It’s hard to make fun of death, but Monty Python and the Holy Grail meets that challenge. In the film, an undertaker yelling, “Bring out your dead!” pulls a cart full of corpses through a plague-ridden medieval village. A local, toting the body of an old man, beckons the undertaker. The body, however, protests that it doesn’t want to go into the cart. The cart-puller refuses to take the emaciated body, noting that the old man isn’t dead yet. “No, but he will be soon, and he’s just taking up room in the house,” argues the local. “I’ll be better tomorrow,” the old man feebly responds. The younger men finally whack the old guy on the head and toss him on the cart.
Michael Hengartner of Cold Spring Harbor (N.Y.) Laboratory finds that macabre scene hilarious. That’s fitting because he studies how animals recognize their dying cells and dispose of them.
This disposal is a vital job. If a healthy cell doesn’t gobble up a dying cell in time, the latter may simply fall apart, triggering inflammation in surrounding tissue or an immune attack upon the cell’s released contents. Indeed, several recent studies indicate that defective removal of dead cells triggers inflammation and autoimmune diseases such as lupus and diabetes (SN: 5/9/98, p. 293).
There are lots of dead cells to get rid of during an animal’s development and adult life. In a process called apoptosis, many damaged or no-longer-needed cells kill themselves. A human embryo, for example, initially develops duck-style webbing between its toes and fingers, but the cells that constitute the webbing commit suicide before a baby’s birth. In the thymus of a young mouse, about 10 million cells a day undergo apoptosis, scientists estimate. And throughout any animal’s life, once immune cells ward off a dangerous microbe, they typically perform this hara-kiri.
“There’s no question that even in health there’s a massive load of dying cells,” says John Savill of the University of Edinburgh.
Most scientists interested in apoptosis study how or why a cell triggers this process (SN: 11/21/92, p. 344). But Savill, Hengartner, and a growing number of other biologists are more concerned with how organisms get rid of cells undergoing apoptosis. Most animals depend upon immune cells called macrophages to engulf and dismantle apoptotic cells, but in a pinch, other cells can also perform this task. The key issue facing investigators is how these undertakers know whether to consume a cell or leave it alone.
“There’s a fundamental biological problem of how you discriminate between ready-to-die and still healthy,” says Savill.
In the past decade, scientists have identified several macrophage-surface molecules that recognize suicidal cells. Researchers also have found a molecule on dying cells that tells macrophages to engulf them. More recently, Hengartner and other biologists have discovered that macrophages facilitate removal of cell corpses by killing cells on the brink of death, an act that echoes the finale of the Monty Python sketch. Savill and his colleagues have also just reported that healthy cells give off a molecular cry of “I’m not dead yet” in order to rebuff macrophages sniffing around for dying cells.
“The field is developing quite nicely,” says Hengartner.
The guiding hypothesis of scientists studying removal of apoptotic cells has been that macrophages recognize so-called eat-me signals on the surface of a suicidal cell. However, biologists have struggled to find these signals.
“We know very little about the surface of the apoptotic cell,” notes Valerie Fadok of the National Jewish Medical and Research Center in Denver.
About a decade ago, researchers led by Fadok and her collaborator Peter Henson, also of the Denver center, offered the first convincing case that a specific molecule was an eat-me signal. In their original paper, the investigators reported that human immune cells undergoing apoptosis display phosphatidylserine on their surface. This invites macrophages to engulf the suicidal cells. The researchers have since determined that almost every kind of apoptotic cell recognized by macrophages flaunts phosphatidylserine.
A lipid, phosphatidylserine is found in animal-cell membranes. However, it isn’t typically displayed on the membrane surface. In healthy cells, “phosphatidylserine is actively kept on the inside [of the cell membrane],” says Robert Schlegel of Pennsylvania State University in State College.
In cells undergoing apoptosis, however, an enzyme rapidly shifts phosphatidylserine to the outer surface, Schlegel’s team has found. Consequently, he says, apoptotic cells “can immediately get the eat-me signal out there.”
