Suhadolnik, R. A., Peterson, D., Reichenbach, N., Roen, G., Metzger, M., McCahan, J., O’Brien, K., Welsch, S., Gabriel, J., Gaughan, J. and N. McGregor. 2004. Clinical and biochemical characteristics differentiating chronic fatigue syndrome from major depression and healthy control populations: relation to dysfunction of the RNase L pathway. Journal of Chronic Fatigue Syndrome 12: 5-35.
It is not often that a paper is described as a ‘landmark’ upon publication but that is how the editors of the Journal of Chronic Fatigue Syndrome, Kenny De Meirleir and Neil McGregor, described this latest offering from Robert Suhadolnik and his colleagues. Even the authors of the paper abandoned some of their normal caution and described it as ‘pivotal’. As this is the first part of a two part paper the full findings are not yet available.
(It’s nice to see Dr. Suhadolnik and his American counterparts back in the ‘hunt’ again after several few years. Dr. Suhadolnik discovered RNase L fragmentation in CFS. The Belgium research group lead by Drs. De Meirleir, Englebienne and Nijs has done the lions share of RNase research in the past five years.)
Objectives of the study
This paper examined symptom presentation, functional disability, RNase L fragmentation, and blood biochemistry in CFS patients, depressives and healthy controls.
One of the first things to notice about this study is that it is large. It consisted of about 180 subjects that were more or less evenly distributed between CFS, depressed and healthy controls. Most CFS studies, particularly exploratory ones are small and, as such, are subject to sampling error. The large size of this one means its results are robust and should stand up over time. The large sample size also allowed the researchers to probe the subgroups and come up with some interesting results. The authors note this study included only significantly impaired CFS patients (Karnofsky score <60) and thus may not reflect the CFS population at large.
Several things stand out in the beginning of the paper. First the authors state that CFS involves a ‘global’ deregulation. This implies a considering broadening of emphasis beyond the immune deregulations this group has uncovered in the past and indicates the authors believe a wider array of cellular regulatory pathways are disrupted in CFS. The authors also note that two kinds of immune dysfunction are regularly found in CFS patients; one involving poor cellular function (NK cell functioning, impaired lymphocytes response to mitogens) and the other involving immune activation (increased T lymphocyte activation, increased cytokine levels).
Having increased T lymphocyte activation but impaired T lymphocyte responses in the same person seems somewhat paradoxical. High levels of activated lymphocytes in CFS patients indicate the body is fighting off an infection. Reduced lymphocyte responses to mitogens such as phytohemagglutin (PHA) and pokeweed indicates lymphocytes in CFS patients respond sluggishly to stimuli. Poor T lymphocytes response to mitogens suggests a defect in the immune surveillance system of CFS patients allows some pathogens to slip by and possibly causing further immune system dsyregulation.
There is some evidence CFS patients display a ‘hole’ in the immune system. Several pathogens (EBV, coxiella, Ross River, parvovirus) appear more likely than others to cause CFS (Lloyd 2003) and CFS patients typically harbor a kind of mycoplasma (M. fermentans) rarely found in healthy controls. There is also evidence for a kind of sequential immune disruption in CFS. Increased rates of mycoplasma infection over time in CFS patients suggests pathogens may be able to exploit a widening hole in the immune system (Nicolson et. al. 1999). Mycoplasma infection did not, however, increase the risk of Chlamydia pneumoniae and HHV-6 infection in another study (Nicolson et. al. 2003).
CFS and depression
CFS patients have long been known to exhibit high rates of depression. Anyone with a chronic illness is at increased risk of depression. Some have suggested, however, that CFS patients exhibit higher rates of depression than expected for those experiencing a chronic illness.
The role depression plays in the pathophysiology of CFS is controversial. While CFS patients experience some symptoms in common with depressives (reduced ability to concentrate, insomnia, fatigue, worry about the future, ‘somatic’ complaints, etc.), many of their symptoms (fatigue after exercise, flu-like feelings, etc.) are not usually found in depressed patients.
