Update Jan 2008

Mal's Musings

Malcolm A Traill

IN  THE  PUBLIC  INTEREST
Updated 28/1/2008

 

LITHIUM

AND

NON-PSYCHIATRIC DISEASE

 

Historical Note

My first interest in lithium and its medical uses was in 1969. I had become associated with the laboratory at Royal Park Psychiatric Hospital, Parkville, Victoria, Australia. Dr John Cade was the Psychiatrist Superintendent, and many readers should be aware that it was Cade who, in 1949, published his findings on the use of Lithium for manic-depressive conditions (now called bipolar disorders). By a combination of serendipity and astuteness, Cade found that Lithium was probably the first effective mood stabilizer. Having discovered this, he left the further experimental work to others, preferring to pursue general clinical psychiatry. However, there was maintained considerable clinical interest in Lithium, including a special outpatients’ Unit for those having Lithium and, with Cade as the Superintendent, a general feeling that we were in the company of God. From that environment I published two items regarding Lithium[1],[2].

In about 1987-8, there were being introduced changes in the structure of pathology in Australia (see the website chapter on Medical/Clinical Pathology). To me, for any Pathologist to have any valued decision-making rôle in the future, required that Pathologists attend and consult with patients. Accordingly, I started understudying an old acquaintance, Dr Roy Bean, a Consultant Physician and Oncologist. He introduced me to Lithium in Oncology; he would give cancer patients one Lithium tablet a day as an “immune stimulator.” (Roy was an original thinker in medicine. He emphasized the need to observe patients carefully, and to monitor and document meticulously. This applied to every individual patient in order to obtain a complete and sound longitudinal study; the patient partly being the patient’s own control.) In 1989, my employment with a laboratory was such that I had plenty of time at my disposal, so I started reviewing the topics associated with Oncology, including the literature relating to Lithium and immunity. Sadly, Roy developed a cancer himself, and I acted as his locum tenens when he became sick. At about that time two events occurred which were memorable :

           i            A female breast cancer patient, probably about 40 years old, was found to have a leukoerythroblastic pancytopenia, which was pretty clearly due to secondary breast cancer spread to the bone marrow. Feeling that Roy’s Lithium protocol was somewhat homeopathic, I set out, with a prescription, for Lithium to be taken in slowly increasing doses. From memory, it was something like one tablet (250 mg Lithium carbonate) per day for three days, then two tablets a day (separated) for two days, and then three tablets (separated) per day. However, there was some misunderstanding involving the Pharmacist and/or the patient, and what actually happened was that she took one tablet three times per day, then two tablets three times per day, then three tablets . . . until there was a frenzied telephone call from the distraught husband to say that his wife was delirious, unable talk, could not sit or stand, and was rolling around on her bed. The serum Lithium was measured at 2.2 mmol/L. All further Lithium medication was ceased forthwith (never to be given to her again) and fluids pushed. She slowly recovered from the Lithium toxicity, with apparently no further harmful effects. But the surprise was that she then went on to survive for an extraordinarily long time, even to Roy’s amazement. (She did have other forms of Chemotherapy such as cis-Platinum.) When the cancer did reappear, it was in an unusual situation, I recall a site such as the spinal cord. Naturally, I wondered if the Lithium could have contributed to the surprisingly good survival.

         ii            Another patient with colon cancer metastasized to the lungs, was given Lithium in a protocol as one would see in Psychiatric outpatients. Roy always liked to measure the basic lymphocyte surface markers. I was less enthusiastic about measuring them, because I never felt that I knew what they indicated (other than gross indicators). I did not feel that therapies did anything much to change them, particularly for the perceived better. So, this patient had lymphocyte surface markers measured from time to time. On one attendance, he seemed to be showing features of elevated serum Lithium levels (coarse tremor, unsteady). Accordingly, his Lithium was stopped. Shortly after this he had another lymphocyte marker assessment, and there seemed to have been noticeable changes. It was then that I wondered if an effect of Lithium may be revealed when the Lithium serum levels fall. From this, I developed the concept that the rise in serum Lithium sets off a cytokine cascade, stimulating the described production of TNFα (and possibly other cytokines) from monocytes[3] which, in turn stimulates the production of GM-CSF from the reticuloendothelial system, which then stimulates granulopoiesis and the production of neutrophils and monocytes from the bone marrow, producing the well-known haematological consequence of Lithium treatment. But, whilst the Lithium is present in the serum, it may be blocking the effect(s) of some of the cytokines (etc.) that may have been produced in the cascade subsequent to the first peak. Whilst the stimulated production of TNFα by Lithium in close proximity to cancer cells seemed attractive in the world of Oncology and a glib justification for patients, it posed a major problem elsewhere, because TNFα, being a pro-inflammatory cytokine, would be expected to make conditions with an inflammatory basis worse. Fortunately, that does not seem to be the case, making the Lithium rôle in such conditions a puzzle.

About this time, a former associate was diagnosed with Multiple Sclerosis (MS). From my reading of the literature and my understanding of Lithium’s immunological effects as described up until 1991 and, given that there was no effective treatment (other than high dose corticosteroids for relapses) I suggested that she may wish to try Lithium. I felt that it was likely to do something. The only problem was that I did not know whether that something would be good or bad, and the published scientific studies showing TNFα production stimulation boded ill. A check of the literature indicated that Lithium had been given to MS sufferers with affective disorders, but no-one seemed interested, or had observed, anything particularly noteworthy regarding the effects (if any) that it had on the actual MS disease process.  She was told of all this, and was prepared to give it a try and, accordingly, was started on a slow release preparation. At the end of a week, she reported no detectable deterioration, but felt there may be of slight benefit, but the effects (if any) were not appreciable. With the possibility that the fall in the serum Lithium level may be important, standard Lithium tablets were then tried in a bolus dosage form, with an interval left for the serum level to fall (later drinking large quantities of water to try to flush it out faster in the urine). She reported that this seemed to abort relapses and, under guidance, she drew-up her own protocol, based upon the principles that have been outlined. (Her story and photographs are in the Chapter on the NH&MRC Submission on the website.) It was about then that I came across the paper by Levine and Saltzman (1991)[4]. This extraordinary study, at last, put some semblance of order into the empirical use of Lithium for non-psychiatric conditions. The authors demonstrated that an alternate-day bolus treatment regime (with some variations) could prevent experimental allergic encephalomyelitis in rats, whereas a daily dosing regime did not. The study seemed to provide an in vivo experimental, animal-based justification for the applied fall in the serum Lithium level, and a delay in a subsequent dose, even though a more detailed explanation, obviously, was wanting.

