stanford medicine


Yin-Yang Factor

The yin-yang factor

It could wreck your immune system. Or save your life

Most mystery stories start with a crime. This one starts with a suspect.

This suspect is a loiterer, a biological leftover that could linger in all of us for decades, doing — well, nobody knows what it’s doing. Some think it’s up to no good, that it instigates cellular riots. Others wonder if it isn’t cleaning up the damage done by the real criminals. Or perhaps it does both, like a neighborhood bully with a heart of gold.

The story begins with blood.

Something was wrong. Diana Bianchi, MD, had spent much of the mid-1990s gathering blood samples from pregnant women, hoping to develop a safe prenatal test for Down syndrome. Now, the data were coming together in computer files and on spreadsheets, which revealed an unexpected pattern.

“We kept finding evidence of male cells in women who were carrying female fetuses,” Bianchi says.

Bianchi, a reproductive geneticist at the Tufts University School of Medicine in Boston, had started this line of research years prior, in the Stanford laboratory of Leonard Herzenberg, PhD, emeritus professor of genetics, who himself has a son with Down syndrome. Upon graduation, she’d traded Stanford’s low-slung skyline for the high-rises of downtown Boston, where she hoped to develop safe methods of diagnosing fetal genetic defects.

But now, a cluster of Y chromosomes had thrown her off track. She quickly followed the paper trail on these deviant bits of DNA, pulling the medical records of her study participants in search of reproductive histories. Sure enough, every last one of them had previously terminated a pregnancy — or had given birth to a son. The boys were years from the womb, and yet something remained, perhaps for a lifetime: Further digging turned up a woman with her 27-year-old son’s cells still in her blood.

“Pregnancy may thus establish a long-term, low-grade chimeric state in the human female,” Bianchi wrote in 1996, breaking the news in the Proceedings of the National Academy of Sciences. In mythology, the chimera is a fire-breathing composite of three animals: goat, lion and snake. In medicine, the term refers to a condition of separate genomes existing in one organism. This could be the result of genetic engineering, or simply because of an organ transplant or blood transfusion. In this case, the semi-permeable placenta facilitated the swapping of fetal and maternal cells. It wasn’t only male cells that seemed to stick around; cells from female fetuses could result in chimerism too, though they were harder to spot without the stand-out Y chromosome.

As these fetal cells were one-in-a-million outliers among masses of maternal cells, Bianchi chose a name to reflect that rare nature: microchimerism. The discovery was bad news for her Down syndrome screens, as she could no longer be sure that circulating cells were from current pregnancies. And now Bianchi had a new problem to solve: Why were these fetal cells hanging around for so long?

While Bianchi was turning up new questions, immunologist J. Lee Nelson of the Fred Hutchinson Cancer Research Center at the University of Washington in Seattle was chasing a very old one. Why, she wondered, are women more vulnerable to autoimmune diseases than men?

Doctors had known for years that these diseases — there are more than 80 — are gender-biased. Between 14 million and 22 million Americans have an autoimmune disease. More than three-quarters of those are women. The disorders occur when the immune system turns against the body, attacking organs and tissue. The consequences can be mild, or they can leave their victims disfigured, fatigued, in chronic pain, even dead.

Nelson, a Stanford graduate with an MD from UC-Davis, began her career at a time when most research into the cause of autoimmune disease focused on hormones. Perhaps, researchers thought, the gender divide was the fault of the primary female sex hormone, estrogen. Some studies suggested that estrogen weakens the body’s suppressor cells, which keep the immune system in check. Researchers also thought that certain diseases, such as lupus, get worse in the presence of estrogen — though for lupus, at least, new evidence throws the hormone’s guilt into question.

Women could have bits of their mothers still inside them as their own unborn children shed a completely new set of cells into their bloodstream. The cells of one child, circulating in the mother’s blood, might become a lifelong part of younger siblings.

Nelson was already skeptical. She knew that some autoimmune diseases, lupus included, hit mostly during the childbearing years, when estrogen production peaks. But other disorders strike later, even after menopause starts, when estrogen declines. If the disorders didn’t correlate nicely with hormone levels, Nelson reasoned, how could a hormonal culprit tie all of the cases together?