The very presence of many unexplained somatic symptoms in CFS patients suggests to psychologists, however, a mood disorder is present. Differentiating symptom presentation in CFS patients from depressed patients would help demonstrate CFS is a unique disorder.
This study statistically examined the prevalence of ‘core CFS symptoms’ in CFS patients, depressed patients and controls. It found that people with depression experienced few of the symptoms or clinical abnormalities considered to be core CFS symptoms (post-exertional malaise, memory impairment, muscle pain, relapsing fatigue, sore throat, poor rest alleviation, enlarged lymph nodes, abnormal reflexes, abdominal pain, Romberg, tandem stance). Thus CFS patients were clearly differentiated from depressives with on the basis of symptoms. A functional status evaluation indicated that the CFS patients experienced a far high degree of disability than did depressed patients.
RNase L fragmentation in CFS, depression and healthy controls – this is the largest study yet to examine RNase L fragmentation in CFS patients and control groups. The finding that RNase L fragmentation is significantly higher in CFS patients than both control groups (p< .001) suggests that RNase L fragmentation is a central component of CFS. It also extends a string of consistently abnormal findings in CFS patients that is, unfortunately, almost without precedent in the annals of CFS research. The authors report that five independent studies have confirmed a deregulated RNase L pathway in CFS. (See ‘Testing RNase L‘).
De Meirleir does not believe RNase L fragmentation is the central feature in all CFS patients. In a 2003 talk he indicated he and his colleagues have found three types of IFN mediated immune dysfunction in CFS; (1) increased RNase L fragmentation and dysfunctional PKR, (2) increased RNase L fragmentation and normal PKR, and (3) normal RNase L fragmentation and dysfunctional PKR activity. The data indicates most CFS patients have increased rates of RNase L fragmentation.
Most intriguingly neither levels of IFN-a, 2-5A concentration or RNase L activity differed significantly between CFS patients and the controls. This suggests RNase L activity need not be increased for RNase L fragmentation to negatively effect CFS patients. RNase L activity level has long been discarded as a potential biomarker for CFS. Yet it is strange that the three such integral aspects of the RNase L system are not activated in CFS patients. If they are not activated – if RNase L is not ‘active’ how then could it play a role in CFS pathophysiology?
A layman’s speculation
RNase L, at least with regard to pathogens, is considered to be quiescent until it is activated by IFN or retinoic acid (via 2-5OAS and 2-5A) and RNA strands of pathogenic origin. If this is so, then how could RNase L whether it is whole or fragmented, normal or abnormal, have any effect on the cell? Since it doesn’t seem that it could then we have to re-examine our premise regarding RNase L activity.
RNase L, it turns out is not only activated by pathogens; its appears to figure in a variety of cellular events including cell growth, differentiation, ubiquitination, red blood cell morphology and apoptosis and probably other as yet unidentified functions. RNase L then is probably often active even when the cell is not under pathogenic attack and RNase L activity need not be increased for RNase L dysfunction to negatively effect the body (see Englebienne overview).
De Meirleir has formulated a theory that illustrates how chronically low levels of RNase L activation could be harmful. In his 2004 AACFS talk De Meirleir stated RNase L fragmentation in CFS patients is due to a low level activation of the RNase L system by RNA fragments derived from apoptotic fragments. He believes that the nucleases responsible for degrading the apoptotic fragments are simply overwhelmed by the high rates of apoptosis in CFS. This results in the production of high levels of shortened RNA fragments that activate the initial enzyme (2-5OAS) in the RNase L pathway.
There are three forms of the 2-5OAS enzyme. The form most prevalent in CFS, p100, is from 10-100 times more sensitive to RNA fragments than the other 2-5OAS isozymes. The p100 isosyme also produces shorter strands of 2-5A (dimers instead of trimers) than do the other isozymes. The 2-5A dimers negatively effect RNase L in two ways; first they bind to RNase L in such a way that leaves it unactivated and secondly they leave it in a configuration that leaves it unprotected from proteases (enzymes that degrade proteins). In CFS Two proteases upregulated in CFS patients, elastase and calpain, attack RNase L and fragment it. Because the normal RNase L inhibitor, RLI, does not bind to one of the fragments it becomes active. It appears possible, therefore, for RNase L to be inactivated (because of 2-5A) but for a fragment of RNase L to be highly activated.