I was interested in Lithium because, whilst its immediate non-psychiatric biochemical effects seemed to be suppressive, as on gene transcription[5] and yet, on many downstream products, there seemed a net stimulation (eg the Lithium-induced neutrophilia, and Th2 responses[6]. This last reference has a brief review of the literature of animal studies involving inflammatory conditions. The study used ex vivo human blood cells in a medium with 5 mmol/L Lithium for 5 days; quite artificial. Shorter term incubations seemed to favour Th1 effectors.) Lithium is quite unlike most medications, in that it produces a net stimulation of some key cytokines. Since the early cases, I have tried Lithium, in the sorts of regimes described herein, on patients with a wide variety of non-psychiatric conditions, chiefly of inflammatory type or malignancy (with the patients’ knowledge and approval). Being in a primary care setting, there was no opportunity to establish trial groups. The patients had to be assessed carefully and individually, in longitudinal studies – it could take years to find a suitable index case. (All were advised of the experimental nature of the treatment and, provided the treatment protocols were followed, could carry effectively no risk. For many of the patients, I carried a supply of lithium tablets, and provided the patients with the exact number needed for the days involved – usually 3 tablets per day on alternate days – so they had no way of developing a toxic blood level. [Any short-term large dose or a large bolus results in vomiting the tablets, making significant toxicity from such doses next to impossible].)  Accordingly, the results of these treatments can only be presented as potential avenues to examine in more detail by those who may wish to pursue the possibilities. The conditions include :

 

               Clinical conditions for reasonable conclusions

Condition        Number*  Outcome (presumptive)

Sarcoidosis

2

Helpful, “Lithium works every time.” èlong remission

Hepatitis C

1

Helpful (2 treatments 3 year apart; see website)

Rheumatoid  arthritis

3

Helpful, can maintain remissions; see Chapter NH&MRC

 Submission. (Observations/trials over ~10 year for one)

Lupus erythematosus

2

Seemed helpful in acute relapses, and may have been helpful in reducing maintenance corticosterone treatment.

Temporal arteritis

2

Unhelpful

Polymyalgia rheumatica

2

Unhelpful

Cancer

1

Helpful (see website)

Multiple sclerosis

1

Helpful. May abort relapses and reduce progression (over ten years)

*The number represents memorable cases, where the cooperation, compliance and follow-ups were of sufficient validity and veracity to permit some attempt to draw a presumptive conclusion in longitudinal assessments. One patient with Rheumatoid arthritis and another with MS were studied closely over ten years or more. Naturally, the critics and cynics will scoff and sneer at these numbers but, in an extended and careful study of patients, major confounding factors such as compliance, matching negative control groups and observer variability are eliminated largely, allowing pointers for further study to be identified.

 

Pause

After that brief, historical account, the emphasis will now turn to the examination and discussion of immunology, using the old and more recent literature. If you are not reasonably acquainted with immunology, then don’t bother – just look at the graphs and tables. A brief listing of the points covered is :

 

Summary

a.      Lithium, when administered for some days to mice, causes cellular dissolution of the thymus gland

b.      Why this occurs is discussed

c.       Lithium is known to block certain signal pathways that use the G-proteins

d.      Lithium does have documented hyperacute and acute effects on some transcription factors. These may explain how it can have effects when administered in bolus doses

e.      There are a number of pathways that Lithium may inhibit; that of the Wnts is currently topical

f.        Current immunology requires an understanding of the regulatory T cells (Tregs & TR1). Much of this work is very recent and current.

g.      An explanation for the enigma that Lithium can, in some contexts be an immune stimulant and, in other contexts, be an immune suppressant, may come from the description of two types of regulatory T cells, those from the thymus (natural Tregs) and those spawned in the lymph nodes associated with local disease (eg cancer; nodal TR1)

 

Lithium effects

Later, Péret-Cruet and Dancey (1977)[7] provided experimental evidence that, under the right condition, Lithium can induce cell death. They administered Lithium chloride twice a day intraperitoneally into 30 g mice for four days, and then examined the thymus glands. The glands showed a dose-dependent weight reduction, with the residual weight, after using the highest dose, being some 12.9% of the untreated weight. The workers did not examine the process at the cellular level. However, we can graph their results :

GRAPH 1

 

There seem to be two levels of sensitivity :

·        Very sensitive cells, which are eliminated by 1-3 mEq (= mMole) and

·        Less sensitive cells, progressively eliminated by doses >3 mMole.

Given that the thymocyte maturation stages are now better understood[8], such differential sensitivity is not unexpected. (The possible changes in progenitor homing and egress of intact cells seem likely to be insignificant.)  Of particular interest, is the scale of the cell dissolution (apoptosis ?), which can hardly be regarded as the negative selection of “forbidden clones;” rather, the wholesale dissolution of cellular contents, to be processed by dendritic cells and macrophages.  These results may be compared with those obtained by Silverstone et al. (1994)[9], Staples et al. (1998)[10] and Zubkova & Mostowski (2005)[11]:

Thymic dissolution by medications

 

                                                       Thymus cells__                                    Thymus cells__                            

Authors            Dexamethasone   Low      At          Oestradiol                  Low        At

Silverstone et al.

5 mg/kg statim

~18%

~3 day

75mg/kg statim (in oil)

~2%

~24 day

Staples et al.

5 mg/kg statim

~2%

  2 day

5 mg/kg statim (in oil)

~20%    

~5 day

Zubkova et al.

12.5mg/kg statim

~7.1%

~4 day

5 mg/kg statim (in oil)

~5.2%

~7 day

 

 Likely cellular signalling factors in the thymus

 

Cell-to-cell (Zubkova, Mostowski et al. 2005)       (Typically produced by epithelial cells, macrophage/stroma, thymo/lymphocytes, as demonstrated by other workers)

            MHC II+, CD45-        MHC II+ CD45+ CD11c+       After Dexamethasone

Cell      Stromal cells                 Dendritic cells   *                      Unknown         change

            IL-7                             IL-7                                         MIG                 x13

            SDF-1α                       TECK                                      RANTES         x12

            SLC                                                                             Fractalkine       x6.7

                                                                                                MCP-2                        x6.3

                                                                                                SDF-1α           x5.4

                                                                                                SLC                 x4

                                                                                                TECK              x3.8

                                                                                                IP-10               x3.2

            Not measured: BAFF/APRIL, Wnts,

  SCF, IL-1α, GM-CSF, IL-4, IFNγ[12]

Intracellular Factors 

            Ras pathway

*Dendritic cells are here included with macrophage/stromal cells

 

Whilst there may be some difficulty equating the changes after Lithium with these other thymus involution-inducing agents, we may assume that the processes involved more closely resemble those for Dexamethasone, rather than Oestradiol, especially given the speed of the reduction; showing a sensitivity very roughly equivalent to Dexamethasone 10 mg/kg. Such cell losses after Lithium and Dexamethasone can hardly be by immunological cellular “attack.” The most likely explanation is that the withdrawal or blocking of one or more cytokines or chemokines induces apoptosis, as seen with cytokine or hormone withdrawal and the cessation of cell-cell contact signals (anoikis[13]). Silverstone et al. (1994) concluded that the Single Positive (SP) thymocyte population (CD3int CD4+; immature) was the most Dexamethasone-sensitive group of cells in the thymus, with CD4+ CD8+ (immature) not much different, making the former and the latter possibly the sensitive and less sensitive thymocytes respectively, as demonstrated in the graph of the results by Péret-Cruet and Dancey (1977) (see above).   Silverstone et al. (1994) and Staples et al. (1998) reported demonstrable evidence of apoptosis on the first day only after the Dexamethasone administration; peaking at 12 hour, largely abated by 16 hour, and that it was blocked in mice with mutant Bcl-2+ thymocytes. This indicated that, in Dexamethasone-induced thymic atrophy, the Bcl-2-inhibiting apoptosis pathway is involved.