So she began to follow her own leads. She knew from her rheumatology experience that pregnancy often ameliorates the symptoms of such diseases as rheumatoid arthritis. Some researchers credited hormonal fluctuations, but attempts to change the disease course with oral contraceptives and other hormone replacements hadn’t proven fruitful. To Nelson, that meant something else was going on during pregnancy — something immunological.

“It’s not that hormones aren’t important,” Nelson says. “But before I started getting up and trying to ring the bell for immunology, people didn’t think about it or talk about it or look into it.”

The April evening was clear and dry, a welcome respite from Seattle’s trademark rain. Nelson was at home relaxing when the phone rang.

On the other end was Nelson’s friend, a transplant researcher from the Hutchinson Center. He was “blown away” by something, Nelson recalls, and he knew she would be too. A friend of his, the head of a lab at CellPro, a local biotech company, was doing a little informal experimentation, the researcher explained. A technician in the lab had a baby, and knowing that fetal cells can cross the placenta, the lab head wondered how long after delivery it would be until those cells disappeared.

He decided to find out, and the technician agreed to periodic blood tests. The baby had just had his first birthday. His cells were still thriving in his mother’s veins.

It was the first time Nelson had heard anything like it. If true, she realized, the discovery could mean that we aren’t as genetically homogenous as once thought. Maternal cells could cross the placenta and lodge in babies for the long term. Women could have bits of their mothers still inside them as their own unborn children shed a completely new set of cells into their bloodstream. The cells of one child, circulating in the mother’s blood, might become a lifelong part of younger siblings.

Nelson clicked the receiver back into its cradle, and her mind turned to a recently deceased patient of hers. This patient had Klinefelter’s, a genetic disorder in which a man has an extra X chromosome. He also had scleroderma, an autoimmune disease that turns the skin and organs hard and swollen. Nelson had been fond of him, but his case was severe. He died quickly.

The man’s disease, with its inflammation, skin rash and organ damage, looked a lot like another condition Nelson knew well, graft-versus-host, which sometimes occurs after bone-marrow transplants. Doctors try to match donors’ and recipients’ human leukocyte antigens, or HLAs, the proteins that help the immune system distinguish self from non-self. If HLA profiles aren’t close enough, the new, “grafted” bone marrow can actually reject the recipient, causing rashes, jaundice, joint inflammation and sometimes death.

With her mind on her scleroderma patient, graft-versus-host and persistent fetal cells, Nelson felt the puzzle pieces falling into place. What if the HLA profile of those lingering cells didn’t match the mother’s? What if microchimerism created complex mixtures of several different people’s cells inside one individual? Would it be enough to send the immune system on a self-destructive spree years after the pregnancy?

“It wasn’t a deliberate thought. A light bulb went off,” Nelson says. “I went, whoa, maybe these lingering fetal cells, a year or two years later — maybe that’s what’s happening in scleroderma.”

The idea was so exciting that Nelson couldn’t sit still. She laced her shoes and charged up a long staircase built into a bluff next to her house. Up and down, up and down, until she realized she was going to be sore the next day.

Within a month, Nelson was hammering out a plan. She started collecting blood from scleroderma patients with sons — the telltale Y chromosomes in fetal cells would provide a convenient marker in a sea of maternal Xs. She met colleagues for coffee and shared her theory. She got the name of the lab head at CellPro, Jeff Hall, PhD, and contacted him. They did some preliminary tests on lung samples, working out the kinks of how to identify evidence for male cells in women who had given birth to sons.

But Nelson and Hall weren’t the only ones on the trail of microchimerism. In Boston, Bianchi had her own evidence and was working on the study that would definitively show fetal cells persisting in maternal blood for decades.

As soon as Nelson heard of Bianchi’s work, she knew they needed to collaborate. It was “natural,” she says, for her to pair with a fellow academic like Bianchi.

“I called up information,” Nelson says. “I got her telephone number and I called her up. And I said, ‘I think these cells that persist might have something to do with autoimmune disease.’”

Bianchi and Nelson wasted no time in setting up a cross-continental collaboration. Nelson drew blood from her scleroderma patients in Seattle and shipped the samples overnight to Massachusetts, where Bianchi’s team analyzed them for fetal DNA. Of the 40 participants, 17 had scleroderma, 17 were healthy and another seven were healthy women who had sisters with scleroderma. All had given birth to sons.

