Natural killer cells, perforin, and glutathione depletion

[richvank]

Hi, all.

As many of you know, the most reliable immune-related marker for ME/CFS is probably the low cytotoxic (cell-killing) activity of the natural killer cells.

As you may also know, natural killer cells normally kill cells that are infected with viruses. They do this by secreting a substance called perforin, which makes a hole in the cell membrane, and then injecting granzymes, which induce the cell to undergo apoptosis (programmed cell death). The CD8 cytotoxic T cells operate in the same way in terms of their killing mechanism.

Some years ago, Dr. Kevin Maher, who was in Dr. Nancy Klimas's group, reported that the natural killer cells in PWMEs are low in perforin. It was also found that the CD8 cytoxic T lymphocytes were also low in perforin. This would, of course, inhibit their cytotoxic activity.

The question then became "Why are the NK and CD8 cells in ME/CFS low in perforin?

In 2007, when I proposed the Glutathione Depletion-Methylation Cycle Block (GD-MCB) hypothesis, I noted that the perforin molecule has a large number (20) of cysteine residues in its protein structure. It is known that in order for a cell to be able to synthesize a protein that contains cysteine residues, it must have sufficient glutathione and a high enough ratio of reduced to oxidized glutathione to keep the cysteine molecules in the cytosol of the cell in their chemically reduced state as cysteine, and not oxidized as cystine. Otherwise, the cell cannot assemble the chain of amino acids properly and join the cysteine residues to their proper partners to form the proper tertiary structure of the molecule. I therefore proposed then that the perforin deficit in NK cells and CD8 cells in ME/CFS is due to glutathione depletion in these cells. If this is true, one would expect that the gene expression of the perforin gene (as measured by the level of messenger RNA corresponding to the PRF1 gene) would not be below normal, because the glutathione deficit would impact the protein synthesis process downstream of gene expression.

Recently, a group at Bond University in Australia (lead author Ekua W. Brenu) published a paper entitled "Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis":

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120691/pdf/1479-5876-9-81.pdf

In this paper, together with a following one:

http://www.translational-medicine.com/content/pdf/1479-5876-10-88.pdf

they reported that the NK cell and CD8 cell cytotoxic activities were consistently low in ME/CFS patients, but the messenger RNA for the perforin gene PRF1 was significantly higher in both cell types in the PWMEs than in the normal control subjects.

I suggest that this new result is consistent with the mechanism I have proposed for low perforin in ME/CFS. It indicates that the NK cells and CD8 cells are "trying hard" to produce perforin by boosting the transcription of the PRF1 gene to messenger RNA, but the protein synthesis process is at least partially blocked and cannot respond. If glutathione is somewhat depleted in the NK cells and CD8 cells, the protein synthesis process would indeed be partially blocked.

I continue to propose, as I did in 2007, that other features of the observed immune dysfunction in ME/CFS can also be explained by the GD-MCB hypothesis. These include the shift to Th2 immune response, the elevated RNase-L and formation of the low-molecular-weight RNase-L, the elevated inflammation, the failure of lymphocytes to proliferate when stimulated with mitogens, the reactivation of viruses and intracellular bacteria, and the accumulation of pathogens over time.

Best regards,

Rich

 

[nanonug]

I continue to propose, as I did in 2007, that other features of the observed immune dysfunction in ME/CFS can also be explained by the GD-MCB hypothesis.

There is one question that has been bothering for quite sometime now regarding the GD-MCB hypothesis. If glutathione depletion were the main culprit, wouldn't simply supplementing with N-acetyl cysteine solve the problem? Although I have no doubt in my mind that glutathione depletion is a problem, I am not yet entirely convinced that it is the problem just by itself when it comes to the persistence of the CFS condition.

As for your astute observation regarding elevated expression of PRF1 RNA, I would like to add another tidbit of data. PRF1 has a bunch of probable-pathogenic polymorphisms. In my case, I am heterozygous for rs35947132 (PRF1 A91V), which is a probable-pathogenic polymorphism associated with reduced perforin function. Wouldn't these polymorphisms explain at least partially the observed immune system screw up?

 

[Sushi]

Hi, all.

...As you may also know, natural killer cells normally kill cells that are infected with viruses. They do this by secreting a substance called perforin, which makes a hole in the cell membrane, and then injecting granzymes, which induce the cell to undergo apoptosis (programmed cell death). The CD8 cytotoxic T cells operate in the same way in terms of their killing mechanism.