The impression is, that the Dexamethasone effect, which has virtually eliminated thymic cellularity by two days and with no recovery until after 3 days, is probably faster in its effects than Lithium, although no time course for the latter was shown. The Lithium effects were appreciably faster than those for oestradiol, which did not appear to involve apoptosis. This means that, whilst Lithium may involve a reasonably fast terminal apoptosis pathway, such as that activated by Dexamethasone, there may be slower points at the initiation of the pathway, as may be expected if the response is activated by the withdrawal of a stimulant.

There are possible ways that Lithium may induce thymic involution :

·        A direct inhibitory effect upon thymocytes. This appears unlikely, because Lithium has been used for in vitro studies for years without any appreciable effect being noted at physiologically relevant levels over days

·        An effect upon surrounding cells, such that there is a net stimulation of target cells by the release of cytokines into their surroundings (paracrine &/or autocrine). There are numerous studies documented, where lithium causes such a net stimulatory effect and, in the case of TNFα, its cytotoxicity effects are also accentuated by Lithium[14],[15]. The work by Wu & Cai (1992)[16], using Lithium both in vitro and in vivo, indicated that it could induce an anticancer effect alone, and that this effect can be augmented by the addition of IL-2, largely attributed to LAK cell activation. Presumably, the Lithium effect is achieved by blocking one or more intracellular inhibitors.

·        The inhibition, or blocking, of stimulatory cytokines that maintain the survival and/or proliferation of cells such as thymocytes[17], (particularly since Pertussis toxin, which blocks G-protein-linked receptors, inhibits the migration of DP thymocytes[18]), but possibly extending to lymphocytes and cancer cells. This effect has not been considered generally, but may be relevant in the thymus, at least, also, possibly in germinal centres and in the stroma-cancer relationship and the cancer stem cell-differentiated cell relationship. For these, the presumption is that Lithium blocks the cytokine (or other) receptor complex at the recipient plasma membrane, depriving the receptor cell of the supporting cytokine or chemokine signal needed for full cellular function and, possibly in some circumstances, triggering apoptosis. Such involvement appears relevant in this analysis.

Most of the chemokines involve receptors that are linked to G-proteins[19], as are the receptors for sphingosine-1-phospate (S1P), groups that are important for thymocyte/lymphocyte egress and homing[20].  The pathways involving G-proteins (GTP-associated) are considered effector sites for at least some of Lithium’s effects, with lithium at 0.6 mmol/L shown to block G-protein activity during 15 min in an in vitro system[21], with the Gαs and Gαi &/or o implicated. In vivo tests were performed but, sadly, the Lithium treatment was for ~2-3 weeks, without considering an acute effect. Other workers followed[22],[23], the latter reporting that Lithium at 2 mmol/L could suppress two pathways in calcitonin-treated osteoclasts; one involves quiescence, and is cAMP-dependent and mimicked by Cholera toxin, the other being Calcium-dependent cell retraction, mimicked by Pertussis toxin. Chronic Lithium treatment was also believed to inhibit a tonic inhibitory action of the Gαi-protein’s effects on adenylate cyclase[24], up-regulate the mRNA of adenylate cyclase and G-proteins of the Gαi1&2 ­type[25]. However, the precise site(s) and mode of action(s) were generally unclear. By 1994-5, there was recognition that there was an appreciable difference between acute Lithium effects (~ ≤ 1 week, vague and relative) and chronic effects (~ 5-6 weeks, vague and relative). There were specified receptor-associated enzyme systems: G-proteins, the phosphoinositide cycle[26], which relies upon an inadequate replacement of inositol transported from the bloodstream to the brain and embryo, and not applicable to joints and cancer. The perivascular inflammation in MS would also be expected to break the barrier to inositol movement[27].) Adenylyl cyclase (producing cAMP)[28] can be inhibited, as may protein kinase C (PKC, with isoenzymes PKCα and PKCε implicated) and gene expression modulations (eg fos) by uncertain means[29],[30]. Whilst most attention was given to the chronic effects, there was mention that early (hyperacute) dosing stimulated some PKC-mediated responses (as ACTH production by transformed and non-transformed pituitary cells[31], plateauing at ~3 h), mimicking the effects of Phorbol ester, but later these effects were down-regulated. There can be proteolytic degradation of PKC with a half-life of 2.4 h in rat glioma cells[32]; reasonably fast, similar to the desensitization process. Such a biphasic response may well be significant in the hyperacute (~<6 h) setting of bolus lithium administration, but has not been studied to any great degree; and there is little to find specifically relating to such administration. However, there seems enough documentation to suggest that hyperacute effects of Lithium on essential cell functioning can be expected and are not implausible. Later attention was transferred to the putative enzymes and genes that are affected, chiefly in the chronic administration protocols[33][34],[35], with the latter providing a recent summary of the literature and recent findings regarding chronic Lithium administration which, presumably, may have some relevance to acute administrations :

Chronic Lithium targets

                                                                    G-proteins

                                                                    cAMP

                                                                    PKA/PKC

                                                                    MARKS

                                                                    GSK3

                                                                    AP-1 ↑

                                                                    AP-2 ↓

There was the conclusion that chronic Lithium dosing is the only treatment form relevant to Bipolar Disorder, which may be correct. This approach was to the degree, that Lithium is not mentioned in two other comprehensive reviews on G-proteins[36],[37]. However, the Lithium-G-protein stage of signal transduction was not ignored by others and, currently, work seems to support belief that there may be a defect in the GRK3 inhibition of the G-protein-coupled receptors in Bipolar Disorder and that these hyper-activated G-proteins/receptors are indirectly down-regulated by Lithium, not only in the human brain, but also in peripheral tissues[38]:

Ø      With GRK3 localizing to the plasma membrane in the frontal cortex of chronically treated rats given oral Lithium for 6 weeks[39].

Ø      With Dopamine D2-like receptor-related activity, PLA2 levels and mRNA decreased in the rat brain after chronic Lithium dosing; the summarized literature indicating that Gαi1 & Gαi2 activities/levels were reduced by chronic Lithium treatment, whilst Gαq was unaffected[40].

Ø      NMDA*-initiated PLA2 activation in the rat brain was inhibited by chronic Lithium dosing[41], considered due to Calcium signalling affecting cPLA2 by  various possible mechanism, and including reduced cPLA2 and mRNA levels[42]. *Subtype of Glutamatergic agonist, ~75 % of mammalian brain synapses

Ø      Chronic Lithium administration to rats caused a significantly increased glucose metabolic activity, particularly in the frontal cortex and basal ganglia, which could be reversed by the acute administration of quinpirole[43], a Dopamine receptor agonist. The reasons for this were unclear. Since chronic Lithium seemed to block the acute quinpirole stimulation of arachidonic acid metabolism involving PLA2 activation[44], there is the possibility of different pathways. However, if chronic Lithium could, by blocking Calcium mobilization away from intracellular stores such as the ER, result in a saturation of Calcium-binding sites, such as Sfi1p, which is located in the centriole, there may be an increase in the organelle’s baseline tone for (say) Calcium-wave driven centrin contraction in the centriole. By this means there could be increased glucose channelled through the Needham pathway (see elsewhere on the website) when Calcium waves stimulated by Insositol-1,4,5-triphosphate are produced in the receptor processing. Such an outcome may be encouraged elsewhere than in the brain, possibly by inhibition of liver Fructose-1,6-bisphosphatase[45], an enzyme in the glucose neogenesis pathway leading from pyruvate.

