Valerie M. Hudson, Ph.D.
Department of Political Science
Brigham Young University
valerie_hudson@byu.edu
Revised version, November 10, 1999
[Acknowledgements: The following individuals generously provided
assistance and information: Myron Seligmann, Keith Crawford, John Richie,
Benjamin Gaston, Jonathan Stamler, Melanie Childers, Paul Linsdell, Larry
Lands, Teri DeWolfe, Cor Veldhuizen, Cindy Ray, George Eckel, and Laura
Gould. This essay is dedicated to the memories of Richard Andrew Young
and Keith Childers, whose hunger for life continues to inspire.]
Several new research findings have prompted a rethinking of the major causes of cystic fibrosis (CF) pathology. This rethinking centers around the role of reduced L-glutathione (GSH) in inflammatory response, immune effectiveness, antioxidant capability, and mucolysis in the human body. Normally, GSH is made by virtually every cell in the body. A redox equilibrium for glutathione is established within each of these cells, and the efflux of glutathione from the cells establishes a redox equilibrium outside of the cells as well. This extracellular redox equilibrium varies by bodily system. For example, the normal level of extracellular GSH in the lung epithelial lining fluid (ELF) is 140 times the normal level of extracellular GSH in blood plasma [47]. The lung is a net importer of circulating GSH [47].
Researchers have long noted a systemic extracellular deficiency of GSH in individuals with CF [52], which is similar to other diseases such as AIDS [66]. The deficit in adults with CF can be quite severe, with GSH ELF levels dropping to 5-10% of normal levels, and GSH plasma levels approximately 50% of normal [11, 59]. (In children, extracellular GSH levels in the ELF are lower, but not significantly lower, than normal, but over time with stress to the lung system, the deficit becomes chronic and significant [60].) Generally speaking, this deficiency was formerly attributed to the abnormally high pathogen burden in CF. However, three new research findings, all dating from 1998 or 1999, have provided a different answer to the puzzle of extracellular GSH deficiency in CF. In addition to explaining that extracellular deficiency more precisely, the synthesis of these three research findings argues for a serious reconsideration of the primary causes of CF pathology and invites novel therapeutic approaches.
Let us discuss each of these three new findings, though we will place them out of chronological order for ease of discussion.
1). Gao and colleagues discovered a normal level of intracellular GSH in CF epithelial cells [10].
2). Lands and his colleagues discovered a decreased level of intracellular GSH in peripheral blood lymphocytes of CF persons [57].
3). Linsdell and Hanrahan discovered that chemical clamping of the CFTR channel resulted in cessation of GSH efflux from cells (baby hamster kidney cells) [1].
Reflection upon these three intriguing findings is very profitable in a theoretical sense. If each of these findings is accurate (and replication studies are in fact under way to make that determination), one begins to realize that a very odd situation can be hypothesized to exist in the bodies, and particularly the lungs, of CF persons. Because CF persons have a missing or defective CFTR channel through which organic anions are normally effluxed, most of their cells should not be able to export GSH very efficiently if at all since GSH is an organic anion. This situation can be presumed to lead, over time, to a severe extracellular deficit of GSH, while simultaneously offering a possible explanation why Gao and colleagues found that CF lung epithelial cells exhibit normal intracellular GSH levels. According to this nascent theoretical framework, most of the CF body's cells will be unable to efflux GSH, thus decreasing the amount of GSH available extracellularly and leading to a build-up of GSH within the affected cell. Of course, this does not preclude simultaneous consumption of any available extracellular GSH by the tremendous free radical burden of the disease CF, which consumption also encourages extracellular deficit. However, this is no chicken-or-egg conundrum: in a theoretical sense, the genetic defect comes first, and underlies the eventual overwhelming free radical burden.
But there is another part to this story. The finding of Lands and his colleagues demonstrates that there are some cells in the CF body still capable of GSH efflux. What cells would those be? Clearly, the only cells capable of normal GSH efflux in CF would be those cells possessing an anion channel redundant to the CFTR channel. The most likely candidate for that redundant channel is the one created by the MRP protein [61], though there are other channels, such as the cMOAT channel, that would also functionally retain this capability. So now the question becomes, what cells in the body normally express the MRP protein? (All cells are ultimately capable of expressing MRP, but only under extreme conditions, such as that of poisoning or chemotherapy. Indeed, the approach of one French research team is to rectify the CF condition by inducing MRP production in normal body cells through use of a hopefully manageable toxin, in this case, colchicine.)