Two years ago, the Denver scientists made another major advance: They discovered a protein on macrophages that can bind to phosphatidylserine. When it does, this phosphatidylserine receptor triggers the immune cell to engulf the signal-bearing cell. The investigators even found that if they added the gene for the receptor to cells that don’t normally interact with apoptotic cells, the genetically engineered cells would recognize and consume the dying cells.
Scientists have suggested a number of other receptors as central to the disposal of cell corpses. For example, macrophages sport a class of proteins called scavenger receptors that enable the cells to bind to and take up a variety of lipids. Biologists have implicated several such receptors in cell-corpse clearance but don’t have a clear idea of their roles. Some of the scavenger receptors can bind to phosphatidylserine, but they also attach to many other molecules.
For other types of receptors on macrophages, scientists don’t yet know the molecular counterpart on the apoptotic cell.
In a model they call “tether and tickle,” Fadok and Henson have suggested that many of these receptors help macrophages initially bind tightly, or tether, to a dying cell. It would then take a tickle–the binding between phosphatidylserine and the receptor discovered by Fadok and Henson–before a macrophage actually engulfs the apoptotic cell.
Studies in animals have identified two other receptors in particular that have attracted considerable interest among investigators. In 1999, Nathalie C. Franc, who is now at University College London, and her colleagues reported the discovery of a fruit fly protein they call croquemort (French slang for undertaker). The investigators found that if the gene encoding croquemort is mutated, a fly’s macrophages don’t ingest dying cells efficiently during the insect’s development. Croquemort resembles scavenger receptors on human macrophages, but there’s no evidence yet that it recognizes phosphatidylserine.
“We don’t know at all what croquemort binds to on the apoptotic cell,” says Kristin White of Massachusetts General Hospital in Charlestown, who worked with Franc.
A similar story has arisen for the worm Caenorhabditis elegans. For many years, H. Robert Horvitz of the Massachusetts Institute of Technology has led a research team working out details of apoptosis in this microscopic worm. The adult C. elegans consists of about a thousand cells, but Horvitz’ team has shown that more than 400 other cells come into being, undergo apoptosis, and are quickly cleared during the worm’s development. Although worms don’t have macrophages, any worm cell can typically engulf a suicidal neighbor.
The scientists have identified a mutant worm strain that does not get rid of many of those dead cells. In a study reported in the Jan. 12, 2001 Cell, Horvitz and his colleagues identified the gene that’s mutated in their cell-corpse-laden worms. The gene encodes a cell-surface protein dubbed CED-1, which resembles the scavenger receptors in people. Whether the protein recognizes phosphatidylserine or another eat-me sign on a worm’s apoptotic cells remains a mystery.
“We’re furiously looking for the signal,” says study coauthor Zheng Zhou of Baylor College in Houston.
Phosphatidylserine is the focal point of another recent discovery concerning cell-corpse removal. In the May 9 Nature, Shigekazu Nagata of Osaka University Medical School in Japan and his colleagues reported that lab-grown mouse macrophages secrete a sugar-laden protein called milk fat globule–EGF-factor 8 (MFG-E8) that binds to cells that are killing themselves. The researchers showed, more specifically, that MFG-E8 binds to phosphatidylserine and that macrophages appear to have receptors that recognize MFG-E8. Nagata’s team found that cells that aren’t macrophages and that lack the phosphatidylserine receptor could nevertheless identify and engulf apoptotic cells if the dying cells are covered with MFG-E8.
Why would macrophages pump out labels for phosphatidylserine when they can already detect the eat-me signal directly through a dedicated receptor? Insurance, says Hengartner. Macrophages may be making it easier for themselves or other healthy cells to spot dying cells.
“This is another clever way macrophages increase their chances of catching apoptotic cells rather than non-apoptotic cells,” says Hengartner.