These effects seems to involve the translocation of PKC, which can be influenced, in turn, by local intracellular Calcium and the Phosphatidylinositol responses involving the Lithium-sensitive enzyme Inositol monophosphatase[46] (Atack et al, 1995, see earlier), which has a Ki of 0.8 mM (Phiel & Klein, 2001, see earlier), or an IC50 of 0.5 mM with Phosphatidylinositol and 0.4 mmol/L for Phosphatidylinositol-4-phosphate[47], being the extracellular Lithium levels assessed using Chinese hamster ovary cells. (More recent studies have drawn attention to the effects that chronic Lithium dosing [6 week] has on membrane lipids, particularly in relation to arachidonic acid and PGE2[48], as opposed to subacute dosing [~1 week]. Rarely is hyperacute/acute in vivo dosing studied. See later).

So, in the thymus, the basic components are present for Lithium to induce apoptosis by “neglect” via inhibition of signal transduction by one or more maintenance growth/differentiation factor(s). The challenge now is to identify the participants and how they operate in the acute situation, as they must surely do!

The studies by Zubkova et al. (2005) of thymocyte regeneration after involution that was induced by Dexamethasone may help identify some cell-to-cell factors that Lithium may affect – Oestradiol effected similar cytokines, but to a lesser degree. Sadly, the workers did not measure BAFF/APRIL or the Wnts. More recently, Ayroldi and Zollo (2007)[49] concluded that the appreciable effects of glucocorticoids were to associate with a receptor (GILZ) and then inhibit the small GTP/GDP-binding protein Ras and Raf’s downstream targets of the ERK pathway. Just how Lithium, initially an inhibitor, could initiate such a pathway is by no means clear, although cross-talk between pathways is possible[50]. However, the unblocking of a Lithium-blocked (G-protein-dependent) pathway, such as the Wnt pathway, could stimulate components of the mitogen activated pathway[51], producing results similar to those starting at GILZ. and, unless some method of activating the receptor(s) concerned can be identified, this pathway, with its dependence on clathrin-coated vesicles, and its variant form with G-protein receptors[52] may best be kept in reserve.

What may be more promising could the Wnt signal pathways which can direct differentiation of embryonic development and stimulate stem cell proliferation and self-renewal[53] (at least). In the thymus gland Wnts would seem very important[54], yet have been difficult to study until recently. In particular, the decreasing levels of Wnt signals from Wnt-1 and Wnt-4 resulted in a progressive failure of thymocyte proliferation. Since the proportions of the various maturation populations stayed essentially the same, the conclusion was that the most immature stages failed to proliferate. Based upon mice with deficiencies of TCF-1, the deduction would be that, without all Wnt signals, there would be close to 0-2 % thymocyte population left, when compared with the wild-type animals. This could provide a basis for the Lithium effect, if the production &/or (more likely) the receptor machinery of Wnts in the thymus are inhibited or blocked, probably by G-protein inhibition (see earlier). Not only would there be a lack of stimulus for the prothymocyte population, but there would, very likely, be the prompt onset of apoptosis resulting from the deprivation of the proliferation stimulus.

Reports on the Wnt pathways are rapidly increasing. Whilst it is of particular interest here, other pathways may be relevant – this presentation is not necessarily dedicated to one. The Wnt pathway will be examined in detail because it illustrates principles that seem important and features that other intracellular messengers may also display.

Further consideration of Lithium, GSK3b and the Wnt receptor system brings attention to certain problems :

a)      The pharmacological range for serum Lithium in humans is generally given to be (with some variations) ~0.5 - ~1.2 mmol/L[55]. Whilst some undesirable effects, such as coarse tremor, unsteadiness, vagueness, polyuria, diarrhoea and nausea may be noticed in the upper reference range and higher (~1.4 – 1.6 or 1.7 mmol/L), levels above this cannot be allowed for any length of time because of the clinically apparent toxicity and potential for renal damage.

b)      The Ki (50% inhibition) for GSK3b in vitro is reported to be ~2.1 mmol/L[56] and another group obtained a value ~ 1.5 mmol/L[57] (~3 mmol/L for the Drosophila homologue; both values considered “physiological !” – for cell culture). The former group notes that the intracellular level of lithium is ~ 5 % of the extracellular level. This, in part, explains why most workers subject in vitro cell systems to 20 mmol/L of extracellular Lithium, a level that rapidly would kill any living mammal exposed to it. Even so, the intracellular level might be only ~1 mmol/L (below the Ki). Where various extracellular Lithium levels are given for work on GSK3b, rarely are levels below 2.5 mmol/L provided. (A few examples: where the augmentation of TNFα cytotoxicity seems to be ~ 50% when the Lithium concentration is as low as ~2.5 mmol/L for a few malignant cell lines[58]; where the stimulated 50% production of b-catenin from the GSK3b inhibition seems to be at an extracellular Lithium concentration of ~7.5 mmol/L[59]. All these levels, if in serum, would be incompatible with intact mammalian survival and seemingly irrelevant to clinical practice.)

c)      All the above comments on lithium levels are difficult to reconcile with the findings by Bosetti, Seemann et al. (2002, and that group’s other publications using shorter dosing intervals) which note that, in the chronically-dosed rats (over 2 week duration), the plasma levels and brain levels of Lithium are essentially the same. If the intracellular level were ~5% of the extracellular level, there would be expected to be a dilution factor with the brain levels due to intracellular fluid. Perhaps the ~5% is only applicable to the acute in vitro situation, in which case, the intracellular level would be likely to be rising at an undeclared rate throughout any experiment. Just how lithium enters the cells is not clear. Perhaps, in the acute setting, enzyme systems (eg GPCR/G-proteins) near the plasma membrane may be the most vulnerable for inhibition by lithium, particularly in an inflammatory condition such as MS. An extension of this thinking would be that hyperacute or acute Lithium effects cannot occur in a normal brain. Such a conclusion would assume that there is a relatively steady state, which, obviously could not be the case at the blood peak following a bolus dose.

d)      The ancient and highly conserved Wnt pathways[60] have recently become an important study, particularly since Lithium can act, as far as the effector end of the pathways is concerned, as a Wnt substitute in vitro. The Wnts have mixed and confusing associations with cancer, including “cross-talk[61], and there is the possibility that some of the G-proteins involved may act as oncogenes[62] :

Breast (in vitro cell line & transformed line)[63]     Wnt5a Increased differentiation and matrix adhesion

Breast[64]: Increased Wnt5a protein expression in DIC; reduced in IDC, associated with metastasis/poor prognosis; no difference in invasive Lobular Breast Cancer

Gastric Cancer[65] in humans may produce Wnt5a and its expression augers a poor prognosis, being associated with cell migration. Whilst it does not seem to be associated clearly with the causation of gastric cancer, the more recent seminal revelations concerning the homing, engraftment and transformation of bone marrow-derived stem cells into the chronically inflamed gastric mucosa of mice[66], emphasize the potential roles of stem cells in cancer, and the importance of the factors that may provide both growth and differentiation stimulation to stem cells and their early progenitors, compared to the cytokines stimulating the more differentiated cell lines: hence the current interest in the Wnt pathway and its ramifications.

