "It suggests that activation is a dynamic process in which antibody and complement undergo conformational changes that are restricted by the solidlike membrane." Conformational changes in a protein involve a variety of contortions along its backbone and among its amino acids, and may take place over the full length of the molecule. If the target cell membrane is important in modulating the complement cascade, can it affect other immune systems? Complement consists of a family of soluble proteins. Other destroyers belonging to the immune system are made of cells. They act with somewhat less bravado than complement but no less effectively. Some of them, such as neutrophil cells, show dependence on the state of their target cells' membrane. "The detailed molecular events are different," he says. But as the body's cells attack invader cells, they find that fluid membranes make better targets. "We are systematically pursuing such events in different immune systems to see if these rules work throughout," McConnell says. "It's reassuring to have anything to hold onto." Because the composition of the target cell (the vesicle) membrane is so readily varied, the effects on the membrane of important substances, such as cholesterol, may be tested. In strictly physical terms, 22
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cholesterol exerts unusual effects, tending to make fluidlike membranes more solid but solidlike membranes more fluid, according to McConnell. It also spaces out laterally lipids and other components of the membrane. "Cholesterol has a large effect on the immune system response to these synthetic membranes," he says. Not only does it affect motion within the plane of the membrane, it makes the antigen there more accessible to incoming antibodies. McConnell and his collaborators at Stanford and at Scripps Clinic in La Jolla, Calif., conclude tentatively that lateral diffusion in the target membrane does not limit the extent of the immune response. However, enhancement of the immune response in fluidlike membranes reflects greater freedom afforded by that membrane for conformational changes in the proteins that bind to it. That interpretation is important because it helps focus on a disagreement among scientists over what triggers the complement cascade and other such destructive immune responses. The antibody molecule is wedged into the middle of this argument—and at either end of it are the molecules it binds, and the roles they play. One end of the antibody, the two arms that form the top of the molecule's Y, binds antigen. The other end, the stem of the Y, partakes in triggering the immune response. Disagreement is over the primacy of the rate of the stem (also called the F c portion). For example, Dr. David M. Segal and his collaborators at the National Institutes of Health, Bethesda, Md., find that a sole antibody molecule binding to a target cell cannot set off the complement cascade. However, when antibody molecules are fastened together into pairs and triplets along their antigen-binding sites (the tops of the Y branches), they become efficient in triggering the complement reactions.
"As you increase oligomer size," Segal says, "efficiency keeps increasing." Thus, an antibody pair or dimer does not make a "unit signal" to the complement cascade. Instead, there's a hierarchy: Monomer is ineffective compared to dimer, compared to still superior trimer, and so on. Dr. Henry Metzger, also of NIH, is interested in another component of the immune system called the mast cell, which is responsible for some allergic responses (an unpleasant and seemingly superfluous side to the immune system). Unlike some of the other immune components, mast cells cart around their own antibodies, called IgE's. These molecules are studded along the cell surface, with their stems pointed down into the membrane and the Y branches brandished outward. "The mast cell is oblivious to its IgE molecules," Metzger says, "until exposed to an antigen. Then, the cell degranulates." Put another way, it destroys itself, releasing histamine in the process— hence, the annoying allergic response. But antigen, seemingly the critical ingredient, may be omitted if dimers of IgE are made, Metzger says. Thus, the antibody molecule seems to carry the essential trigger for the immune response. Bring two such molecules together by fixing a chemical bridge between them, and the mast cell begins to self-destruct. It's more complicated, or more simple, than that. "We can eliminate IgE," Metzger says. IgE stems sit in receptor molecules in the membrane. When those receptors are made to aggregate by chemical means, they will trigger mast cell self-destruction—without IgE. "The role of IgE is critical but passive," Metzger argues. "It permits the antigen to cause the membrane receptors to aggregate." He says that the coalescing of receptors is adequate to explain triggering, though he admits that there's "zero knowledge" of what happens molecularly when two receptors meet. Nor can Metzger say what prevents receptors from meeting spontaneously and thereby accidentally tripping the mast cell's selfdestruct machinery. McConnell makes a suitably martial analogy in disagreeing with Metzger's view of IgE. Components of the immune system, McConnell says, "are like hand grenades set to go off for all kinds of reasons. But the natural way is to pull the pin, not to squash or drop them." There are other apparent inconsistencies in the notion that clustering of cell receptors explains immune triggering. Segal recently has turned his research efforts to another kind of cell killing, executed by the part of the immune system known as cell-mediated killing. "Whereas complement was sensitive to antibody oligomer size," he says, "we see nothing like that with cell-mediated killing." Instead, single antibody molecules are just as effective in triggering cell-mediated killing as are oligomers. Segal's tentative explanation is that killer cells can close in on target cells,
