Immunology feature: Bim, Bid and autoimmunity

It may be ironic, but by ending the life of unwanted or damaged cells during both development and adult homeostasis, apoptosis or programmed cell death becomes an essential part of life itself.

Professor Andreas Strasser and his group at the Walter and Eliza Hall Institute of Medical Research (WEHI) in Melbourne are at the forefront of research into apoptosis, with a particular interest in the mechanisms of its regulation.

Strasser and his colleagues have had a significant effect on the field of apoptosis research, including the discovery of quite a few of the genes that regulate it.

They have and continue to study the function of these genes by making genetically altered mice, both knockouts and knock-in mice.

In fact, Strasser and colleagues have knocked out the largest number of these genes of anyone in the world.

At the ASI meeting, Strasser will talk about the relationship between autoimmunity and some of the genes involved in apoptosis.

"We found out a long time ago that knocking out certain pro-apoptotic genes or abnormally increased expression of anti-apoptotic genes can lead to systemic autoimmune disease in our mice, generally one similar to lupus in humans," Strasser says.

"Recently, we have been studying mechanisms used by the immune system to turn off activation, for example once a pathogen has been successfully cleared. It is important to shut these responses off because a constantly activated immune response can cause unwanted destruction of healthy and normally functioning tissues. I will discuss two aspects of this work at the conference."

Initially, Strasser's group looked for possible mechanisms or pathways mediating cell death within normal tissue with the idea of blocking the destruction in the presence of an overactive immune system, such as in chronic autoimmunity.

Strasser became interested particularly in collateral damage to hepatocytes in the liver, based on evidence from human patients and animal models where fatal destruction of the liver is induced by over-activating either the innate or adaptive immune response.

It remained entirely unclear (prior to their work) how these hepatocytes die, mechanistically, in those circumstances.

"We had a number of suspicions, and it turns out that much of the damage to hepatocytes is mediated by signalling from tumour necrosis factor (TNF) and its receptor, TNFR1, in humans and animal models," Strasser says.

TNFR1 is a classical 'death receptor' for programmed cell death. It has an intracellular death domain and can activate the classical apoptotic pathway.

In addition, the group's findings implicated the protein, Bid, a well-known initiator of apoptosis that can be activated by 'death receptors' in this pathological cell death.

Thus, Strasser made Bid-knockout mice with liver damage and showed conclusively that Bid on its own protects the hepatocytes to some extent - it reduces the pathology - but it does not prevent it and most Bid-knockout mice still die.

Seeking the other factors involved, Strasser followed an educated hunch and first tested Bim, a close relative of Bid, as the other player in apoptosis initiation.

"We made double knock-outs for Bid and Bim, and this produced mice that were entirely resistant to liver destruction. Instead of 100 per cent mice dying, we now had almost all surviving - quite a stunning difference.

"We are just finishing off work to elucidate how TNFR1 signalling activates Bim as this mechanism has no precedent."

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Immune pathologies

The second focus of Strasser's presentation is another TNF-related protein involved in mediating tissue pathology when immune responses are highly activated, called Fas-ligand (FasL). It has one dedicated receptor called Fas, whose activation in any cell type can trigger cell death.

"The FasL/Fas system is critical for deletion of chronically activated (foreign antigen or auto-antigen specific) T and B cells," he says. "Accordingly, mutations in Fas or FasL result in immune pathologies."

However, what regulates FasL post-translationally was unknown. This ligand is synthesised mainly by T lymphocytes, but not transported immediately to the cell surface.

Instead, it is stored in cytolytic granules or vesicles inside the cells, thereby introducing another level of control on the molecule. Upon antigenic activation of the T cells, these intracellular storage compartments fuse with the plasma membrane, placing Fas ligand on the surface where it can be presented to the environment in a highly aggregated form.

FasL on the cell surface can be cleaved by extracellular proteases thereby producing a soluble (trimeric) form.

The controversial issue in the field prior to Strasser's work was which form of FasL is bioactive - is the membrane-bound form used to kill target cells or is it the secreted, cleaved form?

"Half of the world believed one theory and half believed the other," Strasser says.

In addition, some in the field also believed that the secreted form of the ligand does not kill cells, but instead has a separate function to inactivate target cells.

Strasser decided to address all these questions using his group's substantial mutational engineering expertise in mice - making subtle mutations in the endogenous Fas-ligand genes. They made knock-in mice in which Fas ligand could either only be secreted or only be membrane-bound.

"We thought that by putting these animals through their paces we should be able to answer all of the questions on this that are out there ... and we were right. We also found a few entirely surprising things, which was nice."

Strasser's experiments showed that the membrane-bound form of Fas-ligand is solely responsible for initiating cell death in the target cells, and that the secreted form has no such function.

"It also became clear that the cleaved and secreted Fas ligand has a function that we had not been thinking about, so this other group of people were also right.

"All of the mice that only make secreted Fas ligand eventually develop a range of defects and diseases that the animals not able to cleave and release Fas ligand and even animals that completely lack the function of Fas ligand never get - cancer, heart pathology, autoimmune problems, severe dermatitis and so on. What the nature of this activation is, however, we do not know."

Together, these results suggested that membrane-bound Fas ligand guards against autoimmunity, while secreted Fas ligand may play a critical role in tissue damage and tumour suppression.

While striving towards a complete understanding of the interplay between apoptosis and the immune system, Strasser's findings along the way continue to significantly inform the field.

"Beyond the basic research, we always have a bit of a wondering eye on whether anything we see in the mice is relevant to a disease setting, in collaboration with clinicians and other scientists," he says.

"It turns out that a lot of treatments used in cancer therapy also work extremely well for some autoimmune diseases, most likely because both diseases involve defects in apoptosis as an underlying cause. So if you have a drug that reinstates normal apoptosis you can potentially treat either of those disease families.

"A recent example of this is the anti-CD20 antibody reagents (Rituximab) developed by Genentech for the treatment of certain types of B-cell lymphoma - they also work extremely well for treating rheumatoid arthritis."

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Understanding apoptosis

Andreas Strasser did his PhD in Switzerland with Professor Fritz Melchers at the Basel Institute for Immunology, investigating B lymphocyte development. At the end of his PhD, Strasser wanted to pursue research that was more disease-related.

An offer by Professors Suzanne Cory and Jerry Adams of WEHI in Melbourne sounded interesting to the young scientist. So, without even visiting, but with enthusiastic encouragement from his wife who had travelled to Australia previously, Strasser packed his bags and headed down under.

After about three years and an attractive job offer from the Cancer Institute in the Netherlands, Strasser was persuaded to stay at the WEHI to launch an independent research career. Since that time his lab has grown from one person (himself) to around 11 scientists, and Strasser has amassed an extremely impressive CV.

He has held numerous fellowships and special awards, and is currently an NHMRC Australia Fellow and co-head of his division at WEHI with Professor Jerry Adams.

Strasser's discoveries have strongly influenced our understanding of apoptosis signalling and its role in shaping the immune system, and besides appearing in one of the 'big three' journals (Nature, Science or Cell) almost every year of his not-all-that-long career, these achievements have been recognised with a plethora of national and international awards.

Amongst these, Strasser became a Fellow of the Australian Academy of Science in 2003, was recently appointed as co-head of the new ACRF Centre for Therapeutic Target Discovery in Melbourne, and in August, he joined fellow scientists, Professors Alan Cowman and Doug Hilton, in receiving one of the inaugural Australian Fellowships from the NHMRC.