Chapter Six: Immune Cell Apoptosis and Chronic Fatigue Syndrome (By Marc Fremont, Anne D’Haese, Simon Roelens, Karen De Smet, C. Vincent Herst, and Patrick Englebienne)
Cellular apoptosis or programmed cell death is a critical regulatory component that causes the deaths of millions of mostly healthy cells in our bodies every minute. Cell apoptosis occurs when a suicide program is activated in the cell.
When this happens DNA repair stops, the cell shrinks, its DNA fragments, the contents of the cell condense, acids degrade the cellular components, and the cell splits itself into membrane bound fragments that macrophages quickly engulf. Within 30 minutes the cell is no more.
Apoptosis can be induced in many ways. If a cell fails to pass certain checkpoints during the cell maturation cycle the suicide program will be invoked. Physical or chemical agents such as cellular toxins or hormones can also induce apoptosis. (Under or over expression of certain hormones can initiate apoptosis.)
Special signaling molecules (tumor necrosis factor TNFa, FasL) can induce apoptosis by binding to their respective receptors on the cell. (This occurs when, after a search of a cells surface, a cytotoxic T cell (Tc) concludes it is damaged (cancerous) or infected. If this occurs it induces the cells suicide program by punching a hole in the cell and depositing apoptotic triggers called granzymes).
As mentioned earlier the death receptor pathway appears in CFS to be induced by FasL rather than TNFa cytokines. FasL cytokines are produced by cytotoxic T-cells that seek out infected or damaged (cancerous) cells and kill them.
PKR up regulation is also able to enhance the expression of the Fas receptors on the surface of the cell and thus increase the sensitivity of the cell to the FasL cytokine. This suggests that either PKR up regulation or increased cytotoxic T-cell activity could help cause the increased apoptosis seen in CFS.
There is evidence for increased cytotoxic T-cell activity in CFS; a recent study suggested that the cytotoxic T-cells are so chronically activated that they have begun to run out of ammunition (perforin).
Problems inside the cell can also prompt it to kill itself. Mitochondrial problems can induce apoptosis through activation of the Bcl-2 proteins. (This apoptotic pathway was not discovered until 1994!)
Recent evidence indicates that stress in the endoplasmic reticulum (ER) can also induce apoptosis. ER stress may be induced by amyloids (abnormal aggregations of proteins) and the unfolded protein response (UPR). (What an interesting finding given the recent Baraniuk paper suggesting that an amyloidic state may exist in the brains of CFS patients).
The apoptotic process, then, is responsible for clearing the body of developmentally damaged and infected and cancerous cells as well as cells with energy production problems, etc. It’s not surprising, then, that problems with apoptosis can resulting in chronic and sometimes deadly disorders such as autoimmune diseases, neurological disorders and cancer.
The main players in apoptosis are proteolytic enzymes called caspases. These enzymes break down cellular cytoskeletons, shut down repair of the cells DNA, activate enzymes that destroy DNA, and chop up the cell into small apoptotic bodies. Calpain is another apoptotic enzyme (cysteine protease) that is implicated in cell death and the breakdown of the cells cytoskeleton.
RNase L and apoptosis in chronic fatigue syndrome
The finding that calpain can also fragment RNase L suggested that the apoptotic process could be altered in CFS patients. This possibility prompted the authors to investigate the role that caspases play in CFS.
The suicide program can be invoked in three ways; one begins at the cells external receptors (TNFa, FasL binding), one, as was just noted, is initiated in the mitochondria (activated by c-Jun, Bax, calcium), and one, just recently discovered, is activated by stress in the endoplasmic reticulum (ER). All three pathways ultimately achieve the same result: the activation of a caspase called caspase 3 that is capable of cleaving hundreds of substrates.
Caspases and RNase L cleavage
The researchers found that different suicide programs occurred at different levels of RNase L fragmentation. Caspases associated with the death receptor pathway showed increased activity levels at intermediate to high levels of RNase L fragmentation (2-20x’s the 83-kDa RNase L).