killer cells can close in on target cells, bringing along their triggers as they latch onto the targets. Possibly some sort of motion of molecules that reside in the gap between the killer and its victim or along the killer cell's membrane sets off the destructive reactions. He admits, however, that "we were looking for a trigger, and didn't find it." The difference between Metzger and Segal and McConnell's interpretations is not resolved. "And it's not a trivial difference," McConnell contends, although it is a friendly one involving a minor wager. "There's no way under the sun that a gigantic antibody molecule can bind to two antigens on a membrane without undergoing a significant conformational change," he adds. "It's that change that's a crucial element in triggering." Moreover, constrictions imposed on the molecules by membranes force those changes, which don't occur when those molecules are free in solution. The question of exactly what happens during the destruction of the target cell's membrane also has been controversial. Most arguments over how complement completes its task are resolved. "The evidence is overwhelming that channels form because of complement," says Dr. Manfred H. Mayer of Johns Hopkins University's school of medicine. That evidence has grown steadily over the past decade from the work of scientists in Europe, Japan, and the U.S. The complement cascade involves almost a dozen soluble proteins circulating in the bloodstream. The last five in the process form a doughnut-shaped complex that jams into the membrane of target cells, including artificial targets such as vesicles. The hole of that doughnut is the pore through which cell contents may leak. Though the protein complex projects outward from the membrane's outer surface, it does not intrude beyond its inner surface, according to Dr. Sucharit Bhakdi of the Institute of Medical Microbiology in Giessen, West Germany, and Dr. J^rgen Tranum-Jensen of the University of Copenhagen in Denmark. Thus, the protein complex actually is like a doughnut sitting on top of a cylinder that's plugged directly into the membrane. Once formed, the protein complex tends to hold together tightly. For example, a procedure called freeze-fracturing seems to yank such complexes out of membranes. That procedure, used in preparing samples for the electron microscope, splits membranes in two, breaking their inner surfaces away from their outer surfaces. Complement protein complexes remain intact during such procedures, Mayer explains, but they leave nothing to maintain the pore in the inner half of fractured membranes. Because it is made from proteins in solution, the complement pore complex lends itself to molecular description. In a more difficult exercise, several research groups are looking into the perplexing problem of what forms the hole in cell
membranes when a cell of the immune system kills a target cell. Though similar to what occurs when the complement cascade is set off, the problem is considerably murkier. A killer cell riddles its victim when the two are in intimate contact. No holes form in passing target cells. "It's not like a bomb going off," says Dr. Pierre Henkart of NIH. If anything, it's more like an intimate mugging. Moreover, the killer cell is orders of magnitude more complicated than the admittedly complicated complement system. Several years ago, Henkart and his colleague Dr. Robert Blumenthal noted that, in the presence of appropriate antibodies, one such killer cell, the lymphocyte, damages lipid membranes, making them permeable to ions. Since then, Henkart and collaborators and others, including Mayer, have filled in some of the gaps in knowledge of how killer cells destroy their targets. However, the nature of the molecules involved still is unknown. "Proteins may be released in some kind of specialized secretory process," Henkart speculates. "And there's a suspicion that some complement components might be involved in lymphocyte killing." One thing is sure: No particular proteins need be in the target cell membrane, as any membrane is a potential victim. The size of the hole made by the lymphocyte seems sharply defined, suggesting that it is a true pore rather than a variably sized tatter. Henkart has turned from using lipid bilayers as a target to redblood-cell ghosts. Such ghosts may be loaded with a range of different-sized molecules tagged with either radioactive or dye labels. When lymphocytes poke holes in loaded ghosts, the largest molecule that slips out is a globular protein of about 500,000 molecular weight. That corresponds to a pore with a diameter of 120 to 165 A, Henkart says. By this same method, complement forms a pore of about 50 A that permits passage of molecules of about 40,000 molecular weight. Henkart and Mayer admit that the search for the molecular components of these pores—both agree that the damage
Segal: looking for a trigger
genuinely consists of pores—is frustrating. But says Henkart, understanding of the cell-mediated phenomenon "is just in its infancy." Other similar damage processes exist in nature, and one seemingly parallel example involving components attacking bacterial membranes was described recently by scientists Stanley J. Schein, Bruce L. Kagan, and Alan Finkelstein working at the Marine Biological Laboratory in Woods Hole, Mass. Bacteria, such as Escherichia coli, sometimes harbor genes for factors called colicins that can kill similar bacteria (but usually not the colicin's host cell). Though several colicins interfere with DNA or protein synthesis, others kill cells by poking holes in their membranes, according to the Woods Hole researchers. Colicin K, for example, is a protein that can form an ion-permeable channel in a phospholipid bilayer. The molecule acts alone—that is, a single molecule may form a channel, or pore. Through that channel, potassium and sodium ions can travel. The colicin pore is considerably smaller than either that of the complement or the one formed by killer cells of the immune system.