Malignant melanoma.  Wnt5a stimulation through Frizzled has been implicated in metastatic aggressiveness[67],[68], apparently via the non-canonical pathway and involving PKC. The effects seem to change actin orientation and cell shape towards a more spindle (sarcomatous) outline. In the cell lines with induced Wnt5a production, blocking Frizzled negated the stimulus, meaning that an involved G-protein-involved pathway might be susceptible to Lithium in this context. 

Colon cancer[69] may produce increased amounts of NFAT, an intermediate in the Fz-2/Calcium (non-canonical) pathway[70], usually involving the G-protein components Gα0 & Gαt2 (Malbon & Wang, 2001, see earlier)  stimulating COX-2, which goes on to stimulate production of prostaglandin E2 (PGE2) – not a desirable product in cancer treatment, because PGE2 has a pronounced immune-suppressive effect, especially at the transcription of IL-12[71]. (Neuronal NF-κB may also be involved in brain COX-2 transcription[72], possibly also involving, or influenced by NFAT and Calcium[73],[74]). Since Gαo is mainly found in the brain and Gαt2 in the retina, this pathway may not be well expressed in other tissues, and may be less relevant with respect to Lithium modulation. Interestingly, the level of the PGE2 end product was still rising at 24 h but, sadly, measurements were stopped then. This type of delayed end product rise could, in part, explain the need for the alternate day (~48 h) Lithium treatment regime that was tested by Levine & Saltzman (1991, see earlier) and raises the issue of the potential importance of effector precedence, which could involve the promoter AP-1[75] (an NFATn), and the cell types involved – for example, in lymphocytes which express Fz5 and receive Wnt 5a signals[76], the pathway involves NFAT (which is a general gene “switch” with a corresponding intranuclear NFATn) and produces IL-2, an immune stimulant, but this is produced earlier than the colon cancer cells could produce PGE2 in appreciable amounts. Perhaps the earliest NFAT product may take effector precedence by modulating other genes, such as that for Fas ligand[77] in Jurkat T cells and breast cancer cells, (which aids apoptosis), although chronic lithium treatment of rats caused an inhibition of PGE2 activity, considered to be due to post transcriptional or post translational changes (Bosetti et al. 2002[78], & Basselin et al. 2005 & 2006, see earlier). This means that, in vivo, the undesirable rebound PGE2 production that may follow an acute bolus of Lithium may be suppressed, particularly if it involves plasma membrane receptor mechanisms upstream of cPLA2 and COX-2, the latter being located in the Endoplasmic reticulum, at the nuclear membrane and intranuclear[79]; not near the plasma membrane where the initial receptor activity all happens.

 

GRAPH 2

Graph 2. Wnt stimulated transcription activity (luciferase; red) shows a rapid increase at ~2 h, a plateau from ~3 to ~8 h, then a decline (possibly biphasic) to a level which may represent the steady state (Ma & Wang 2006, see later). The desensitizations due to GRKs and arrestins are usually described as occurring in the first 2 hour, and do not seem to be operating here. The production of PGE2 (lavender) shows an initial rise similar to the NFAT luciferase transcription, but later becomes more pronounced, probably because the COX-2 (the transcribed protein) activity becomes less dependent upon transcription for rate activity (Duque & Fresno 2005). (cLPA2 and COX-2 may be indirect targets for Lithium in the more chronic setting at least - Bosetti et al. 2002 & Basellin et al. 2005 & 2006). The phosphorylated and activated DDR1 (light green) shows a rapid rise, with no appreciable evidence of desensitization. It is still ~80% active at 16 h, and probably still reasonably active at ≥ 24 h (Jönsson & Andersson 2001[80]). The Lithium-activated binding of AP-1 and CRE are shown (dark blue & dark green), the former showing an hyperacute rise (Ozaka & Chuang 1997[81]). The Lithium-activated product of AP-1 (Yuan et al. 1998) is shown (dark red). It shows a slower rise, and still rising at 24 h. It, and NFAT could combine to present a late activating transcription combination. The Lithium-stimulated c-Jun (sky blue) activity starts to rise by 4 h, with an appreciable rise by 10 h (Yuan et al. 1999[82]). The serum lithium levels are followed with time, and show what may be expected after a bolus dose of 750 mg Lithium carbonate in an overweight adult human (blue), with levels at 0.8 mmol/L and 0.4 mmol/L indicated. The peak is at ~1 h, after which the level falls to ~0.4 mmol/L at 4 h (Reiss, Haas et al. 1994[83]). The level is in a range that may inhibit G-protein-associated functions or induce hyperacute effects over an interval of ~3 h.

Significantly, the actions of Wnts may differ at differing times of early development[84], a feature that may be extrapolated to cancers, which are usually considered to represent primitive levels of differentiation.

e)      Relatively unknown is the effect of Lithium on the receptor molecules involved in the Wnt signalling. Frizzled-1 & Fz-2 molecules are the main cell surface receptors[85],[86],[87], which associate with Dishevelled and G-proteins in the initial reception process. Since Lithium is considered to block some G-protein-associated pathways, interest can be drawn to the Wnt pathways, both canonical and non-canonical. In the cGMP/Calcium Wnt pathway with Frizzled-2, there is activation of NFAT, a lymphocyte activation transcription factor (with more general actions), apparently downstream of a suppressed PKG and elevated Calcium, reaching a peak over about 7 h and returning by 20 h. This study supports the delay seen after Cyclosporin A-treated lymphocytes are stimulated by Wnt from apposed endothelial cells, where there is a delay of 8 – 12 h for IL-2 production, being the result of NFAT activation (Murphy & Hughes 2002, see earlier). Just where this time delay occurs is not clear, but it would seem to be well down the pathway, probably in the suggested steps of Calciumè Calcineurinè NFAT. The sensitivity (if any) to Lithium is not tested, but there was sensitivity to Pertussis toxin, an inhibitor of the Gα family members, of which Gαt2 and Gαo (more associated with retina and brain respectively) seemed targets in this study, with stage times :

 

                                                                     Stage activation times

                                                                     _____Time to_____

Stage -activated            To                    Change           Peak

Wnt5a/Frizzled                cGMP ↑           ~25 min            ~40 min

R-Frizzled                       NFAT ↑           ~120 min         ~8 h

R-Frizzled                       PKG ↓             ~5 min              120 min

R-Frizzled/Propranolol     PKG ↑             ~30 min            120 min

R-Frizzled/PTX               PKG ↑             ~10 min            120 min

Wnt5a/Frizzled                Calcium ↑         ~4 min              ~60 min

R- refers to the activated Chimera receptor complex[88] using isoproterenol; PTX = Pertussis toxin

 