The cells in the body which normally express MRP include hepatic cells (in which are also found the cMOAT channel) and all cells derived from hemocytoblasts, including such cells as erythrocytes, neutrophils, monocytes (and thus macrophages), and lymphocytes (including both T-cells and B-cells). It appears that the cells most heavily involved in transport in and/or detoxification of the body are endowed a wide variety of redundant channels with which to fulfill their functions effectively, even under conditions of duress.
With this understanding in mind, we can now theorize how it might be that Lands and his colleagues found decreased intracellular GSH levels in peripheral blood lymphocytes of CF persons. These lymphocytes are MRP-expressing cells, and it is reasonable to assume that they will attempt to efflux GSH in order to help achieve redox equilibrium of extracellular GSH. It is also reasonable to assume that because the extracellular deficit becomes so large over time (not only because of the genetic defect, but also due to the heavy consumption of GSH because of the free radical burden characteristic of CF), these MRP-expressing cells eventually prove unable to fully rectify that deficit by themselves. If these assumptions are correct, then the end result would be that the MRP-expressing cells of the CF body are likely to find themselves in a chronic state of intracellular GSH depletion. In the remainder of the essay, we will focus on those MRP-expressing cells that are part of the immune system, in particular neutrophils, macrophages, T-cells, and B-cells [44, 45, 46]. However, we pause to note that GSH depletion in hepatic cells is strongly correlated with cirrhosis of the liver, a common pathological development in CF [78. 79, 80, 81].
At this point, we can recap what the theoretical framework leads us to believe about GSH levels in CF bodies. If this theoretical framework is for the most part accurate, most cells -- those which do not normally express MRP (at least at the apical surface) -- will exhibit normal intracellular levels of GSH because the defective CFTR channel does not permit them to efficiently efflux GSH, if at all. Over time, a chronic and severe extracellular deficit of GSH should and does appear. This deficit is further encouraged by the consumption of GSH due to the heavy free radical burden that develops because of the initial genetically-caused deficit. Nevertheless, MRP-expressing cells will still be able to efflux GSH, but since these cells are in the minority they will be unable over time to fully rectify the extracellular GSH deficit. However, their persistent efforts to do so will result in chronic depletion of intracellular levels of GSH in these MRP-expressing cells, including the immune system cells.
This is what the theoretical framework leads us to believe we will find.
The results presented by Gao et al., Lands et al., and Roum et al. provide
prima facie empirical evidence to support this conceptualization.
However, while waiting for replication of their findings, we can provide
additional support for the theoretical framework by examining the types
of pathology that would be expected to arise from this abnormal situation,
and ask whether the predicted pathological consequences bear any similarity
to typical CF pathology. If so, a strong theoretical foundation will have
been laid for a new therapeutic approach to CF. Let's take each aspect
of the situation in turn.
Though GSH conjugation of cytotoxins is still taking place, it is unclear whether the lack of GSH efflux will affect the clearing of such conjugates from the cell. We hope this issue will be empirically investigated.
2) MRP-expressing cells will develop chronic intracellular GSH depletion over time.
Here the findings are much more numerous. The effects of chronic GSH depletion in T cells, B cells, macrophages, and neutrophils have been the subject of much research. What is the effect of chronic GSH depletion in these types of cells? Chronic, excessive inflammation coupled with immunodeficiency. Intracellular GSH levels in these inflammatory-regulating cells are the major "switch," if you will, of inflammatory response. GSH deficiency in such cells is related directly to increased transcription of NF kappa-b [30, 31], as well as elevated levels of IL-8 [32]. GSH deficiency in these cells is also linked to elevated levels of IL-4 [34], also linked to an immunodeficient Th2-type host response that promotes the adhesion of bacteria like Pseudomonas a. [35, 36]. Indeed, such excessive inflammation accompanied by immunodeficiency is a hallmark of chronic GSH depletion in immune system cells. To give but one example, GSH deficiency leads to T cell inactivation and apoptosis [42, 43]. The signal to "kill" is not well transmitted in the context of GSH depletion, and pathogens will not be effectively cleared [77]. Immunodeficiency leads to creation and recruitment of larger numbers of NK cells, leading to increased oxidant burden and elastase damage [52]. A normal intracellular level of GSH seems to be required for optimal PMN activity. Neutrophils themselves are modified and crippled when the GSH level is abnormal [53]. Leukocytes in CF patients show oxidant damage, and in one study were shown to be unable to kill bacteria as effectively as normal leukocytes [54]. Other studies show that a depleted intracellular GSH level decreases parameters of PMN activity, such as superoxide generation, release of lysosomal enzymes, depressed leukotaxis, phagocytosis, and even migration of PMN [55, 56]. The above observations do not begin to exhaust the list of all immune system dysfunctions linked to GSH deficiency. Furthermore, excessive inflammation accompanied by immunodeficiency has been found to be independent of pathogen burden -- this syndrome is constitutive in CF patients, found to be beginning even in the youngest uninfected infants [48, 49, 50, 51].
