Friday, February 23, 2007

Cytokine storms

Question: What do the following life-threatening medical problems have in common: avian flu, SARS, and anthrax? If you guessed "cytokine storms", you're correct. What was your clue?

In view of the medical problems in which cytokine storms have been implicated, the topic is obviously of high importance. However, because they represent a malfunction in the immune system, which is quite complex, cytokine storms are presently not at all well understood, so it's hard to make definitive statements about them. Indeed, cytokine storms are more of a symptomatic condition and could occur in varying forms, involving a number of different mechanisms. "Storm" may be an appropriate metaphor, acknowledging a variety of mechanisms in a variety of circumstances. What different examples have in common is certain components of the immune system becoming seriously out of control and causing life-threatening problems.

As background for this discussion, you might want to read (or reread) our previous article on T cells. One of the key players in this drama is the subtype of T cell known as helper T cells (also known synonymously as Th or CD4+ cells). As the name implies, Th cells assist other types of immune system cells in performing their function, so a number of other cell types may also be involved in a cytokine storm.

Immune system cells (as well as various other cell types) communicate among each other with chemical messages known as cytokines, which are proteins or peptides (small proteins). The list of known cytokines is large and continually growing. To make matters worse, the nomenclature is not well-standardized and consistent, but some examples you may have come across (if you ever read medical literature) are various kinds of interleukins, interferons, and tumor necrosis factor (TNF).

Although cytokine storms are not well understood, here's a general overview of what happens. Th cells appear to play a central role. The reason they are called "helper" cells is that they produce cytokines which in turn affect the behavior of other types of immune system cells. Which cytokines can be produced, and when, are quite variable, depending on circumstances. Likewise, which other immune system cells are affected, and in what ways, depends on the circumstances and the cytokines in the immediate vicinity. All these variables is what accounts for the complexity of the process.

One possible effect of a cytokine is to cause proliferation of a particular type of cell. Some types of cells can even generate cytokines that cause proliferation of the same type of cell. A cytokine that does this is called an autocrine. Th cells provide an important example. Immediately after a Th cell becomes activated (upon enountering an antigen), the cell secretes interleukin-2 (IL-2), which acts on the Th cell to make it (and its progeny) divide rapidly. Clearly, such a positive feedback loop has the potential to start a runaway chain reaction, so there are also mechanisms that eventually slow it down or stop it – if everything goes as it should.

Of course, Th cells also affect other cell types, such as B cells – the sort of immune system cell that produces antibodies. Cytokines from Th cells activate B cells and cause them to proliferate and begin to produce their antibodies. Other cytokines from Th cells may cause a different type of T cell – cytotoxic T cells (Tc for short) – to be activated and proliferate. At the name implies, Tc cells produce toxins that kill body cells (if they've been infected by a virus associated with an antigen that activates the Tc cell).

Other cytokines can activate additional immune system cell types, such as macrophages and neutrophils. All this activity can lead to inflammation, which may cause a variety of problems of its own.

Clearly, the immune system can do a lot of damage if it slips out of control. It's surprising problems don't crop up much more often than they actually do. In some of the research to be mentioned below, a few of the mechanisms which keep things in check will be noted. It may be possible to harness some of these as therapies to treat conditions where cytokine storms play a part – such as avian flu.

In some of the news articles to be discussed below we'll learn about a few specific examples of known cytokine storms. Here are some additional general links dealing with cytokines and cytokine storms:


Avian flu and Spanish (1918) flu



By now, is there anyone who isn't aware of avian flu, caused by the H5N1 influenza virus? You have probably also heard or read about another type of influenza virus (H1N1), which was responsible for the 1918 Spanish flu that may have killed as many as 50 million people. (An influenza virus is said to be of type HxNy depending on the variants present of two virus coat proteins, haemagglutinin (H) and neuraminidase (N).) In 2004 it was discovered that a normal flu virus modified to look like the 1918 virus could cause similar severe symptoms in mice, by inducing a cytokine storm. Background: here, here, here.

Just a year later, in 2005, the entire 1918 virus was recreated in the laboratory. It proved to be, as expected, quite lethal to mice. Background: here, here, here, here, here, here, here, here, here.

But the important question remained: Why was the virus so lethal? In September 2006 reports of the research appeared that gave a partial result:

1918 flu virus's secrets revealed
Dr John Kash, lead author of the study and assistant professor of microbiology at the University of Washington, said: "What we think is happening is that the host's inflammatory response is being highly activated by the virus, and that response is making the virus much more damaging to the host.

"The host's immune system may be overreacting and killing off too many cells, and that may be a key contributor to what makes this virus more pathogenic."

Dr Christopher Basler, a co-author from Mount Sinai School of Medicine, New York, said: "Our next step is to repeat these experiments, but deconstruct what the immune system is doing so that we can understand why it is reacting so strongly, yet failing to fight the infection."