Studies showing 2-5A dimers are more prevalent in the early stages of RNase L activation suggest the p100 isozyme is used to ‘wake up’ the system. De Meirleir appears to suggest that by continually ‘waking up’ the 2-5OAS system, the poorly digested apoptotic fragments in CFS patients generate large amounts of fragmented RNase L. Alternately the inadequate apoptotic process could produce the types of RNA (very short) that the P100 isozyme is particularly sensitive to. Some of these fragments could contain viral segments that were harmless when integrated into our genome (i.e. HERV’s), but may be pathogenic once released (see Chapter X CFS ABA.).
While it is clear RNase L activity need not be increased for RNase L fragmentation to have negative effects on the cell, it is very possible RNase L should be increased in many CFS patients but isn’t. There is evidence the same increased proteolytic activity that fragments RNase L in CFS patients also fragments the STAT I protein responsible for carrying the signal for IFN activation into the nucleus. This would leave CFS with inhibited IFN-a production and low RNase L activity. See below.
It is intriguing that one of the activities that monocytes/macrophages, the only immune cells RNase L has been found in to date, are engaged in ‘garbage cleanup’. As such they seem like prime candidates to encounter RNA fragments left behind by poorly degraded apoptotic cells.
RNase L and reduced NK cell functioning – One of the singular findings was that RNase L fragmentation was highly correlated with NK cell dysfunction (p<.006) in CFS patients. Reduced NK cell functioning is one of the few immune abnormalities consistently seen in this disease. The tight correlation between the two suggests they make up part of the same ‘pathogenetic’ mechanism that underlies CFS.
This is a significant finding. NK cell counts and activity, like some other immune parameters, can be altered by stress. Some researchers suggest the immune abnormalities seen in CFS simply reflect the stressful conditions they find themselves in and indeed some CBT programs have resulted in enhanced immune cell activity (both in CFS and AIDS).
This studies finding – that NK cell functioning is correlated with RNase L fragmentation suggests NK cell functioning in CFS has an immune basis. The authors noted reduced nitric oxide activity was associated with poor NK cell functioning in CFS patients in one study. Reduced NO mediated NK cell function could be tied to reduced protein kinase R (PKR) activity in CFS.
PKR, which forms another branch of the IFN mediated innate cellular response to viruses, bacteria and toxins activates NO via NF-kB. Reduced PKR therefore could conceivably result in reduced NK cell function. A more direct connection to RNase L, however, involves RNase L’s mRNA regulation of a cytokine, ISG 15, involved in T-cell activation. Since T-cells are involved in activating NK cells, destruction of ISG 15 mRNA by a dysfunctional RNase L could result in decreased T-cell activation and decreased NK cell activation (See Englebienne 2003 Overview)
RNase LfFragmentation and blood chemistry in CFS
Using blood chemistry tests, which are almost always normal in CFS – to help explain CFS pathophysiology is a new and intriguing approach. Many of the ‘normal’ ranges of blood chemistry are quite large. The normal range for alkaline phosphatase, for example, is from 30-120.
Using an age and sex matched control group gives a truer indication of ‘normal’ than do ranges that reflect a healthy but heterogeneous population in terms of age, sex, weight, etc. The authors used an ANOVA statistical test to determine small but significant shifts in blood chemistry that indicate abnormalities in CFS patients relative to healthy controls.
Correlation analyses were then used to determine which blood chemistry measures were correlated with RNase L fragmentation levels. While a clinician might look at these blood chemistry panels and find nothing abnormal the ANOVA and correlation analyses enable the authors to determine patterns of change that may be of clinical significance.