3) A chronic and severe extracellular deficiency of GSH will develop over time.
This circumstance leads to a number of harmful effects in the extracellular environment, including loss of antioxidant capability, loss of mucolytic ability, depletion of lung surfactant, degradation of the antiprotease system because of increased oxidant burden, lowered inhibition of lipid peroxidation, and so forth [47, 37, 38, 39, 40, 41]. Other, less investigated dysfunctions also arise: for example, extracellular GSH deficiency helps create a deficiency of S-nitrosoglutathione (GSNO), an important "reservoir" of NO for the body and a potent bronchodilator in its own right [12, 13, 14, 15, 16]. Without the reservoir of GSNO for NO produced by iNOS, NO is rapidly metabolized into nitrites and nitrates [33]. Though cells can still produce GSNO, the production of GSNO extracellularly is also very important in keeping GSNO levels normal. Thus we can see that there are important independent effects of extracellular GSH deficiency.
Let us take a closer look at some of these effects. First, let us understand the role of extracellular GSH:
a) being the major small molecule water soluble antioxidant in the lungs, GSH neutralizes harmful oxidants introduced into the lungs or released by cells such as neutrophils into the lungs.
b) GSH protects the antiprotease system. Alpha 1-antitrypsin (a1-AT) is the major antiprotease of the lower respiratory tract and secretory leukoprotease inhibitor (SLPI) is the major antiprotease of the upper respiratory tract. Since oxidants can inactivate antiproteases, the antioxidant activities of GSH serve to shield the antiproteases from degradation [37].
c) GSH is necessary to maintain lung surfactant, probably through its role in the maintenance of adequate levels of phosphatidycholine, the main component of lung surfactant [40].
d) When there is ample GSH in the ELF, GSH is regenerated to provide long lasting antioxidant protection. This regeneration process involves the reduction of oxidized glutathione (GSSG) through the activity of glutathione reductase and NADPH. (Glutathione reductase, NADPH, and glutathione peroxidase are present in at least normal quantities in the blood of CF persons [53, 18].
e) Mucolysis is greatly reduced. GSH is one of the body's most powerful mucolytic agents, capable of cleaving disulfide bonds. Indeed, end-stage AIDS patients, whose ELF GSH levels drop to levels comparable to CF adults, develop a jelly-like consistency to their mucus [25].
f) There may be other important activities of GSH as well, those these
have not been as thoroughly investigated. For example, an abnormal level
of GSH in the ELF may affect the level and diffusion of NO species, in
turn affecting both the oxidant burden and antimicrobial capacity [62].
Additionally, glutathione deficiency may cause decreased numbers of type
2 lamellar bodies, lamellar body damage, mitochondrial degeneration, capillary
endothelial swelling, damages skeletal muscle, and decreased amounts of
intraaveolar tubular myelin [40].
What can we expect in a condition where a chronic and severe deficit of extracellular GSH develops over time?
a) The lung antioxidant system is crippled. Though other elements of the system (e.g., catalase) may be present, small molecule oxidants are not effectively neutralized. These oxidants damage lung tissue directly.
b) The lung antiprotease system is degraded. The small molecule water soluble oxidants, unchecked by sufficient GSH, inactivate a1-AT and SLPI, creating an imbalance between neutrophil elastase (NE) and the antiprotease system [38, 39]. NE then attacks the elastin of the connective tissue in the lung. Barbero notes, "The pathological effects of NE, demonstrated in a number of in vitro and in vivo investigations, include cleavage of fibronectin, lung elastin, immunoglobulins, and immune complexes, complement receptors in neutrophils, and other receptors on T-cells and B-cells. Furthermore, NE inhibits ciliary beating and stimulates mucus production from goblet cells and facilitates P. aeruginosa adherence. Finally, by cleaving receptors for interleukin (IL)-1 and IL-2 or the T cell antigen receptor, it may hypothetically inhibit message transmission and immune recognition. Thus, besides destruction, [free NE in CF] may lead to acquired immune suppression [63]."