Some years ago, Dr. Kevin Maher, who was in Dr. Nancy Klimas's group, reported that the natural killer cells in PWMEs are low in perforin. It was also found that the CD8 cytoxic T lymphocytes were also low in perforin. This would, of course, inhibit their cytotoxic activity.

The question then became "Why are the NK and CD8 cells in ME/CFS low in perforin?

In 2007, when I proposed the Glutathione Depletion-Methylation Cycle Block (GD-MCB) hypothesis, I noted that the perforin molecule has a large number (20) of cysteine residues in its protein structure. It is known that in order for a cell to be able to synthesize a protein that contains cysteine residues, it must have sufficient glutathione and a high enough ratio of reduced to oxidized glutathione to keep the cysteine molecules in the cytosol of the cell in their chemically reduced state as cysteine, and not oxidized as cystine. Otherwise, the cell cannot assemble the chain of amino acids properly and join the cysteine residues to their proper partners to form the proper tertiary structure of the molecule. I therefore proposed then that the perforin deficit in NK cells and CD8 cells in ME/CFS is due to glutathione depletion in these cells. If this is true, one would expect that the gene expression of the perforin gene (as measured by the level of messenger RNA corresponding to the PRF1 gene) would not be below normal, because the glutathione deficit would impact the protein synthesis process downstream of gene expression.

Recently, a group at Bond University in Australia (lead author Ekua W. Brenu) published a paper entitled "Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis":

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120691/pdf/1479-5876-9-81.pdf

In this paper, together with a following one:

http://www.translational-medicine.com/content/pdf/1479-5876-10-88.pdf

they reported that the NK cell and CD8 cell cytotoxic activities were consistently low in ME/CFS patients, but the messenger RNA for the perforin gene PRF1 was significantly higher in both cell types in the PWMEs than in the normal control subjects.

I suggest that this new result is consistent with the mechanism I have proposed for low perforin in ME/CFS. It indicates that the NK cells and CD8 cells are "trying hard" to produce perforin by boosting the transcription of the PRF1 gene to messenger RNA, but the protein synthesis process is at least partially blocked and cannot respond. If glutathione is somewhat depleted in the NK cells and CD8 cells, the protein synthesis process would indeed be partially blocked.

I continue to propose, as I did in 2007, that other features of the observed immune dysfunction in ME/CFS can also be explained by the GD-MCB hypothesis. These include the shift to Th2 immune response, the elevated RNase-L and formation of the low-molecular-weight RNase-L, the elevated inflammation, the failure of lymphocytes to proliferate when stimulated with mitogens, the reactivation of viruses and intracellular bacteria, and the accumulation of pathogens over time.

Best regards,

Rich

Thanks Rich,

It is very interesting to see perforin's role in this.

Just a clinical note: in Jan 2011 my perforin mRNA expression (is this the same as messenger RNA for the perforin gene PRF1 ?) was measured at 2062 with a range of 250 - 750 copies/ml. After a year of treatment (which included elements that might well help a methylation block and thus raise glutathione), my perforin mRNA expression was 433.

Thanks for your thoughts.
Sushi

 

[aquariusgirl]

Wow, you are doing something right.

 

[richvank]

Hi, nano.

According to the GD-MCB hypothesis, which was based on research done in autism, glutathione depletion is indeed not the whole story. It's part of a vicious circle mechanism. For five years, from 1999 to 2004, I encouraged PWMEs to try to boost glutathione directly, and NAC was one of the approaches. It didn't work, and I didn't know why, until I read the paper in late 2004 by S. Jill James et al. The problem is that the glutathione depletion is associated with a partial block of methionine synthase, which is upstream, and it's necessary to correct that in order to bring glutathione up on a permanent basis. A brief outline of the whole GD-MCB hypothesis, as it currently stands, is pasted below.

Thanks for the information on your PRF1 polymorphism. I don't know if that would account for low perforin production, or whether it would just produce less functional perforin molecules. I think that either would lower the cytotoxicity. The original paper by Kevin Maher et al.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440524/pdf/cei0142-0505.pdf

actually measured the amount of perforin using anti-perforin antibodies. I don't know whether these antibodies would bind to perforin molecules that have the polymorphism you mentioned, so I guess the question you raised is unresolved.

It may be unlikely that a large fraction of the PWME population would have this polymorphism,and if so, it could not explain why the NK cell cytotoxicity is low in such a large portion of the PWME population. Do you know what the frequency of it is in the general population?