A correlation analysis found several abnormalities in blood chemistry findings in CFS patients (increased – GGT, ESR, alkaline phosphatase; decreased – NK cell function, calcium, iron, chloride). Of these ESR, alkaline phosphatase and chloride were correlated with RNase L fragmentation. Alkaline phosphatase and ESR were significantly increased (p<.009, p<.02) and chloride was significantly decreased (p<.02). None of the readings were out of the normal range. The authors noted this topic will be further explored in the second part of this paper.
Since not all of these readings have an immune basis the effects of RNase L fragmentation appear to extend beyond the immune system. If this indeed turns out to be a pivotal study; i.e. one that causes researchers to ‘pivot’ and change direction, this may be one of the findings that causes them to do this.
While the authors do not comment on two findings in the paper it is interesting to note that hemoglobin and hemocrit levels were significantly lower in CFS patients as well (p<.005, p<.002). Dr. Cheney has long noted that inadequate oxygen carrying capacity of red blood cells in CFS. Likewise, low levels of hemocrit, an analogue for red blood cell mass, have been associated with the low blood volume and red blood cell mass seen in CFS as well.
Blood chemistry measures significantly different in CFS patients relative to controls and significantly correlated with degree of RNase L fragmentation.
Erythrocyte sedimentation rate (ESR)
Interpreting the significance of the ESR test has become, oddly enough, more difficult over time. A very simple test, the ESR measures the distance erythrocytes fall after a hour of being suspended in a tube of blood. ESR is directly correlated with fibrinogen levels. The conversion of fibrinogen is into fibrin by thrombin causes blood coagulation.
While the ESR levels of neither CFS patients nor controls were out of the normal range, the ESR rate in the CFS patients was almost double that of the controls (6.7-3.7). It is interestingly given the interest in abnormal red blood cell morphology in CFS that abnormal red blood cell morphology usually leads to decreased not increased ESR levels. On the other hand increased ESR levels may be indicative of increased blood coagulation in CFS, a subject that has been of interest in CFS for several years.
Increased ESR is associated with acute phase of the immune response and inflammation but is an important diagnostic tool for only two diseases. Interestingly both diseases have features (polymyalgia rheumatica – severe aching and stiffness of the neck and shoulder girdle; temporal arteritis – extracranial vasculitis) that may show up in CFS. Dr. Hyde believes many of the symptoms in CFS are generated by a widespread vasculitis. Vasculitis is inflammation of the blood or lymphatic vessels (see Hyde 2004 talk). Abnormal alkaline phosphatase readings are frequently present in temporal arteritis.
It appears, then, that increased ESR could be indicative of increased infection, inflammation, hypercoagulation and/or a vascular condition in CFS.
Alkaline phosphatase levels in CFS patients were well within normal range (85; 30-120) in CFS patients but were nevertheless significantly higher in CFS patients than healthy controls (65, p<.006).
Significantly increased blood alkaline phosphatase levels can occur when blocked bile ducts due to liver disease cause liver alkaline phosphatase levels to leak out into the blood. Vasculitis of the veins leading to the liver after organ transplants causes increased alkaline phosphatase and GGT levels. Interestingly this is associated with capillary leakage into the extracellular spaces (orthostatic intolerance?) and weight gain; two processes that may be occurring in CFS. Dr. Hyde believes vasculitis is an essential feature of CFS. Because vasculitis is also associated with increased fibrin deposition there is a connection with hypercoagulation is as well. (Thanks to Mark London for added info).
Because growing bones require alkaline phosphatase any condition requiring increased bone growth (childhood, Paget’s disease, rickets) also result in increased alkaline phosphatase levels. Alkaline phosphatase is also increased in inflammatory bowel disease, hypervitaminosis D, EBV and cytomegalovirus. De Meirleir et. al. have suggested CFS may be at risk from osteopenia.