More: here, here, here, here.

But notice that this report is still a little vague. It talks about a strong "inflammatory response" and how the immune system is "overreacting". As we know, the immune system is quite complex and has a large repertoire of responses for combating an infection. Is it possible to be more specific about what the system seems to be doing here? And does a similar effect occur not only in mice, but also in humans, or at least primates similar to humans?

Answers to these questions appeared in January of this year, with details of experiments performed on macaque monkeys.

1918 Killer Flu Tested on Monkeys
The macaque experiment was supposed to last 21 days, but after eight days the monkeys were so sick – feverish, in pain, and struggling to breathe – that ethical guidelines forced the researchers to euthanize them.

"There was some surprise that it was that nasty," University of Washington virologist and study co-author Michael Katze said. "It was the robustness of the immune system that helped victimize them."

The virus is very good at replicating itself, said Peter Palese, chairman of the microbiology department at Mount Sinai School of Medicine in New York. Its effect on the immune system "triggers what one refers to as a cytokine storm," he said. Cytokines transmit messages among cells in the immune system. Palese wasn't part of the study but has worked on the resurrected virus before.

No other flu virus is deadly to monkeys, and the speed in its spread and the overwhelming immune system response is similar to those in the H5N1 bird flu, Kawaoka said.

So in fact, what this 1918 H1N1 virus appears to be doing is raising a cytokine storm that winds up destroying a lot of the victim's lungs. And that seems to be the same thing that happens with humans who have contracted the H5N1 avian flu virus. Nasty stuff.

More: here, here, here, here, here.

Obviously, there is an urgency to discovering countermeasures to lethal influenza viruses like H1N1 and H5N1. One biotech company has already announced testing of a drug, in rodents, which may be able to control excessive immune system reactions caused by flu virus infection. The account mentions one particular cytokine, IL-6 (interleukin-6), which has been associated with other immune system disorders, and which the experimental drug appears able to control.

ImmuneRegen's Viprovex Demonstrates Immune Response Potential In Treatment Of Avian Influenza And Spanish Flu
Cytokine storm occurs when an infected individual's immune system remains activated against the virus beyond the point of being helpful to where the immune response turns deadly. Persistent, highly elevated levels of pro- and anti-inflammatory cytokines induce a complex, dysregulated condition resulting in massive pulmonary inflammation and fluid accumulation, vascular dysfunction and eventually shock and death. Thus, in cytokine storm, the body's immune system fights to rid itself of the virus, but somehow escapes from the normal controls that prevent an overzealous immune system from killing its owner.

As noted in the Nature publication, there are other disease conditions in which a hyperactive immune system is involved, and other drugs under development for treating those conditions might be beneficial in treating a pandemic influenza infection that could trigger cytokine storm. Specifically mentioned as central to regulation of the immune system, inflammation and hematopoiesis is the cytokine interleukin-6 (IL-6).

Normal production and release of IL-6 is integral to functioning immune and hematopoietic systems, activating lymphocytes and increasing B cell antibody production, but its generation has also been implicated in a number of other diseases, such as rheumatoid arthritis, multiple sclerosis, Alzheimer's Disease and AIDS dementia.


That's encouraging. But it's necessary to remember that studies of the efficacy and safety of new drugs in humans normally take the better part of a decade to perform. Even if this particular drug, or others like it, works in humans, we're hardly out of the woods yet as far as avian flu is concerned. In particular, the human immune system isn't necessarily all that similar to another mammal's, as the next section will demonstrate.

Additional references:


TGN1412 drug trial



Undoubtedly you recall the story about a rather disasterous initial human trial of a new drug that, ironically, was intended to to treat rheumatoid arthritis and other autoimmune disorders. The trial took place in March 2006, and a report on what seems to have happened came out in August:

Mystery over drug trial debacle deepens
Doctors who saved the lives of six men who nearly died in a UK drug safety trial in March have revealed full clinical details of what happened during the first 30 days.

However, far from explaining how the drug caused multiple organ failure in all six men, the results have simply added to confusion over how the drug affected their bodies.

The healthy volunteers were given an experimental antibody drug called TGN1412 in its first human trial to test for safety on 13 March 2006 in London. Within the hour the six men injected with the drug were reportedly writhing in pain. Two others who were given a placebo were unaffected.

The biggest mystery is why the men’s white blood cells – called lymphocytes and monocytes – vanished completely just hours after the drug was injected. This is the opposite of the effect observed in animal trials.

Far from having a calming effect on the immune system, as intended and as was observed in animal tests, TGN1412 seems to have provoked a cytokine storm:
All [experimental subjects] initially suffered from a so-called “cytokine storm” – a flood of inflammation-triggering chemicals pumped into the blood by activated white blood cells. This storm is what eventually led to multiple organ failure, the report says.