The first blood tested was that of a close friend and patient of Nelson’s, a Native American woman with a limited form of scleroderma who had given birth to a son. (Members of the Choctaw tribe, as well as African-American females, have a higher-than-average risk of the disease.) Bianchi and Nelson masked their samples, but the results were obvious almost from the beginning: Patients with scleroderma, including Nelson’s friend, had 20 times the number of fetal cells in their bloodstream as their healthy counterparts. Some had more fetal cells than the average pregnant woman. Women with scleroderma also seemed more likely to have at least one child with a very similar HLA profile to their own. That suggested fetal cells that match maternal cells too closely might confuse the maternal immune system, turning it against its own tissue.

The work triggered a flurry of other studies that also showed increased microchimerism in other autoimmune disorders, including multiple sclerosis, rheumatoid arthritis and Grave’s disease. But the picture became more complex as well.

In the microscope room of Bianchi’s lab, a postdoctoral fellow waved the researcher over. She bent over his microscope, peering at a cross-section of cells. Chills ran down her back.

She was looking at a sliver of thyroid removed from a 48-year-old mother of two. In the light of the microscope, the cells glowed, but Bianchi expected that. Her lab technicians had tagged the tissue with fluorescent markers that lit up the sex chromosomes lurking in each nucleus: red for X, green for Y.

This woman’s thyroid should have been solidly red, the colors signaling the female XX pattern inside. Even a few scattered green dots wouldn’t have shocked Bianchi; she was looking for evidence of microchimerism in the organs of people with autoimmune thyroid disease, a study that had grown out of her collaboration with Nelson.

But the green-spotted cells under the microscope weren’t like the circulating fetal cells Bianchi had seen before. They were fully differentiated thyroid cells, a miniature island of male tissue working in harmony with the larger female whole.

“That was one of those goose bumps moments,” says Bianchi. “You knew that you were seeing something important for the first time.”

The thyroid was the first evidence that fetal cells might be doing something good — not triggering the immune system, not sitting idly by, but moving in to repair damaged tissue. Also interesting was that the thyroid’s owner was a control subject in the study. Her thyroid had been partially removed because of a benign tumor. She didn’t even have an autoimmune disorder.

Other research began to trickle in. Higher levels of microchimerism were found in the cervixes of women with cervical cancer compared with healthy women, though it was impossible to say whether the cells caused the cancer or were proliferating in an attempt to repair the cancer’s damage. In women with breast cancer, on the other hand, microchimerism seemed protective, with fewer fetal cells in more severe cases. Other researchers found that women with pre-eclampsia, which causes high blood pressure in late pregnancy and can kill both mother and child, had five times the microchimerism of healthy women. Fetal cells, it turned out, are complex characters.

Similarly, the autoimmune case file remains a jumble of sometimes-provocative, sometimes-conflicting evidence. Bianchi’s recent mouse research has indicated that undifferentiated fetal cells do travel to wounded organs and specialize, dividing and morphing into the building blocks of repair. Other studies continue to finger fetal cells as autoimmune instigators. In 2001, for example, researchers injected mice with a compound known to trigger scleroderma and found that mice that had given birth had a 48-fold increase in microchimerism and experienced sclerodermalike symptoms. Virgin mice injected with the same compound stayed scleroderma-free, prompting the researchers to propose that microchimerism is a necessary link in the chain of the disease.

“It’s a fascinating hypothesis with some very good, tantalizing data,” Robert Carter, MD, the deputy director of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, says of the autoimmune-microchimerism link. But, he cautions, “we’ve yet to find proof that microchimerism contributes to disease.”

Whether helpful or harmful, a deeper understanding of microchimerism could lead to treatments for autoimmune or other disorders. If the fetal cells are causing trouble, Nelson says, researchers could look for ways to inhibit or remove them. If they turn out to play an important role in organ repair, however, stimulating the cells to multiply could prove beneficial for some patients.

we exist over a lifetime. as we age and are exposed to more and more environmental triggers, cells that once were beneficial could become a liability, providing a bridge for the immune system to cross over from attacking foreign to non-foreign tissue.