Increased alkaline phosphatase levels in CFS patients relative to healthy controls could then be due to a number of factors including infection, liver malfunction, bowel problems or very high levels of vitamin D. Questions regarding Vitamin D levels in CFS have come to the fore lately.
Chloride levels in CFS patients were decreased in CFS patients relative to controls. Chloride effects cellular integrity via its influence on osmotic pressure. It is involved in the acid-base balance and water balance. Chloride levels in the body are regulated by the kidneys and are influenced by the hormone aldosterone. Reduced aldosterone levels are found in CFS and may be responsible for the low blood volume sometimes found (See Orthostatic Intolerance III).
Sodium is mainly responsible for water retention and the serum osmolality level (concentration of solutes in the serum). The chloride ion frequently appears in combination with the sodium ion. Elevated chloride levels are found in acidosis and when too much water crosses the cellular membranes. Interestingly given the belief that an overly acidic environment prevails in CFS, CFS patients in this study had decreased levels of chloride relative to controls. Reduced chloride could be conceivably be due to a mild channelopathy as well
Blood chemistry measures significantly correlated with RNase L fragmentation but not significantly different in CFS patients versus controls
The authors did not discuss any of these findings presumably because they did not differ significantly between CFS patients and controls. That they are nonetheless positively correlated with RNase L fragmentation is intriguing.
Gamma glutamyl transferase (transpeptidase) (GGT) – Table 5 indicates GGT levels were strongly positively correlated (p< .001) with the degree of RNase L fragmentation. GGT transfers amino acids across the cellular membrane. Most importantly it transfers the amino acids involved in glutathione (GSH) manufacture into the cell.
GSH transport from the plasma into the cell is complicated. First GSH is broken up into its constituent amino acids by GGT and is then transported across the cellular membranes where it is synthesized (in the y-glutamyl cycle) back into GSH by glutamylcysteine synthetase (GCS).
The GGT test is used to detect liver, kidney and bile duct disease. Higher than normal GGT levels are found in bile duct congestion, cirrhosis of the liver and hepatitis. As noted this study does not indicate that GGT levels are out of the normal range in CFS patients.
This is the first finding that could link the apparent GSH deficiency noted by Cheney and others and described by Rich Van Konyenburg with RNase L fragmentation (click here). Depleted cellular GSH levels would presumably be the source of increased GGT production. Thus this finding suggests increased RNase L fragmentation results in increased oxidative stress and depleted cellular GSH levels.
Since the synthesis of prostaglandin G2 to H2 requires GSH, one wonders if increased prostaglandin synthesis could be occurring in CFS as well. Prostaglandins regulate the inflammatory cascade. Since the inflammatory cascade releases free radicals GSH could be depleted during prostaglandin synthesis and during free radical detoxification. That GGT levels did not differ significantly from controls argues against this interpretation, however.
Both calcium and iron were also significantly correlated with the degree of RNase L fragmentation (p < .024, p <.04) as well.
The authors stated these blood chemistry findings will be discussed in a later paper.
Tying down immune dysfunction in CFS? RNase L and the IFN system
An even more interesting finding – and one which will also be discussed in a later paper – concerns differences found in immune measures between CFS patients and the control groups. Surprisingly the healthy control group had significantly higherrates of RNase L fragmentation than did the depressed control group.
This prompted the authors to break up the control group into controls that had been in contact with CFS patients (‘contact controls’) and that had not been in contact with CFS patients (non-contact controls) and to re-examine the clinical data gathered previously. Suhadolnik appears to have gathered a substantial number of his healthy controls from health care providers that treat CFS and/or family members of CFS patients.
Laboratory measures indicated that the contact controls, while still healthy, exhibited signs of an IFN mediated response (increased RNase L ratio, IFN-a, sodium, liver enzymes, glucose) to an infection that may have been picked up from CFS patients. Since the study took place over many months no single pathogen was believed responsible. The authors noted a 2000 study found increased RNase L fragmentation in contact controls as well.