c) Lung surfactant cannot be maintained at appropriate levels, providing increased opportunities for pathogens to assault the lung.
d) When GSH in the ELF is deficient, the body decreases reduction of GSSG back to GSH, and instead further oxidizes GSSG into chloramines, which are long-lived oxidants. Thus, though GSSG levels in CF ELF are in the low normal range 52], the reduction of that GSSG is not favored by the body in a GSH deficient environment [64, 65]. Indeed, in CF persons an elevated level of glutathione reductase (implying non-use of the enzyme) has been noted [17].
e) Decreased mucolysis leads to thickened mucus, especially in the context of vastly increased recruitment of neutrophils and other immune system cells.
f) Increased lipid peroxidation will occur due to the increased oxidant burden in CF. Since a marked lipid imbalance favoring arachidonic acid has been noted in CF, increased lipid peroxidation due to GSH deficiency will worsen the consequences of this imbalance. [82]
g) Other, less studied consequences include lowered NO levels coupled
with higher levels of nitrites and nitrates [12, 13, 14, 15, 66, 33], plus
lower levels of GSNO.
-- chronic inflammation
-- immunodeficiency
-- increased recruitment of neutrophils and other immune cells
-- thickened mucus
-- decreased antioxidant capacity, with increased oxidant damage
-- decreased antiprotease capability, with increased elastase damage (including inhibition of ciliary beating)
-- decreased lung surfactant
-- decreased NO and GSNO availability, causing decreased effectiveness of pathogen killing coupled with decreased availability of the body's natural bronchodilator
-- increased lipid peroxidation
-- impaired immune message transmission and signalling
-- increased damage to connective tissue
We submit that all of these pathological events can be found in CF. There is a high correlation between the effects of GSH depletion in immune cells and in the extracellular environment in non-CF cases and the pathology of CF itself. We believe that this is a strong theoretical foundation upon which to rethink effective CF therapy.
We respectfully submit that given these facts, it is reasonable to propose the hypothesis that augmentation of extracellular GSH is essential in CF -- both in and of itself and in conjunction with therapeutic approaches designed to normalize intracellular GSH levels in immune system cells.
Extracellular augmentation of GSH has been accomplished through intravenous (or injection) administration of GSH, oral ingestion of GSH, and inhalation of nebulized GSH. All have been shown to elevate not only blood levels of GSH, but also to elevate GSH levels in other areas of the body, such as the lungs. This is accomplished through the body's importation of circulating GSH into areas where the loss of redox equilibrium is most drastic. Since the deficit of GSH in the ELF of CF persons is so severe, elevation of blood levels of GSH insures that transport of GSH to the ELF will occur, and this has been documented in studies mentioned below.
Intravenous (or injection) administration of GSH. This therapy is not used as often in the United States, but is very common in Japan and is also practiced in Europe. When used in the United States and Europe, GSH sodium salt solution is used intravenously to combat chemical or radiation poisoning. In Japan, injectable GSH is used in allergy treatment, as well. To our knowledge, it has never been used to treat disorders such as CF. We urge examination of this approach. Though one study examining the use of IV GSH suggests that the effect is short-lived, this has been disputed by other scholars [2]. IV GSH was proven to raise not only blood levels of GSH, but also ELF levels of GSH, as well [2].
Oral Ingestion of GSH. The oral ingestion of GSH has often been overlooked as an effective route of augmentation of extracellular GSH for a number of reasons. The first centers around the dispute over whether GSH is cleaved or destroyed in the digestive tract, or whether GSH can be taken up intact from the duodenum and jejunum and transported into the bloodstream. Fortunately, the number and sophistication of recent research articles demonstrating that GSH is taken up intact from the small intestine outweigh those denying that such uptake occurs. [68, 69, 70, 71]. Some researchers have asserted that oral GSH must be accompanied by oral ascorbic acid in order to assist in that uptake, and this should be investigated. Furthermore, blood elevation of GSH after such ingestion has been noted to be of 3-4 hours in duration [25]. Remember that blood elevation of GSH does in fact lead to ELF elevation of GSH [2].