Complement pores in red blood cell membrane in electron micrograph by Dr. Robert M. Dourmashkin of CRC Clinical Laboratory, Harrow, England Jan. 22, 1979 G&EN
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Henkert: it's not like a bomb
Close examination of membranes as targets and signaling sites for the immune system has entailed building a new technology for measuring physical and chemical properties. One of the biggest challenges is obtaining a reliable measure of diffusion in membranes. Lipids may be either solidlike or fluidlike at 37° C. Their state affects the motion and action of larger molecules, particularly proteins, in the membrane. "In ordinary biochemistry, small molecules diffuse to enzymes in solution," McConnell says. "In membranes, the locations of those molecules may be controlled." Thus the membrane introduces a kind of fudge factor that has been extremely difficult to decipher. Yet, it is
crucial for establishing the sequence, say, of the triggering reactions in an immune-recognition sequence. Several years ago, Dr. Mark S. Bretscher of MRC Laboratory, Cambridge, England, postulated that "continuous and rapid oriented flow of lipid molecules" in membranes could account for the coalescence of larger molecules there. He also postulated "a molecular filter" to exclude membrane proteins while membrane lipids are absorbed into the cell in a recycling process. Bretscher still holds to that idea. "In cells membrane is moving from one place to another," he said in a recent interview. "There's always a problem of diffusion of proteins against a flow of membrane. Diffusion coefficients seem boring, but they're the key to all this." No group has been more dedicated to measuring diffusion coefficients than McConnelPs. Typically, he and his colleagues have used several methods, including paramagnetic resonance of molecules tagged with several distinct spin labels and also laser bleaching of dyetagged molecules. "With several independent methods, we always get the same results," McConnell says. , He and Dr. Barton A. Smith recently have improved one method for following diffusion of large, dye-tagged molecules in membranes. One such change involves passing a laser beam through a grid and then onto a membrane sample mounted in the light microscope. The beam bleaches only the dye-carrying molecules that it reaches, missing membrane regions protected by the grid pattern. After a pulse of light, the bleached and unbleached regions diffuse together. Measured this way, the rate of diffusion of a
Bretscher: diffusion coefficients are key
large molecule is corrected for contributions from lipid flow. Smith and McConnell have turned the grid pattern into a checkerboard. With(it, they have seen "anisotropic motion" of molecules in living mouse fibroblast membranes. "We don't know yet if we are seeing simple diffusion or directed flow," McConnell says. Diffusion, even if it goes more readily along one axis of a cell than along another, is still a kind of random motion. But oriented flow is precisely one of Bretscher's postulates, part of a continuous membrane cycling and filtering system. "We haven't demonstrated flow," McConnell cautions, "but it may be there." D
The mammalian immune system is as complex as grand opera The mammalian immune system is like grand opera, with a cast of versatile performers, each playing a discrete role. In it, the performance is played before, indeed it is about, an unruly audience. For it is the immune system's place to recognize and then to remove members of the audience—termed antigens—that wander onstage where they don't belong. As in opera, the themes of the immune system often are death and destruction. Immunity is the body's main line of defense against invading foreign objects, be they certain toxic molecules, bacteria, viruses, or other cells. To cope with such invaders, the immune system divides its defense into two main acts. The first such act, recognition, involves a class of molecules called antibodies, or more formally, immunoglobulins. There are several classes of immunoglobulins, but all are proteins made up of several (typically, four) polypeptide chains. The chains of immunoglobulins contain regions that show little variation from molecule to molecule, and other
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regions that are hypervariable. The latter regions help form the part of the molecule that binds specifically to a particular antigen. Antigens may be a free molecule or part of a cell. Critically, the immunoglobulin binds to a small portion of the antigen. If that antigen is part of a large entity, the recognized portion may still be recognized and bound when it is separated from the rest of the molecule. This small antigen portion is called a hapten. By themselves, antibodies bind antigens but cannot destroy them. The task of recognizing and binding is, by itself, vast. It requires distinguishing foreign molecules from friendly ones. Antibodies rarely err. The second act of the immune system is to destroy or at least to remove invading antigens. One such scenario consists of actions by a set of soluble proteins in the blood, called complement, that is alerted to an antigen's presence by the antibody molecules bound to it. Though the intermediate steps of the complement cascade involve interesting biochem-
istry, it is the first and last steps, involving recognition and destruction, respectively, of the antibody-tagged antigen that are of critical importance. In the first step, a protease is activated, setting off a series of modifications, culminating in the insertion of a multiprotein poreforming complex into the antigen's cell membrane. The new pores cause the antigen's contents to leak out and thus the antigen is destroyed. Other scenarios for destroying antigens are mediated by cells of the immune system, the real villains in this dramatic onslaught. Some of these cells, such as macrophages, literally engulf the antigens to destroy them. Other cells, including neutrophils and other lymphocytes, act more subtly but are no less deadly. They bind to the stems of antibody molecules that are bound to antigens. Then, for example, lymphocytes cause the invading cells, the antigens, to lyse. This act of destruction probably also involves pore formation in the antigen's membrane. However, little is yet known about the molecular events involved in such pore formation.