This provides an interesting, and relatively slow response to Wnt stimulation, which may be relevant to the Lithium effects; for example, in the in vivo situation, where there may be a constitutively active Wnt5a signal, bolus Lithium, when present about its serum level peak, may block this signal. This block would cause a fall in intracellular Calcium (as it returns to ER stores or is lost from the cells) and a fall in Calcineurin/NFAT activity, with less blocking of b-catenin effects[89]. With the Lithium level falling after the peak, the Wnt5a receptor/Frizzled-2 stimulus can reactivate the pathway, but with a ~6 - 7 h delay to the peak NFAT response (with b-catenin less opposed), and a ~20+ h delay before the NFAT level has fallen to ~22% of the activity seen in an inactive pathway, as it did with the continuous Wnt stimulation in vitro. (The reason for the fall is not clear – probably the typical agonist-induced desensitization [Morris & Malbon, 1999, see earlier], but possibly also the reduced Calcium flux, substrate exhaustion, Calcium store depletion, or some other negative feedback loop. Usually the desentizations due to GRKs and arrestins are apparent in the first 2 h, so this, and other delayed falls, [such as that shown by DDR1], are not typical.) There may need to be a pause after the Calcium (or other) response (of over ~ ≥ 24 h), before the PKG/Calcium response can be repeated; the subsequent repeat perhaps being able to boost  the overall activation of (say) NFAT. After the inhibition, the constitutionally expressed Wnt would continue to provide a constant signal for receptors freed of agonist desensitization. This could mimic the situation when cells have constitutive Gαq-producing signals inserted by plasmid transfection, and this can lead to Bcl-2 sensitive apoptosis within 48 h of transfection in vitro[90], a pathway involving PKC and Phospholipase C. Such a situation may be mimicked by rebound sensitivity after an interval of Lithium G-protein inhibition. There is a similar delayed fall in the response of the G-protein–dependent DDR1 receptor phosphorylation[91] following the rapid response to Wnt5a (Jönsson & Andersson, 2001, see earlier), with a peak at ~2 h, and falling to ~79% of the peak by 16 h. Clearly, it would have appreciable phosphorylation activity for considerably longer, possibly extending past 24 h, and illustrate a persisting G-protein receptor effect that may justify an alternate-day dosing schedule.. However, the picture becomes more complicated when the existence of the other constitutional Wnts operating via Frizzled-1 is considered. Overall, when looking to biochemical pathways only for any explanation (ignoring immune-based mechanisms), the non-canonical Wnt/Calcium/NFAT pathway would seem the most promising explanation for the anti-inflammatory rôle of bolus Lithium (as described by Levine & Saltzman, 1991), and the anti-tumour (?) stem cell effect noted after bolus Lithium treatment (see elsewhere on the website. Also on the website is the hypothesis that Calcium, by causing Centrin in the centriole to contact, may activate the glucolytic pathway of Needham, producing lactate and NIR.) Clearly, more experimental work is required.

f)        The thymic atrophy induced by Lithium shown by Pérez-Cruet & Dancey 1977  may be explained by the Wnt pathways – the canonical pathway using Fz1 (or other) receptor, GSK3b, b-catenin (and other factors), because Fz1 has been demonstrated to be a G-protein-linked receptor in mammals[92], producing a transcription peak at about 7 h, and falling away to low levels at ~13 h. This makes possible the involvement, by Lithium, with the Fz1 receptor and its G-protein complexes (with Gαo & Gαq). The canonical pathway seems to be the main pathway involved in the thymus[93], and it “cross-talks” with Notch, another thymus/bone marrow stimulus[94]. The serum levels of Lithium are almost certainly too low to affect GSK3b appreciably (see earlier). Blocking the other likely non-canonical pathway would involve blocking the Fz-2, G-proteins, Dishevelled, PKC(or PKG), Calcium, Calcineurin and NFAT, leading to a IL-2 &/or PGE2 &/or other production sequence (see earlier). If Lithium has an appreciable effect upon the G-protein-linked function, it could bring about a Wnt1/Fz2 failure of IL-2 support when the level is over an inhibition threshold, as it would have been most of the time during the animal experiment. These seem very attractive potential explanations for the 48 h dose-to-dose Lithium protocol; however the authors found that Wnt 1 and, to a lesser extent Wnt 4, were the main thymic Wnts, and that the receptors found were Fz5, Fz7 and Fz8, all involved in the canonical b-Catenin pathway (see earlier). We will have to assume that Fz1 will behave essentially similarly to Fz5, Fz7 and Fz8. Perhaps there are ligands and receptors that have not been identified or studied sufficiently yet. Redundancy seems to be a feature of the Wnts and their receptors, so that Wnt1 may activate both the canonical pathway and the Calcium/Calcineurin pathway. Lithium, by blocking the receptor stage for the canonical pathway in the thymus, could precipitate an agonist-withdrawal apoptosis without recourse to other pathways.

g)      The Wnt receptor pathway seems to involve endocytosis, with the Wnt carried first to endosomes for signalling and later to lysosomes for degradation[95]. The authors were surprised when the stabilization of b-Catenin by Lithium required an intact endocytic Wnt pathway upstream of GSK3b. (An explanation was suggested that GSK3b was also involved in vesicular trafficking, perhaps independent of the Wnt pathway).

h)      Given the above, lithium may inhibit Wnt signalling by inhibiting the G-protein receptor pathway complex and also by inhibiting GSK3b-dependent vesicular trafficking (at low levels), yet stimulate Wnt signalling by blocking the GSK3b degradation of b-Catenin (at high levels that are unphysiological).

i)        There remains the possibility that extracellular Lithium, by becoming included within the newly-formed signal-related endosomes, may gain access to Lithium-sensitive sites at concentrations greatly in excess of the level in the cytoplasm generally. These would be expected to be the G-proteins and the receptor complex APC, Axin, Frizzled, dishevelled and GSK3b, within a compartment separate from the GSK3b that is involved in the degradation of b-Catenin. Just where and how Lithium can enter cells now appears an important consideration.

j)        The canonical Wnt pathway cannot be used as an explanation for Lithium’s peripheral actions in vivo. Lithium may be a useful tool for studying the pathway but, other explanations for Lithium’s actions in therapy must be sought.

k)      There have been those who considered that a major site for Lithium’s action is at the inhibition of Inositol monophosphatase[96], which upsets Calcium controls in the phosphoinositol cycle. Some effects may be similar to the effects of some Wnt isoforms[97],where Wnt5A, involving Frizzled-2, was associated with greater than additive increased intensity and incident of Calcium transients at about the time of cell divisions in the early Zebrafish embryo[98]; a response that was G-protein dependent, requiring the Gbγ subunits. The Calcium, apparently, came from the Endoplasmic reticulum stores in response to myo-inositol, there being the augmentative effect of Wnt5A and activated Frizzled-2. Since the stimulation by Wnt5A (± others) is constitutional from certain cells, as in the Thymus and cancers, the potential for Lithium to block IMPase and, to some limited degree GSK3b, the non-canonical Wnt Calcium pathway may be the major means that Lithium has its effects.    

l)        Testing the Calcium dynamics in human B lymphoblast cells from volunteers and subjects with bipolar disorder[99] indicated that acute (24 h) Lithium exposure at the pharmacologically relevant level of 0.75 mmol/L produced a small (but considered insignificant) drop in Calcium mobilization and stimulated entry into the cells, but an increase in these parameters after chronic (7 day) exposure, considered significant. There was speculation as to the site(s) of action. The interest may be in the seemingly biphasic response over the 7 day interval; a pattern that may be explained by the non-canonical Wnt pathway, with the initial Calcium change resulting from a blocking effect of Lithium on this Wnt pathway, and later a stimulatory rôle.

m)    In the thymus, stimulation by multiple Wnts seems constitutional (Mulroy & McMahon, 2002)

n)      In gastric cancer, Wnt5a (whose receptor is Frizzled-2, which is linked to a non-canonical Wnt pathway, see above) is related to aggressiveness and invasion[100].