This provides evidence for an infectious component in CFS that has been noted before at least in some instances (epidemic CFS) One wonders if their poor immune surveillance causes CFS patients to be excellent vectors for whatever pathogen is present. It may be the epidemic form of the disease occurs when CFS patients happen to pick up a pathogen that is a) highly infectious and b) particularly apt at inducing CFS.
Several pathogens (Brucella, EBV, coxiella, Ross River) appear to produce high rates of CFS (@10%) . Several pathogens associated with CFS (Brucella sp., Borrelia burgdorfii (Lyme disease), Coxiella burnetii), share an ability to survive in the highly acidic environment of the macrophage phagolysosome (Lloyd 2003). Monocytes/macrophages are the only immune cells RNase L fragmentation has been observed in.
Also intriguing were the differences between the contact controls and CFS patients. While all the findings were not given the authors stated a normal IFN mediated immune response did not take place in CFS patients; in particular, IFN-a and platelet levels were not correlated with RNase L fragmentation. This is not the first time IFN levels were not increased in CFS patients. Type 1 IFN levels were not correlated with 2-5OAS levels in another study.
This seems almost paradoxical since one of the central features of CFS – RNase L fragmentation – is activated by type I IFN’s. It is possible, however, that degradation of the STAT proteins noted in ‘Chronic Fatigue Syndrome A Biological Approach‘ that transfer the IFN signal into the nucleus could be responsible for the reduced IFN activity seen in CFS patients.
The inability of CFS patients to mount an IFN mediated response to pathogens would constitute a major hole in the intracellular immune response. Since IFN mediated PKR activity is a source of NK cell activation (via nitric oxide) it is also possible the reduced IFN response seen in CFS patients is responsible for the reduced NK cell activity seen.
Several research groups are trying to model CFS . One is following a cohort of patients with a viral illness known to cause CFS in some people. By measuring immune changes in patients that successfully resolve the infection and in those that do not and come down with CFS, researchers are hoping to identify specific stages in the immune response that go awry in CFS patients.
Another group is identifying immune changes in hepatitis patients given interferon, a cytokine known to cause CFS-like symptoms. Suhadolnik and his colleagues demonstrate here that IFN-a, the main inducer of the intracellular response to viruses, bacteria and other pathogens, is not enhanced in CFS patients that are presumably under pathogenic attack. This suggests a breakdown of the IFN mediated immune response in CFS.
This large study helped to settle some issues and raised several new questions regarding CFS. First a statistical analysis indicating depressed patients do not commonly suffer from the core symptoms characteristic of CFS provided further evidence the two diseases have different etiologies.
Significantly increased levels of RNase fragmentation in CFS patients vs. depressed patients and healthy controls substantiated the above finding and confirmed RNase L fragmentation as a potential biomarker for CFS. Significantly decreased NK cell activity in CFS patients vs. controls provided further confirmation NK activity is reduced in CFS.
Perhaps more importantly NK cell activity levels were negatively correlated with RNase L fragmentation; this is the first time these two immune abnormalities have been linked and again suggests the centrality of RNase L disruption in CFS. Most interestingly several blood chemistry measures that were significantly different in CFS patients vs. controls were also correlated with RNase L fragmentation.
Since some of these do not have an immune basis this suggests RNase L dysfunction may effect other than immune functioning in CFS patients. Intriguingly, in contrast to controls not in contact with CFS patients, healthy controls in contact with CFS patients displayed several abnormalities (increased RNase L fragmentation, IFN activation, etc.) suggesting infection and immune activation.
That CFS patients did not display several of the measures of immune activation (increased IFN-a activity, platelet counts) found in the healthy contact controls suggests an area of immune deficiency in CFS patients. The last two findings will be explored further in a later paper.
*Update 9/08 – Later paper? What later paper? Four years later no paper has appeared. (So much for Landmark!)
Lloyd, A. 2003. Post-Infective Fatigue. In Handbook of Chronic Fatigue Syndrome. 108-122. Ed. Leonard Jason, Patricia Fennel, Renee Taylor and Charles Lapp.