Best regards,

Rich


Here's a brief outline of the whole GD-MCB hypothesis, as it currently stands (always subject to modification as we learn more):


1. Genetic predisposition is present (perhaps including SNPs in genes coding for enzymes related to glutathione that cause it to be more easily depleted, as reported in autism by Bowers et al., 2011).

2. Stressors (physical, chemical, biological and/or psychological/emotional, the mix varying from one case to another, based on patient histories) deplete glutathione by various means, some by oxidative stress, some by conjugation, some by lowering its rate of synthesis. The depletion of glutathione is demonstrated by the methylation pathways panel.

3. The state of oxidative stress worsens as a result of the depletion of glutathione, and peroxynitrite rises, due to reaction of rising superoxide with existing nitric oxide.

4. Glutathione depletion lowers the affinity of the CblC complementation group for cobalamin (as reported by Jeong and Kim, 2011), producing a functional B12 deficiency, thus lowering intracellular methylcobalamin and adenosylcobalamin. Anecdotal observations of elevated urine methylmalonate in the presence of normal or elevated serum B12 in ME/CFS patients confirm the presence of a functional B12 deficiency.

5. The lowered methylcobalamin inhibits the methionine synthase reaction, since it is the necessary coenzyme for this reaction.

6. The methyl trap mechanism continues the conversion of other forms of folate into methylfolate, but the lowered rate of the methionine synthase reaction decreases the demand for it.

7. The elevated peroxynitrite catabolizes methylfolate, preventing its rise in the plasma.

8. The above process depletes the intracellular folates in general (as inferred from measurements with the methylation pathways panel).

9. Homocysteine drains into the transsulfuration pathway, since its conversion to methionine is inhibited, and over time, methionine therefore becomes depleted (as found by Bralley and Lord,1994), leading to dysregulation and depletion of the sulfur metabolism in general.

10. The above combination of steps produces a stable vicious circle mechanism, and this is the reason ME/CFS is chronic.
11. Treatment must include a high dosage (relative to the RDA) of a form of vitamin B12 delivered to the bloodstream, such as sublingually or by injection, together with an RDA-level dosage of folate, which can be given orally. The high dosage of B12 is necessary to compensate for the greatly lowered affinity for cobalamin of the CblC complementation group, so as to overcome the functional B12 deficiency, and the oral route is not adequate to supply this necessary high dosage (first reported by Lapp and Cheney, 1993 and 1999). The folate is necessary to compensate for the loss of methylfolate from the cells due to the peroxynitrite catabolism reaction. The B12 is best given as hydroxocobalamin or methylcobalamin. The folate is best given as methylfolate, though folinic acid works for some patients. There are individual differences in genetic polymorphisms that determine the best forms of B12 and folate for individual patients. If there are deficiencies in cofactor vitamins and minerals or in necessary amino acids, these must be supplemented in addition. Replacement of oxidatively damaged essential fatty acids is also needed. If toxic metals levels are high enough to significantly block enzymes in this part of the metabolism, chelation may be necessary before this treatment will be successful.

12. This treatment is directed at the core of the pathophysiology. However, it does not directly treat the etiologies ("stressors" in step #2 above) that brought about this pathophysiology, nor does it directly treat pathogens and toxins that may have accumulated since the onset of ME/CFS, while the body's immune and detoxication systems have been dysfunctional as a result of the dysfunction of the sulfur metabolism. Additional treatments are needed in most cases to deal with them directly, to work toward achieving full recovery, because even though the immune system and the detoxification system may be largely restored, they are often not able to overcome these etiologies and accumulated factors on their own. Some of the etiologies and accumulated factors in various cases are Lyme disease and its coinfections, biotoxin illness, entrenched viral infections (and perhaps retroviral infections), and high body burdens of toxins.

 

[richvank]

Thanks Rich,

It is very interesting to see perforin's role in this.

Just a clinical note: in Jan 2011 my perforin mRNA expression (is this the same as messenger RNA for the perforin gene PRF1 ?) was measured at 2062 with a range of 250 - 750 copies/ml. After a year of treatment (which included elements that might well help a methylation block and thus raise glutathione), my perforin mRNA expression was 433.

Thanks for your thoughts.
Sushi

Bravo, Sushi! Yes, that's the same. Thanks for the information. It fits very well. Can you tell whether your defense against viruses has improved?

Best regards,

Rich

 

[richvank]

Wow, you are doing something right.

Hi, AQ.

Maybe so! It's always nice when predictions come true!