Thus, there is the potential for Lithium to be involved in blocking either the production of a stimulating cytokine or the intracellular receptor pathway for any of a number of cytokines which, when unblocked, could result in the activation of an apoptosis pathway.

 

Specific Diseases

Possibly Important Factors (out of many)

 

Condition        Cells                                                    Factors______________________

Sarcoid[101],[102]   T cell CD4 CD8 Macro                 IL-1     IL-2     IL-6     IL-8     IL-12 

                                 Treg*                                             IL-18   MIP1α MCP1  TNFα   sIL-2R

                                                                                       sTNFRII          MIP2   IFNγ    TGFb 

RA[103],[104],[105],CD4     B cell    T cell    Macro      TNF     FKN    IFNγ    IL-6?   IL-7   

[106], [107],[108]      Treg*                                             BAFF? BCMA?           TACI? IL-1b

   IL-17?

MS[109],[110],[111]CD4     CD8αb                             Macro   IL-6     IL-6R   IL-12   CD25* p75R

 (See below)              γ/d T    Mono    B  cell    Plasma cell        Block TNFèworse  p55R     IL-17   G-CSF

                                Treg*                                              NF-IL6            NF-κB   MMP

EAE[112],[113]       Any?    Dendritic                             IL-12  IL-2      IFNγ      TNFα   LTX

                                Treg     IgG(non-specific)                 H1R(?) IL-10  Vb8.2     G-CSF

MG[114]                 Treg*                                               (CD4+CD25hi)

 

*defective suppressive function.  

p55R, p75R = p55 & p75 receptors for TNF. Several pathways lead to MAPK; (blue=inhibitory propensity)

EAE – inhibition/block of a considerable number of factors decreases activity

For MS/EAE, MAPK seems important

 

Regulatory T cells (Tregs) seem to feature with most of the conditions considered here.

Sadly, on looking at the listed factors, there does not appear to be an obvious common one - and there is the impression that these are only phenotypes. There needs to be examination of upstream events.                                                                                      

Information on the factors actively involved in the induction of MS is scanty, and there is some evidence that, in many examples, the inflammatory changes may be secondary to degeneration of the oligodendrocytes[115],[116] and loss of their processes (inner tongues). This possibly indicates mitochondrial respiration defects which may be associated with a deficiency of MAG[117], and setting off a myelin-associated disease process probably set and determined by host characteristics[118] and, based upon mouse models, the extent of myelin involvement and age of the individual[119]. Such an early change would direct interest to the genes and genetic controls (extrinsic and intrinsic), of which the Wnts could be included, rather than the downstream inflammatory cytokines that may be involved secondarily. Some pointers to the early changes may be gleaned from the upgraded genes in the apparently normal appearing white matter[120] of MS patients. This study has produced a long list of genes and the changes recorded. On scanning the large list, some notable features can be noted  :

Up-regulated genes.  

Adenylate cyclase type 1*

            Calmodulin-dependent Calcineurin A subunit α

            Calcium-Calmodulin-dependent protein kinase type IV catalytic subunit

            Calcineurin B1

            Calcium-Calmodulin-dependent protein kinase IIb

            Protein kinase Cb1

            Signal transducer and activator of transcription 6 & 3 (STAT6 & 3)

            GSK3b*

            c-Myc

            PKCε*

            APC

            PKCα*

            NSE

*May be influenced by Lithium

 

Not especially remarkable (little or effectively no change)

            AP-1

            MAP kinase kinase 4 (many of the other MAP factors seem increased)

            RANTES

            RANTES Receptor

            PLCγ1

            MAPK p38

            SDF1-Receptor (CXCR4)

            b-catenin

c-Maf

CREB

            TNFR1

 

The down-regulated genes are small in number, and generally do not excite attention.

The genes for the Wnts and NFAT did not seem to have been included in the screen.

The NFATn factors listed by Wu et al. 2007 (see earlier), include AP-1, GATA4, Jun, Fos, Foxp, MEF2, and suggested that there are probably more that are currently unknown. From all of the above, there may be an indication for the involvement of the non-canonical Wnt pathway, because of the Calcineurin, and APC (which generally decreases b-catenin stability). Supporting this are the non-appreciable rises of the gene activities for b-catenin and GSK (which may be suppressed by NFAT[121]), but the significant increase of c-Myc seems to go against the pattern, (if the correlation between b-catenin and c-Myc in thymocytes is relevant[122]). A number of those listed above may be influenced by Lithium. PKC/adenylate cyclase may also be involved. AP-1 is not appreciably increased, which would indicate that other components would have to fulfil any NFATn function. Some of the MAP pathway(s) are up-regulated (not specifically listed above). The absence of an appreciable rise in the activity for the TNF receptor and the SDF1 receptor seems to diminish the significance of the corresponding ligands. The upgraded NSE may point to some disturbance of glucose handling, the Embden-Meyerhof-Parnas pathway and/or the Needham pathway, the latter, possibly indicating a disturbance of centrosomal/microtubular function (see elsewhere on the website).

The ability for Lithium, on an alternate day dosage regime to suppress EAE (Levine & Saltzman 1991, see earlier), draws attention to recent work[123] indicating that LF 15-0195, when administered to rats at the time of receiving MBP, conferred a long-lasting tolerance that could be transferred, having some similarities to a predictive hypothesis of 1968[124], which was presented for general medical readers[125]:

 

The hypothesis revolved around two main observations, the incidence of various diseases at different ages compared to the thymic medulla and cortex relationships[126]: the thymic cortex:medulla ratio with age showed strong similarity to the age incidence of acute leukaemia and intrinsic asthma, whereas the medulla:cortex ratio matched the age incidences for extrinsic asthma, lupus erythematosus and myasthenia gravis. The second observation was from a prospective assessment of tumour tolerance: Neonatal thymectomy was performed on all of one litter of an outbred strain of laboratory mice. At about adolescence (~6 week ? – it was a long time ago !) each received a subcutaneous Millipore chamber implant: in 4, each implant contained a thymus from the same strain of about the same age, and in each of the other implants was a spleen. They then received an intraperitoneal injection of Ehrlich ascites tumour and the weight gains of the groups compared; the weight changes taken to be measures of tumour rejection (lack of tolerance) – a basic assumption[127].

 The actual graph presented in 1968 is shown below left, and demonstrates that the mice receiving the chambers with the thymi were not restored back to near normal – rather they were more different than those receiving a spleen in the chamber ! This result was not in accord with expectations based upon the then current publications and thinking, but was consistent with the proposition that the cortex was involved in active tolerance dissemination. The observations concluded that the thymic cortex was a major factor for tolerance, whereas the medulla, was largely involved in basic immunity (ie non-tolerance).