Grab and stab
When a cell undergoes apoptosis, it typically activates enzymes called caspases that rip apart the cell’s DNA and other molecules. Most scientists had thought that once caspases are released inside a cell, it’s doomed. Yet last year, two research groups reported that cells can sometimes recover after releasing their caspases–as long as another cell hasn’t gobbled them up.
The scientists propose that the engulfing cell is more than an undertaker. It ensures the death of apoptotic cells, they say. Hengartner, who was a member of one of the teams, uses “grab and stab” to describe this active killing role for the engulfing cell.
This new view could have medical importance if physicians learn how to prevent the rapid engulfment of seemingly dying cells. Take stroke, in which brain cells starved of blood start to destroy themselves. If brain cells undergoing apoptosis aren’t immediately eaten, perhaps some could recover, suggests Hengartner.
The aggressive nature of macrophages may explain why some cells have developed the means to repel them. In the July 11 Nature, a research group led by Simon Brown of the University of Edinburgh suggests that healthy cells actively fend off macrophages. In a field dominated by the notion of eat-me signals on dying cells, this is a new concept, says Savill, a coauthor of the report.
In studies of macrophages streaming by cells called leukocytes, the scientists noticed that macrophages briefly grab on to the healthy cells but then release their hold. They, however, hold on to dying leukocytes. The investigators determined that the initial attachment occurs between copies of a cell-surface protein called CD31 that sits on both macrophages and leukocytes.
CD31 was previously known as a molecule that propels leukocytes through layers of cells to the site of an infection, so it’s not surprising that it serves to push macrophages off of healthy leukocytes, says Savill. He refers to CD31 as a Greta Garbo molecule. In one of her films, the Swedish actress uttered, “I want to be alone,” an apt line for the famously reclusive star.
In dying cells, however, CD31 sheds its Greta Garbo impersonation to become one of the proteins onto which macrophages tether. To explain this dramatic switch, Savill and his colleagues are studying what changes in CD31 occur when a cell begins to die.
The party’s not over
While the study of how organisms dispose of their dying cells is a young field, researchers have begun to find evidence that their work could have medical payoffs. There’s even a small biotech firm in Royston, England, called Apocyte, that seeks to develop therapies to speed the removal of apoptotic cells in some situations and slow the process in others.
Fadok notes that the proper disposal of a dying cell is vital to regulating inflammation. First, if a macrophage consumes a dying cell before it bursts, it can prevent inflammation from ever starting. Moreover, when macrophages swallow up cell bodies, they secrete chemical signals that suppress other immune cells involved in inflammation.
The chronic inflammation that afflicts the mucus-filled lungs of a person with cystic fibrosis may stem from macrophages doing a poor job of getting rid of dead cells. In the March Journal of Clinical Investigation, Fadok and her colleagues reported that the sputum of people with cystic fibrosis contains abnormally large numbers of apoptotic cells. Macrophages may be unable to see these cells because a protein-cleaving enzyme abundant in the lungs of the people with cystic fibrosis destroys the macrophage receptor for the eat-me signal phosphatidylserine.
Such findings raise the notion that phosphatidylserine or cells bearing it could prove useful in curbing inflammation. In the January Journal of Clinical Investigation, Fadok, Henson, and Mai-Lan N. Huynh of University of Colorado Health Sciences Center in Denver detailed the first test of that theory. They deliberately provoked inflammation within the lungs of mice and subsequently injected apoptotic cells into those lungs. The inflammation cleared up more quickly than it did in untreated mice or in mice receiving dying cells that don’t make phosphatidylserine.
“We’re high on phosphatidylserine as an anti-inflammatory signal,” says Fadok. “It suggests some novel drug targets to control inflammation that haven’t been looked at yet.”
With results such as Fadok’s, biologists investigating the disposal of dying cells are certain to gain more attention from traditional apoptosis researchers. Until recently, says Hengartner, most biologists considered his field boring, the equivalent of studying the workers who clean up Times Square the morning after the New Year’s Eve party. Perhaps now his colleagues will begin to appreciate how important it is to pick up the garbage.
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