The second reason for overlooking oral ingestion of GSH lies in the nature of the disorders for which such augmentation is usually recommended. In other types of diseases involving glutathione depletion, such as AIDS, there is no CFTR problem. Thus, increased production of GSH is favored over rectification of specifically the extracellular deficit of GSH because all cells are physically capable of effluxing GSH as needed. Thus it is easier to simply boost production and allowed that increased production to solve the extracellular deficit in natural fashion. However, in CF, most of the cells of the body cannot efflux GSH well if at all. Boosting production for the MRP-expressing cells is all well and good -- and hence our advocacy of cysteine augmentation as well -- but the MRP-expressing cells by themselves cannot rectify the type of severe extracellular deficit experienced by CF adults. It is necessary to simultaneously overcome that extracellular deficit, else the added cysteine will never result in intracellular redox equilibirum of GSH in the MRP-expressing cells. Thus, not only is oral ingestion of GSH arguably effective, it is also theoretically justifiable. We urge closer examination of this route of delivery of GSH, as well.
Inhalation of Nebulized Glutathione. Because of the uncommon use of IV (or injected) GSH in the United States, and because of the disputes over the bioavailability of orally ingested GSH, some researchers have turned to an investigation of direct inhalation of aerosolized GSH as an alternative approach to augment extracellular GSH levels in the ELF.
There have been eight in vivo studies of inhaled GSH, one a murine study and the other seven human studies [2, 3, 4, 5, 6, 7, 75, 76]. Human subjects ranged in age from 4 years old on up to mature adulthood. Only one small in vivo study of seven subjects has specifically examined CF patients [76]. This study found that certain inflammatory markers significantly decreased after three days use of inhaled glutathione. In addition, one in vitro study using CF sputum found that the addition of GSH caused the reduction of baseline O2- by approximately 90%, and reduction of PMN-induced burden by approximately 46% [29]. These two studies provide some encouragement that should a larger in vivo trial on CF patients be performed, at least some of the desired and predicted effects would be forthcoming.
The studies cited above demonstrated a significant and transient elevation of GSH in the ELF. Both degree and duration of elevation was dose-dependent [22]. When a smaller dose was used (600 mg), elevation over baseline persisted for approximately 3 hours. However, when a much larger dose was used (anywhere from 2-4 grams of GSH), GSH level was still many fold times above baseline at the 3 hour point, and did not return to baseline for some time afterward [2, 7, 22]. Six of the seven studies used free acid GSH in an isotonic saline solution (150 mg/ml). One study used pH-adjusted GSH sodium salt in isotonic saline solution [7]. All studies used pharmaceutical grade GSH (minimum 98% pure).
Bronchoconstriction was noted in one study [6], which was eliminated by the prior inhalation of salbutemol. Another approach to the bronchoconstriction problem is the use of pH-adjusted GSH sodium salt. Free acid solution GSH has a pH of approximately 3.0. Understanding that inhalation of low pH substances can induce lung irritation [8], the use of the sodium salt is worthy of investigation. In one in vivo study, a 2.4 gram dose of inhaled GSH could be tolerated when the pH was adjusted to 7.0 by use of the sodium salt [7]. Other possibilities for modification include delivery by liposomes to lengthen duration of elevation [72, 73, 74], as well as the inhalation of a slower release crystalline form [2]. Maintenance of isotonicity of the solution is no doubt desirable as well [9].
The inhalation of free acid GSH was put under use patent by the United States government [21]. However, that patent lapsed in 1997 and was not renewed. As of this writing, it appears the use of inhaled reduced glutathione is in the public domain. There is some evidence that there is still a use patent in effect for Europe, which would require the payment of a small fee. GSH itself, of course, is not patentable, as it is a substance naturally produced by the body. Pharmaceutical grade free acid GSH can be obtained for approximately $40 for 25 grams.
A group of twenty-four CF individuals used inhaled GSH for periods ranging from 2-12 weeks. Their experiences, including both adverse reactions and positive health benefits observed, can be viewed in the paper, "Preliminary Results of the Use of Inhaled Reduced Glutathione by Twenty-Four Individuals with Cystic Fibrosis," available at http://members.tripod.com/uvicf/gsh/results.html
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