At very high levels of fragmentation, however, this activity of this pathway was normal but the caspases associated with the mitochondrial pathway are blocked.
How might this strange pattern of apoptosis occur? The authors noted that as the levels of the 37-kDa RNase L increase another fragment appears – not of RNase L but of caspase; it appears that caspase 9 is fragmented as well as RNase L, and they suggest the cause might be the same – increased calpain levels.
How to explain, though, the diminishment of caspase activity at the very high levels of RNase L fragmentation? Several possibilities exist. The authors suggest that the 37-kDa enzyme could end up degrading different types of RNA than the native enzyme.
It could, for instance, degrade mRNA’s that code for proapoptotic proteins. This is after all what RNase L does – it degrades mRNA. Accumulations of the new fragment could also, independent of it’s catalytic activity, impair the functioning of apoptotic regulators.
The apoptotic inhibition seen could also be the result of dysregulation of other apoptotic pathways than those concerning RNase L. The authors state that finding out the cause of the apoptotic inhibition seen at high level of the 37-kDa enzyme will require further examination of the fragment.
*Update 2/07 – This question does not appear to have been addressed in the five years following the publication of the book but look at a presentation given by Toni Whistler of the CDC at the 2007 IACFS conference concerning the Dubbo study project. Her gene expression studies found that mitchondrial apoptotic activity was decreased in people with Post Infective Fatigue Syndrome (or CFS). This is an important independent validation of the Dr. De Meirleir’s findings.
Calpains in chronic fatigue syndrome
Calpains are cysteine proteases that require calcium for activation. Calcium’s charge and its ability to bind readily with many substrates enables it to easily bond with and change the shapes and thus the activity of many proteins. Because of this high reactivity, cytosolic calcium levels are kept low and calcium itself is kept compartmentalized in organelles.
Calcium channels on these organelles (and on the plasma membrane) are opened or closed in order to turn on or off different cellular functions. The possibility that disrupted channel functioning or a ‘channelopathy’ may account for some symptoms in CFS is explored in Chapter five.
The suspicion that calpain was a probable agent in RNase L fragmentation lead the authors to investigate the relationship of calcium levels (a putative analogue of calpain activity) in the PBMC’s of CFS patients with RNase L cleavage (37-kDa/83-kDa).
Just as with caspase (8, 3) activity, calcium concentrations significantly increased (p<.001) until the 37-kDa fragments reached the highest levels (>20x’s 83 kDa), at which point they began to decline. This, of course, suggests that calpain is a important source of RNase L fragmentation.
Caspase 9, the caspase we noted earlier is fragmented in CFS patients, is activated through cytochrome c release in the mitochondria. Since calpain fragments caspase 9, this apoptotic pathway, therefore, would probably be down regulated during increased calpain activity.
This is precisely what is seen in CFS. Caspase 9 activity exhibited a directly opposite pattern to that evidenced by calpains. This suggests that calpains are the most immediate source of increased caspase activity in.CFS. So far tests indicate that calpain can fragment RNase L, G-actin, and may be responsible for STAT 1, p53 and RLI fragmentation (!).
Since increased intracellular calcium levels activate calpain, the source of the increased intracellular calcium in PBMC cells in CFS patients would probably be of great interest. Indeed, the authors suggested at the end of Chapter Two that regulators of calcium homeostasis may be beneficial in CFS.
Calpain activation is just one outcome of high intracellular calcium levels. High calcium levels also activate iNOS, which produces NO in neurons. NO production can stimulate apoptosis by causing DNA damage or by altering protein structure.
If NO binds with the superoxide ion, peroxynitrite, a highly reactive radical is formed that can damage DNA. Because NO stimulates calcium production through S-nitrolysation of the synaptic proteins, it appears a positive feedback cycle between NO and calcium may be formed in the nerves.
Increased calcium levels also allow solutes to pass through pores in the mitochondrial membranes. When this happens the mitochondria swell and eventually rupture releasing the apoptotic signaling factors cytochrome c and the apoptosis inducing factor (AIF). Calcium can also induce apoptosis through activation of PLA2 and the subsequent production of O2-, a free radical.