Still, persistent fetal cells are just one of many suspects under investigation for instigating autoimmune attack. Hormones are still on the list, but researchers now see them as part of a bigger gang. The Epstein-Barr virus and the bacterium Helicobacter pylori have been linked to lupus and rheumatoid arthritis. Some thyroid and liver autoimmune disorders may result from a phenomenon known as skewed X chromosome inactivation. Most women, in need of only one chromosome’s worth of genes, randomly inactivate one X chromosome. In women with some autoimmune diseases, however, those patterns are weighted more heavily toward one X or the other, instead of a roughly equal inactivation of each. These patterns, researchers theorize, could lead immune cells to attack self cells. Finally, phase-3 clinical trials of the drug Benlysta suggest that an out-of-control immune-stimulating protein could worsen lupus. If Benlysta, which inhibits the protein, makes it through final trials, it could be the first new drug approved for lupus treatment in 50 years.

“This is a really tough issue,” says Eliza Chakravarty, MD, a Stanford professor of immunology and rheumatology, who says that microchimerism is “one potential component” of autoimmune disease. “The planets have to align in a certain way,” she says.

As those planets click into line for tens of thousands of new autoimmune patients each year, Nelson and Bianchi often act as the yin and yang of microchimerism, Bianchi on the East Coast finding rays of regenerative possibility and hope, Nelson out West pointing out potential harms. Yet both are quick to say that these positions are not contradictory.

“We do not exist over a single point in time,” Nelson says. “We exist over a lifetime.” As we age and are exposed to more and more environmental triggers, she says, cells that once were beneficial could become a liability, providing a bridge for the immune system to cross over from attacking foreign to non-foreign tissue. Other triggers are no doubt involved, Nelson says, but microchimerism may affect whether an initial immune hiccup becomes chronic. Bianchi and her team have found that in mice, fetal cells in the mother are comprised of different cell types. They speculate that undifferentiated fetal cells may have regenerative properties, while more differentiated cells may provoke an autoimmune attack.

A new journal, Chimerism, is due out in early 2010, with Nelson as editor and Bianchi on the editorial board. Nelson hopes the journal will pull multiple fields of research together and push discovery forward. The field is new, but technology is advancing, she says. And it’s no longer an uphill battle to get scientists to look beyond hormones at the other possible culprits orbiting just out of reach.

How microchimerism will fit into the final constellation of autoimmune causes remains to be seen, but Nelson has only to look at her autoimmune patients as a reminder of the importance of her work. A few stick out in her memory, like that first Native American scleroderma patient who donated blood for the original microchimerism tests. Last Nelson heard, the woman had developed pulmonary hypertension, a dangerous constriction of the blood vessels that feed the lungs. Such complications are to blame for more than half of scleroderma-related deaths. A few years ago, patients with the condition fared even worse.

“They were usually dead within a year,” Nelson says. As to her patient’s fate, Nelson doesn’t know. The woman disappeared from Nelson’s life, leaving only a disconnected phone number and, perhaps, fragments of herself in the bodies of her children.


Stephanie Pappas is at


Autoimmune lineupWhat’s possibly at play

Microchimerism is just one of many suspects in the search for autoimmune disease culprits. Here are some others:

Female hormones: Animal studies have suggested that estrogen worsens autoimmune disease. But women with some disorders find that pregnancy — a time of high estrogen production — brings temporary relief. Now, researchers think typical levels of estrogen stimulate immune response while sky-high levels suppress it.

Male hormones: These chemicals seem to alleviate lupus in both mice and humans, but no one knows exactly why. “We never really did and still don’t today understand the hormonal effects on lupus,” says Stanford professor of pathology and of medicine Edgar Engleman, MD.

X-inactivation: Humans need only one chromosome’s worth of genes per cell, but in most cases they have two chromosomes. So women, who have two X chromosomes (men have just one), inactivate half their X genes at random. Recent research has suggested that women whose patterns of inactivation are weighted heavily toward one chromosome or the other are more likely to have autoimmune diseases of the liver and thyroid.

The environment: Viruses, bacteria, everyday or industrial chemicals — we’re exposed to them all, and some may be sending our immune systems into a self-destructive spiral.






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