A strangely silent allele

Ralph Kettritz's group discovers a unique mechanism by which white blood cells silence a gene involved in autoimmune diseases.

If you think of the immune system in terms of war, then white blood cells called neutrophils are the body's infantry. They fall in such massive numbers that every day, 100 billion fresh recruits are called up from reserves of stem cells in the bone marrow and pressed into service. After rapid training and specialization, the cells are released into the bloodstream to scout for invaders. Upon catching the scent of a virus or bacteria, a neutrophil slips through the lining of the blood vessel, tracks down its prey, and engulfs it. Successful or not, the neutrophil will be destroyed within hours or days. Otherwise it may become a mercenary and attack the body in an autoimmune disease.

For many years Ralph Kettritz has pursued one of these conditions with his lab at the Experimental and Clinical Research Center, a cooperation between the MDC and Charité on the Buch campus. ANCA-associated systemic small-vessel vasculitis begins when neutrophils produce a protein called PR3. In contrast to healthy individuals, patients develop autoantibodies called ANCA against this molecule, and these ANCA activate the white blood cells inappropriately while still inside blood vessels. This damages the vessel walls.

"A protein called CD177 plays an important role in this process," Ralph Kettritz says. "CD177 is produced by neutrophils, which are the only cells in the body that bear CD177 on their surfaces. CD177 causes PR3 to be exposed in large amounts on the cell surface, where it is visible to the ANCA autoantibodies."

So understanding the causes of systemic small-vessel vasculitis, he says, requires explaining the process by which a neutrophil activates the CD177 gene and uses it to produce messenger RNA and the protein. Gene activation and silencing are so crucial to the functions of cells and tissues that they have been a central theme of biology for many decades. Every part of this system that has been discovered so far has turned out to play a role in health and disease. So finding a new type of control – as Ralph Kettritz and his colleagues have done – is likely to have many implications. Their work appears in The Journal of Experimental Medicine.

A choice between parental alleles

Recently Ralph Kettritz's lab teamed up with the group of Fred Luft to figure out what governed the production of CD177 in neutrophils. They began by drawing blood from a group of 167 healthy people, isolating the neutrophils, and testing them for the presence of the CD177 protein. Not suspecting that the project was about to take a strange turn.

An unusual pattern emerged from the neutrophils of the healthy subjects. In a small proportion of the group, about five percent, neutrophils didn't produce any CD177 at all. In the rest of the test subjects, the scientists found cells bearing the protein. But not every neutrophil did so; they also had cells that lacked it. In other words, they had two types of cells. The ratio varied widely from individual to individual: in some subjects, nearly all the neutrophils produced CD177, leaving only a small proportion of cells without, while in a few cases it was the other way around. Unusually, while the ratios differed between individuals, in a single person it seemed to be fixed for life.

"Our group found earlier that patients with small-vessel ANCA-vasculitis had a higher percentage of neutrophils that presented PR3 on the surface compared to controls and the higher this percentage the worse the clinical outcome," Ralph Kettritz says. "It turned out that these cells were identical to the neutrophils that express CD177, which means that understanding the systems that control the production of this molecule have a high clinical relevance."

Obviously the person's neutrophils could produce the protein, but some of the cells didn't – why? A closer study of the cells revealed something even more unusual.

A neutrophil granulocyte with  CD177 (green) and ANCA Antigen Proeinase 3 (red) on the cell surface. Image: Ralph Kettritz, MDC

Each of the subjects' neutrophils – and the rest of their cells as well – carried two copies of the CD177 gene, one inherited from each parent. In most cases if a gene is active in a given cell, then both copies – called alleles – are used to generate RNAs and proteins. Shutting down one copy typically silences the second allele as well. But Ralph Kettritz and Claudia Eulenberg discovered that in any given person, only one allele of CD177 is ever used, and it's always the same one. The other allele is permanently silenced.