Best regards,

Rich

 

[nanonug]

The problem is that the glutathione depletion is associated with a partial block of methionine synthase, which is upstream, and it's necessary to correct that in order to bring glutathione up on a permanent basis.

I guess one of the problems I have is with the qualitative (so far) nature of the hypothesis. Unless there is a "bistable mode" in the dynamics, I would expect increased intracellular levels of glutathione, just by themselves, to eventually correct the methylation block due to reduced oxidative/natrosative stress. Would you not? If not, why not?

Thanks for the information on your PRF1 polymorphism. I don't know if that would account for low perforin production, or whether it would just produce less functional perforin molecules. I think that either would lower the cytotoxicity.

Based on my readings, that particular polymorphism would lead to reduced perforin functionality. My immune system would therefore be shooting a lot of blanks.

It may be unlikely that a large fraction of the PWME population would have this polymorphism,and if so, it could not explain why the NK cell cytotoxicity is low in such a large portion of the PWME population. Do you know what the frequency of it is in the general population?

According to Varview info on Pubmed (link on my original message) the risk allele 'A' has a population frequency of 2.1%.

 

[Sushi]

Bravo, Sushi! Yes, that's the same. Thanks for the information. It fits very well. Can you tell whether your defense against viruses has improved?

Best regards,
Rich

Oh good! It is great when theory and clinical experience dovetail.

It is hard to know about defense against viruses, although my reactivated EBV has gone down to nearly in range. I was never a patient who caught things--in fact I was one who never caught anything--on the noticeable level--though I'm sure my immune system had its guns drawn.

I no longer feel "yuck" very often--which could be a sign of better defense against viruses, whereas before treatment, "super yuck" was my default state in the morning. My immune panels are mostly in the normal range now. So far so good!

Thanks,
Sushi

 

[richvank]

***Hi, nano.

I guess one of the problems I have is with the qualitative (so far) nature of the hypothesis. Unless there is a "bistable mode" in the dynamics, I would expect increased intracellular levels of glutathione, just by themselves, to eventually correct the methylation block due to reduced oxidative/natrosative stress. Would you not? If not, why not?

***I've wondered about that a lot. It may be that it is just very difficult to get glutathione up high enough by direct boosting, perhaps because ongoing inflammation produces enough oxidative stress to keep it from going up enough.

***Or, it may be that there is sort of a "bistable mode" due to the complex feedback regulation within the sulfur metabolism.

***One thing that intrigues me is the positive results that some of the patients had in the Norwegian Rituximab study. If the important effect of the Rituximab in this disorder is that it diminishes the inflammation for a while, which is what I suspect, then that would suggest that getting rid of the load on glutathione would allow it to rise enough to correct the block, as you suggest. There were also a couple of people who reported that going to bowel tolerance dosage of vitamin C brought them to recovery. That might be another way to raise glutathione sufficiently. I don't know.

***What I do know is that treating to lift the partial methylation cycle block does bring glutathione up in most PWMEs, because we measured it.


Based on my readings, that particular polymorphism would lead to reduced perforin functionality. My immune system would therefore be shooting a lot of blanks.

***That does sound like a problem for your defense against viruses and maybe tumor cells, too.

According to Varview info on Pubmed (link on my original message) the risk allele 'A' has a population frequency of 2.1%.

***Well, that's larger then the prevalence of ME/CFS, and this polymorphism would be important early in the pathogenesis for people who have a viral onset, so I can't dismiss this as clearly an unimportant factor. I guess we will have to see if other PWMEs have this polymorphism, too (or another one that renders perforin ineffective).

***Best regards,

***Rich

 

[richvank]

Oh good! It is great when theory and clinical experience dovetail.

It is hard to know about defense against viruses, although my reactivated EBV has gone down to nearly in range. I was never a patient who caught things--in fact I was one who never caught anything--on the noticeable level--though I'm sure my immune system had its guns drawn.

I no longer feel "yuck" very often--which could be a sign of better defense against viruses, whereas before treatment, "super yuck" was my default state in the morning. My immune panels are mostly in the normal range now. So far so good!

Thanks,
Sushi

O.K., Sushi. Thanks. Indeed, so far so good!