Another effect of calcium induced pore opening is the loss of membrane potential in mitochondria. The flow of electrons in the mitochondrial membrane drives the hydrogen pump which propels protons across a membrane thus forming a proton gradient.
As the protons return down the gradient they produce the energy needed to couple ADP and P and form ATP. When membrane potential is lost the energy needed to complete the process disappears and oxidative phosphorylation, the culmination of the energy production process, is uncoupled from the citric acid cycle and ATP levels drop.
Too much calcium in the wrong places of the cell is bad news and can induce apoptosis in at least three different ways.
*Update: Reducted calpain levels were shown to result in reduced proteolytic activity in CFS patients. This study further implicates calcium and calpain in RNase L fragmentation. (2001. Journal of Chronic Fatigue Syndrome, vol. 8, 63-82)
More protein fragmentation – actin and chronic fatigue syndrome
Unlike the skeleton of a human, the cytoskeleton inside a cell changes its shape frequently. The main factor in those changes is actin – the most abundant protein in most animal cells. Actin not only makes up most of the framework of the cell, it is also directly involved in maintaining membrane integrity; problems with actin causes cells to become more permeable to outside substances.
Actin also plays an important role in two immune processes; T-cell activation and phagocytosis. The most critical event in T-cell activation is the T-cell’s recognition of an foreign antigen on the surface of an antigen presenting cell.
Upon activation the T-cell releases cytokines that induce other T-cells to come to the alert, and increase the number of antigen receptors they have on their surface. Actin reorganization is necessary for the creation of these antigen receptors.
Actin reorganization also occurs when macrophages ingest invaders in order to display their peptides on its cell surface. (Problems with actin could therefore impair immune recognition of intracellular pathogens or the ability of phagocytes to ingest pathogens.
Actin is present as a monomer (G-actin) or as a polymer (F-actin). Only polymerized actin is incorporated into the structures that make up the actin cytoskeleton. Monomeric actin, on the other hand, which makes up about half the actin found in cells, forms a large pool of unutilized actin – this actin is available to be formed into polymerized actin when the need arises.
Two actin ‘capping’ proteins, villin and gelsolin, prepare monomeric actin for polymerization. When cells die both G and F actin are released into the bloodstream where they undergo polymerization.This results in increased plasma viscosity.
These researchers found that the levels of the G or monomeric actin fragments in the PBMC’s of CFS patients were positively correlated with levels of the 37-kDa fragment. Tests showed that caspase 3, m-calpain, and PBMC extracts from CFS patients were all able to fragment G-actin in vitro.
This suggests that actin, along with RNase L and the other proteins, is being fragmented in the cells of CFS patients. Given actins immune activities the authors believe that actin fragmentation ‘undoubtedly’ further contributes to the immune problems seen in CFS. G-actin fragmentation could, by taking away the supply of usable actin, negatively impact membrane integrity, phagocytosis, cell adhesion and T-cell activation.
Reduced membrane permeability could ultimately lead to apoptosis if severe enough. A recent report indicated that simply the act of severing actin can induce the apoptosis process (see update).
*Update: Not only was actin fragmentation correlated with RNase L fragmentation in CFS patients but a distinctive LMW actin fragment was formed that the authors suggested could be used as a low cost screening tool for CFS. It was not clear whether the authors believed that the fragment was unique to CFS patients. (2001. Journal of Chronic Fatigue Syndrome, vol. 8, 63-82.)
*Update: Rather than being purely an outcome of cell death, a study indicates that actin fragmentation in epithelial cells can induce cell death. (2001. AM J. Resp. Cell Mol. Biol. (24) 282-294.)
PKR and apoptosis regulation
Interest in Protein kinase R (PKR) was sparked because PKR is induced by and regulated in a similar fashion as RNase L. It, too, is induced by IFN-a and is activated by types of ds or ssRNA that have the stem loop structures characteristic of viruses. Upon activation PKR activates eIF-2a which inhibits the translation of viral RNA.