This wasn't the first time scientists had observed the lifelong shut-down of one allele. It also occurs during imprinting, a process that limits the number of RNAs and proteins that are produced from certain key genes. This affects a few hundred genes whose output needs to be restricted. It can also have another effect: one copy of the gene may have a defect that can cause disease, and the other may be healthy. In this case, the selection of an allele plays a crucial role in health.

But imprinted genes adhere to a strict pattern that holds true for all of a family's children: either the father's allele is silenced or the mother's; whichever is the case, all the offspring inactivate the same allele.

With CD177 that wasn't the case. When the scientists examined families, they found that one child's neutrophils silenced the paternal allele of CD177, while a sibling silenced the maternal gene. Once the child's body produced its first neutrophil, every cell that followed made the same choice.

A gene and its neighborhood

There are other patterns by which cells are known to silence genes, but these lead to a random patchwork in which one allele is silenced in some cells, yet is expressed in others.

Gene silencing is based on epigenetic mechanisms that usually involve the way the enormous DNA molecule is packed into the tiny cell nucleus. The packing makes some sequences accessible to other molecules, while others are hidden and inaccessible. Generally a strand of DNA has to be "open" so that a machine of molecules can bind to it and transcribe it into RNA. If it is closed, the molecules that make up the transcription machinery are unable to bind or don't have enough room to operate.

This packing happens in stereotypical ways in each type of cell, leaving some sequences open and others closed. The pattern changes over time and alters the molecules a cell produces, powerfully influencing its features, specialization and activity. If epigenetic mechanisms were responsible for silencing genes in neutrophils, the best way to observe them would be to watch how the cells developed from the hematopoeitic stem cells (HSCs) that give rise to neutrophils and other blood cells.

A human's first neutrophils arise around the time of birth. The scientists obtained HSCs and neutrophils harvested from the umbilical cord blood of a newborn. This provided an opportunity to determine exactly when and how neutrophils silence one of the alleles for CD177. The researchers extracted stem cells from the blood and grew them in laboratory cultures. They found that before HSCs specialize, they actively transcribed both alleles. But by the time differentiation produced the first neutrophils, one of the alleles had been shut down. The same selection was made by cells in culture and those in the newborn child.

The scientists were able to trace this effect to a particular chemical process by which cells close off regions of their DNA. Enzymes in the cell nucleus attach small chemical tags called methyl groups to DNA sequences and to proteins associated with nearby genes. This usually pulls the DNA into a tightly bound package and blocks sites that need to be accessible for the RNA transcription machinery.

Specific molecules called transcription factors need to gain access to sequences near the CD177 gene to help build the machine that produces its RNA. One of the neutrophil's alleles never permitted the machine to assemble, because the binding sites for two transcription factors called c-Jun and c-Fos were blocked by methylation. Within some cells, the epigenetic mechanism was more active, causing access to both alleles to be blocked. This explained why some neutrophils were unable to produce either RNA or proteins from the CD177 gene. The result is two distinct populations of neutrophils.

While CD177 is the first neutrophil molecule known to behave this way, Ralph Kettritz says there may be others. "It's often difficult to distinguish between the RNAs and proteins produced by paternal and maternal alleles, so other cases may have gone unnoticed so far," he says. "There may be many more. For now, our discovery of this unusual form of gene silencing clears up a number of questions related to neutrophil-mediated autoimmune diseases."

Further Information

Claudia Eulenberg-Gustavus1, Sylvia Bähring1, Philipp G. Maass2,3, Friedrich C. Luft1, and Ralph Kettritz1,4: “Gene silencing and a novel monoallelic expression pattern in distinct CD177 neutrophil subsets.” The Journal of Experimental Medicine. doi:10.1084/jem.20161093

1Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max-Delbrück Center for Molecular Medicine at the Charité and 2Max-Delbrück-Center for Molecular Medicine, Berlin, Germany, 3Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA, 4Nephrology and Intensive Care Medicine, Campus Virchow, Medical Faculty of the Charité, Berlin, Germany

Featured image: Neutrophil white blood cells (leukocytes) by Dr Graham Beards. Licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.