Best regards,

Rich

 

[alex3619]

Hi Rich, how many of those cysteines in perforin are involved in sulfide bridges? Misfolding is a likely result if reduced glutathione is depleted, and this will probably inactivate the perforin that is present. In other words, perforin levels could be normal or near normal and still have low perforin activity. Bye, Alex

 

[richvank]

Hi Rich, how many of those cysteines in perforin are involved in sulfide bridges? Misfolding is a likely result if reduced glutathione is depleted, and this will probably inactivate the perforin that is present. In other words, perforin levels could be normal or near normal and still have low perforin activity. Bye, Alex

Hi, Alex.

I don't know how many of the cysteines are involved in sulfide bridges, but I suspect that quite a few, and maybe all of them, are. And yes, glutathione depletion will likely cause misfolding of the protein structure. I should note that there is a quality control system built into the endoplasmic reticulum of cells, so that many of the misfolded proteins are detected and recycled via disassembly of the amino acids in the proteasomes, but some are probably released, and yes, they might have low activity.

Incidentally, I have invoked this same mechanism to explain why some other secretory proteins, such as some of the peptide hormones, are deficient in ME/CFS. I think that vasopressin and oxytocin are in this category, and I think that ACTH is, also. All of these have cysteine residues that have to form particular disulfide bonds to produce the proper tertiary structure of their molecules. Any secretory protein that normally has disulfide bonds and is formed in a cell that is depleted in glutathione will have this problem. I think the hypothalamus and pituitary are depleted in glutathione. On the other hand, it's likely that the pancreas is not, so insulin is properly formed in ME/CFS. The reason the pancreas is not likely to be depleted in glutathione is that it is one of the few vital organs that have a complete transsulfuration pathway, and thus are able to make cysteine from methionine, so that they can maintain glutathione synthesis when the body as a whole is having problems maintaining its glutathione inventory.

I also think it's possible that some of the autoimmune activity in ME/CFS may result from the immune system trying to clean up these misshapen proteins. To go a step further, that may be why Rituximab helped some people. By knocking out the B lymphocytes, the antibodies will be decreased over time, and that should lower the autoimmune reactions and the accompanying inflammation with its oxidative stress. Maybe that allows glutathione to come back up and restore things.

Best regards,

Rich

 

[Little Bluestem]

I should note that there is a quality control system built into the endoplasmic reticulum of cells, so that many of the misfolded proteins are detected and recycled via disassembly of the amino acids in the proteasomes,

So we are spending some of our limited energy making and disassembling misfolded proteins?

 

[heapsreal]

is there anything on interferon as interferon is said to improve nk function, does the glutathione depletion theory been studied on its effects on interferon. Dr chia has had some success with interferon therapy as well.

cheers!!!

 

[Snow Leopard]

is there anything on interferon as interferon is said to improve nk function,

Were you thinking of these trials?

http://www.ncbi.nlm.nih.gov/pubmed/8675231
http://www.ncbi.nlm.nih.gov/pubmed/8354926

 

[heapsreal]

Were you thinking of these trials?

http://www.ncbi.nlm.nih.gov/pubmed/8675231
http://www.ncbi.nlm.nih.gov/pubmed/8354926

That will do, good find.
a third of patients improved in a study mentioned in a book i have(reviving the broken marrionette), not sure if it was referring to those studies or not. A third suppose to improve with ritux. I wonder if the cfs criteria is only good at diagnosing a third of what we know as cfs/me??
it mentions that the cfs group improved in nk function, do u know if this improved cfs symptoms etc?

 

[globalpilot]

My frtustration in reading this is that it's so darn expensive to get NK cell function tested. I just did mine at UNEVX and it was over $300 plus shipping plus blood draw. Ideally, I"d like to have it tested everytime I test glutathione or isoprostane but it just isn't feasible for me and probably most of us.

 

[heapsreal]

My frtustration in reading this is that it's so darn expensive to get NK cell function tested. I just did mine at UNEVX and it was over $300 plus shipping plus blood draw. Ideally, I"d like to have it tested everytime I test glutathione or isoprostane but it just isn't feasible for me and probably most of us.

My nk test was free, i got my tests done in the study the rich posted the link to. no commercial nk function tests in australia

 

[mellster]

Great thread - I am also wondering whether any of the efforts of the body to compensate for this could lead to the increase in cytokines, something I still don't understand. I noticed when I take a lot of immune stimulant I also experience more inflammation which is not always pleasant. Now that energy/exhaustion is not really an issue for me anymore I am beginning to wonder whether there are some tweaks to be made to get to full recovery. I also still don't understand why a lot of patients never catch any of the common circulating seasonal illnesses, of which most are of viral nature as well so the Th2-shift cannot explain this.