PKR also plays a major role in regulating gene transcription through its phosphorylation of the important nuclear transcription factors, NF-kB, p53, and the STAT. PKR activation also results in the up regulation of the Fas genes (Fas receptor binding initiates apoptosis), p53, and Bax genes involved in apoptotis; PKR is an important apoptotic regulator.
PKR activation is not dependent upon the presence of viral RNA. The ability of such diverse elements as calcium ionophores, lipopolysacharides (LPS’s), IFN’s, or TNF-a (see below) to activate PKR suggests that PKR functioning is not limited to inhibiting the translation of viral RNA.
It can activated by a wide variety of conditions associated with cellular stress, including, as was noted, dsRNA (viral infection), cytokines, and growth factor deprivation. In the context of CFS PKR’s most important role appears to be that of an apoptotic regulator.
Calcium ionophores increase calcium passage through membranes. LPS’s are found on the outer membranes of many bacteria. Tumor necrosis factor is produced by monocytes/macrophages and other cell types (T, B cells, NK cells, neutrophils, etc.) in response to inflammatory and environmental challenges.
TNF-a activates the ‘respiratory burst’ in neutrophils and macrophages that creates so many free radicals. A recent article (2000) identified a protein that activates PKR in response to stress, induced by among other things, hydrogen peroxide. PKR then can be activated by inflammation, increased intracellular calcium levels and cellular stress.
An analysis of PKR and eIF-2a activity in CFS patients indicated it was likely that increased PKR activity is a least partly responsible for the increased apoptotic activity that is seen as RNase L fragmentation increases. A close look at the EIF-2a expression indicates that as RNase L fragmentation increases the amount of the native 38-kDa EIF-2a protein decreases and a 36-kDa EF-2a fragment becomes more and more apparent.
The authors suggest that the increased apoptosis found in the PBMC’s of CFS patients is first induced by the upregulated PKR/EIF-2a pathway but declines later when EIF-2a itself becomes fragmented.
PKR may also induce apoptosis through activation of the tumor suppressor protein p53. P53 (a focus of much recent cancer research) responds to cellular stress or DNA damage either by stopping a cells progression through the cell cycle or by inducing the cell to kill itself.
P53 expression in the PBMC’s of CFS patients was consistent with pattern of cell suicide seen in CFS patients; P53 is found in cells with 37kDa/83kDa ratios below 20 but disappears in cells with higher rates of RNase L fragmentation.
In these cells the researchers found a 30-kDa fragment is detected that is probably caused by breakup of the p53 protein. Since p53 induces apoptosis, its disappearance of p53 would most likely lead to reduced apoptosis.
The cause of the p53 fragmentation is still unknown. Calpain, however, is not only able to cleave p53, but calpain cleavage of p53 in one study generated a 30-kDa fragment that may correspond to the one reported here. This suggests that calpain may be responsible not only for RNase L fragmentation (and others) but for p53 degradation as well.
Calpain concentrations decline, however, as RNase L fragmentation peaks. When calpain levels are at their highest p53 is still largely intact.
P53, at this point, appears to be the most likely to be responsible for the reducedapoptosis found at the highest levels of RNase L fragmentation. Disruption of the p53 protein might be viewed with some dismay by CFS patients since p53 inhibition is found in many cancers.
Since p53 fragmentation occurs only at the highest levels of RNase L fragmentation, it appears, however, that only the very sickest CFS patients need have this concern. A 2006 paper investigating mortality rates in CFS did not find increased rates of cancer.
In conclusion, not only do most of the pathways involved in PKR mediated apoptosis appear to be disrupted in CFS but the disruption follows a similar pattern; first increased apoptotic activity followed by below normal levels of apoptosis when RNase L fragmentation is at its greatest.
This suggests that PKR contributes via multiple mechanisms to the apoptotic problems found in CFS. Of special interest is calpains apparent ability to operate in multiple pathways and fragment both RNase L and p53. The central question in all this – the cause of the apparent calpain activation, in CFS is, however, unknown.