The experimental subjects received a dose 500 times smaller than used in animals. But that wasn't enough of a safety margin.
The injected antibody was unusual, because it was capable on its own of provoking lymphocytes, called T-cells, into becoming as active as they would be if they had to fight an infection. It normally takes two signals, not just the one provided by the antibody, to awaken T-cells. The antibody – known as a superagonist – was designed to be able to activate any type of T-cell without requiring the usual secondary signal. It works by binding to a receptor called CD28 on the T-cell surface.

In earlier trials on animals, the antibody initially triggered multiplication of T-cells, but a specific subset, called regulatory T-cells, ended up multiplying fastest and taking control. The regulatory T-cells calmed the immune system. TeGenero hoped that this immune “calming” process offered potential therapeutic benefit, perhaps easing symptoms of diseases like rheumatoid arthritis, where normal T-cells attack the body’s own tissues.

Unfortunately, the reverse happened. Around 60 to 90 minutes after the men received their injections, their bodies were flooded by a surge of inflammatory chemicals called cytokines, which combat severe infections like those seen in patients with blood poisoning. The cytokines caused severe inflammation.

Note, in particular, that the antibody was expected to activate a special type of T-cells, regulatory T cells (Treg). Until just a few years ago, immunologists weren't even sure Treg cells existed. Fortunately, they do, because their purpose in life is to suppress immune system activation, to prevent activity from getting out of control. What is "supposed" to happen is that Treg cells provide a needed negative feedback to the system, to counteract the positive feedback loop driven by other types of T cells. Treg cells are being actively studied at present, and we'll write more about them in another article. Unfortunately, in the TGN1412 trial, things didn't go as expected.

In December, another report came out that offered some hypotheses about why things went awry:

Horror clinical trial in test tube recreation
The new results presented today suggest that to send immune cells berserk, the antibody has to be tethered to a “surface” in the body rather than be free-floating. The team was only able to replicate the excessive cytokine response in the lab that the patients had experienced by effectively sticking the antibodies to a surface.

But that doesn't seem to be the final word on the subject. About a month ago there was a further report:

Researchers propose reason for severe side-effects of Northwick Park clinical trial
The research shows that stimulating the molecule CD28 on cells that mediate the immune response, known as T cells, can have an adverse effect if these immune cells have been activated and altered by infection or illness in the past.

The scientists found that when they artificially stimulated CD28 on these previously activated 'memory' T cells, this caused the cells to migrate from the blood stream into organs where there was no infection, causing significant tissue damage. CD28 is an important molecule for activating T cell responses and the TGN1412 drug tested on the human volunteers strongly activates CD28.

Around 50% of adult human T cells are memory cells, having been activated by infections and illnesses during the course of a person's life. However, animal models, such as those used to test TGN1412 before tests were carried out on humans, do not have many memory T cells because they are deliberately kept in a sterile environment where they are shielded from infections.

Here we see that yet another kind of T cells has been implicated – memory T cells. Undoubtedly there is still more of this story to be discovered. The immune system is a maze of twisty little passages...

Septic shock



One thing that is clear enough is that there must be negative feedback loops in the immune system, as well as positive ones. The latter enable the system to react quickly to serious infections. The former are needed to keep the system itself from spiraling out of control.

The immune system disorder known as septic shock is another example of the system gone berserk. As mentioned in the Wikipedia article, our old friend, the cytokine IL-6, is among those implicated in this condition. Recent research (last November) has identified a specific gene that seems tasked to protect against septic shock:

Gene Tied to Out-of-Control Immune Response
A gene called auf1 seems to protect against septic shock in mice, a new study finds. Animals lacking the gene were more likely to undergo shock, suggesting that the gene helps keep the immune system's response to infections in check. Researchers hope to discover whether different forms of auf1 and related genes make people more likely to suffer autoimmune disease or life-threatening reactions to infections such as anthrax or flu.

Infectious organisms trip specialized immune cells in the body and cause them to pump out proteins called cytokines, which produce inflammation and other hallmarks of infection, such as chills and fever. The body must carefully regulate its cytokine response, however, because "if it isn't turned off it can lead to septic shock and rapid death," says microbiologist Robert Schneider of New York University. Septic shock, which causes 9 percent of deaths in the U.S. each year, occurs when the immune reaction to a bacterial infection grows out of control, shutting down organs and sending blood pressure plummeting. Researchers think similar effects contribute to death from anthrax and pandemic flu.

It appears that the gene codes for a protein which may interfere with messenger RNA that leads to production of cytokines IL-1β and TNFα – thereby putting a brake on production of those cytokines if they are about to run amok.

Undoubtedly, there are a number of chapters yet to be written in the story of how cytokines (and there are many more than have been mentioned here) are regulated. Stay tuned.

More: New study finds on/off switch for septic shock

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