Conclusions and prospects
It is apparent that unusual patterns of cell suicide are found in the immune cells of CFS patients. Both the apoptotic regulators (p53, NF-kB, PKR) and the apoptotic enzymes (caspases 3, 8) exhibit similar patterns: apoptotic activity is high until the levels of the 37-kDa fragment reach a certain peak at which point apoptotic activity declines or is even inhibited (caspases 2, 9).
How RNase L cleavage and increased levels of cell suicide are related is not as yet clear. The fragmentation of the native RNase L protein and the consequent generation of a more active RNase L fragment, could result in increased apoptotic activity. Conversely, increased proteolytic activity resulting from an upregulated apoptotic system could cause RNase L fragmentation.
Which comes first? Does a deranged RNase L fragment cause increased apoptosis or does an apoptotic system run amok fragment RNase L? Upregulated apoptosis could result in increased levels of calpain and RNase L fragmentation. What is causing the increased apoptosis seen in CFS?
A Th1 deficient system that allows in intracellular pathogens? Increased levels of toxins and/or free radicals? Disruption of mitochondrial activity by nitric oxide or other factors? Altered hormone levels? A lot of things could!
Explaining the apoptotic blockade seen at the higher levels of the 37-kDa fragment (>20’s the native enzyme) is more difficult. The decreased apoptosis seen at the height of RNase L fragmentation suggests that pro-apoptotic enzymes (calpain, caspases) are not causing RNase L fragmentation.
Instead, as we saw in Chapter 2, it appears that RNase L cleavage at least in its initial stages is due to increased levels of the inflammatory enzymes elastase and/or cathepsin G. Explaining, on the other hand, how RNase L fragmentation could cause the apoptotic blockade, is even more difficult.
Further investigation is needed to clarify RNase L’s role in apoptotic regulation and to understand the possible effects the 37-kDa fragment may have on RNase L activity, specificity, or cellular localization.
Another possible factor in the apoptotic problems seen is the high number of opportunistic infections CFS patients have. Viruses, chlamydia and mycoplasma are all able to inhibit apoptosis. We will see later that mycoplasmas also appear to be able to fragment RNase L.
Opportunistic infections can also aggravate the pathogenic process in CFS by disrupting metabolic pathways and cytokine production. G-actin cleavage, in turn, most likely exacerbates the inflow of opportunistic pathogens through its inhibition of the antigen presentation process and phagocytosis.
(CFS patients and apoptosis: CFS patients may be subject, depending on the degree of RNase L fragmentation found, to either increased or inhibited apoptosis. Increased apoptosis has been implicated in the pathogenesis of several diseases.
Mitochondrial defects, increased rates of oxidative stress, and neurotoxic agents are believed to cause increased rates of cell suicide in the nerve cells in Parkinson’s, Alzheimer’s, ALS, and retinitus pigmentosa. Its interesting that all three factors (mitochondrial problems, oxidative stress, neurotoxic agents) may also be present in CFS.
It’s also interesting, and perhaps not entirely surprising to CFS patients, that aging is associated with increased apoptosis in lymphocytes.
Only a subset of the most severely ill CFA patients appear to suffer from what appears a more serious problem; apoptotic inhibition. Inhibition of apoptosis results in an excess accumulations of damaged cells and may result in increased rates of cancer.
It may also predispose people to increased rates of auto-immune diseases such as lupus. Because apoptosis can also be triggered in infected or otherwise damaged cells, the inability to removes such cells could result in increased rates of infection.
Proteolytic Activity in CFS. The list of fragmented proteins continues to grow and grow. Not only are RNase L and RLI broken up but so are STAT I, p53, G-actin, caspase 9 and eIF-2a. That is a lot of protein degradation going on. Only for RNase L and caspase 9 can we explain why. Are the other proteins degraded because of high protease levels? Is this due to chronic inflammation or infection? Is this unique to CFS patients? What is going on??