               Murine experiment 1968                                                     Hypothesis (1968)

 

 

In the hypothesis, the cortical thymocytes were viewed as bags of largely nuclear material, which was released upon cell breakdown (see above right). Prior to this, there had been exposure to antigens (+/- via dendritic/antigen presenting cells) and gene expression modifications associated with “antigen factors.”  These “factor(s)” were multiplied (amplified) and released at breakdown, to be recycled into the new progeny arriving from the bone marrow. This amplification process came from the belief that Nature would not evolve such a proliferation of cells with cell death and wastage merely to create somatic mutations, in order to feed clonal selection by positive and negative selections; the likelihood of some more creative and primitive function seemed intuitive. This conclusion seemed a great idea at the time, but needed reworking with new experimental evidence. Accordingly, this idea will be examined in what follows, with the knowledge that such a mechanism is looking for plausibility.

In 1968, Sir Macfarlane Burnet ruled, and such thinking was heresy. The “antigen factor(s)” were like “magic factor(s),” not easily explained by the knowledge of the time, and only partly explained today under the newer terms “immature dendritic cells” and “regulatory T cells[128]”. However, (at least) two tolerance mechanisms do exist and are not mutually exclusive. Such tolerance transfer by lymphocytes has been claimed (with varying recognition) from 1971[129] to the present, initially by a few, but more recently by growing numbers of workers.

Thymectomized mice with the Millipore chambers, each with a thymus within, could produce humoral factors (cytokines) to improve immunity, without the active cortical thymocyte proliferation (amplification) that would help confer and disseminate tolerance under the hypothesis. The outcome would be likely to produce a heightened immunity, less ascitic fluid build-up and weight gain – as was shown experimentally. (A journal referee’s criticism at the time was that the numbers were too low for statistical significance, not being aware that statistical methods inherently assess probabilities by incorporating the numbers involved !) An extension of the hypothesis was that lymphocytes associated with cancers may, in fact, be providing tolerance towards the cancers (as has been demonstrated later – see further on).

More current findings support the notion that thymic dendritic cells derived from prothymocytes and committed to niches[130] are involved in the thymic negative selection[131] of potential autoantigen-bearing thymocytes, which probably occurs in the medulla and the cortico-medullary region, and involving macrophages[132] for uptake of the more certainly apoptotic cells (cortical macrophages F4/80+, medullary MAC-3+; dendritic cells were not examined), whereas the more mobile and peripatetic dendritic cells are involved with regulatory function (Treg - tolerance) – the CD4+ CD25+(ie hi) commitment believed to be in the cortex[133], where positive selection involves Calcium waves that induce stationary thymocytes afflicted with jitters[134] and up-regulated NFAT signalling; the jitters possibly due to centriole writhing in response to the increased Calcium, thereby influencing microtubular cytoskeletal forces and up-regulating the Needham pathway (see elsewhere on the website). Thymocytes destined for programmed dissolution (generally small, with “sub-diploid DNA” !) up-regulated their surface antigens in vitro, with b/CD3 TCR expression moved from the cytoplasm to the cell surface[135]: a move which may involve more than the TCR and may signal to dendritic cells prior to the thymocyte demise; a process unlikely to be allowed full expression when the dissolution is precipitated by corticosteroids, irradiation or CTL attack[136] (perhaps similar to apoptosis induced by staurosporine[137] in pancreatic b-cells) – organizing affairs and drawing up the last will and testament are aborted  – merely the last rites (if that). There may be two types of both dendritic cells as well as macrophages in the thymus, with the F4/80+ type said to transfer antigens into the thymus. This may have some relevance to an earlier hypothesis that, in the periphery, there may be two types of dendritic cells, a short-lived group that phagocytoses the breakdown products from exposed cells (eg the gut epithelium) and transports the material, then to be presented to longer-lived immature dendritic cells in the mesenteric lymph nodes, to induce tolerance[138]. The 1968 thymus hypothesis diagram has some similarities with a proposed scheme derived by the study of tolerance to pancreatic self-antigens[139]. Their proposal was that only in the mesenteric lymph nodes close to the pancreas and the source of specific auto-antigens, is there maintained a pool of resting CD4+ CD25hi CD62Lhi natural Treg possibly determined by an unknown signal in the thymus between the DN-DP maturation stage[140], from which arise a cycling sub-set of CD4+ CD25hi CD44hi CD62low Treg lymphocytes[141] responding to the pancreatic self-antigens. The tolerant subset (presumably, but not necessarily, originally from the thymus[142]) proliferated (amplified), maintaining itself, the tolerant Treg pool and the tolerant outcome. The excess numbers were considered likely to die there (dissolution) or recirculate in the spleen. No suggested cycling of nuclear or other dissolution products (“antigen factors”) were proposed to influence newcomers (stem cells or progenitors, CD62Lhi) to maintain the dedication of the activated CD4+ CD25hi CD62Llow subset for the specific self-antigens relevant at the sentinel site. (What happens to antigens that are not neutralized or adsorbed there seems unknown.) There is the possibility for such a recycling on location there &/or in the thymus. Within such nodes, CTL lymphocytes have exocytosis inhibited by Tregs, involving TGF-b and other factors[143], of which a suppressive NIR signal from the reorientated MTOC beneath the synapse, which could affect sensors, as may occur in rods and cones (see the Chapter - the Centriole and the Retina, this website), could change the state of D-LDH-related enzymes (GAPDH-like; CtBP/Ribeye/other) molecules in the plasma membrane, vesicle transport and exocytosis[144] &/or nuclear transcription promoter function. (Both Treg and tumour cells may use NIR to inhibit the plasma membrane synapse recruitments[145] and vesicular transport of synapse components[146].) The earlier work with the CTL failure to lyse tumour cells[147] also pointed to a failure of exocytosis, attributed to a defective Calcium and PKC-dependent orientation of the MTOC[148], for which active proteosomal function was necessary for recovery; perhaps microtubular-associated GAPDH may have been inactivated by the calcium wave[149]-stimulated NIR activation of D-Lactate, and this needed to be eliminated before microtubular function and polarization of the MTOC could be effected. 

The EAE suppression did not seem to involve the inflammatory cytokines, but was associated with the up-regulation of Foxp[150],[151], an immune suppression factor with poorly understood initiation, but which modifies cell signalling, provides positive auto-stimulation and appreciable inhibition of the Pde3b gene for cyclic nucleotide phosphodiesterase 3B, cGMP-inhibited, an ER-associated enzyme involved in some receptor pathways[152] (eg insulin). Also, it operates at the NFAT transcription level (at least), which may confer long-term tolerance to activated CD4 T cells (via regulatory T cells[153],[154][Treg~TR]), B-cells[155] and plasma cell exposed to the autoantigen at the time of LF 15-1095 exposure and for 30 days. However, initial excitement over the specificity of Foxp3[156] may be dampened now[157],[158] - it is likely to be only one of a number of factors involved, and more recently, there have been described the immunoglobulin-like transcripts [ILTs] and the “T suppressor cell cascade” which may be important in “infectious tolerance.”[159]) Cardiac transplantation[160] and treatments against experimental allergic myasthenia gravis for 15 days blocked the production of the rat IgG2b (said to be IFNγ-dependent) well past the treatment cessation, but the other antibody fractions showed appreciable augmentation[161] and, in the transplant model, IL-10 production from the mixed lymphocyte response was only mildly reduced, the general pattern being of an inhibition of Th1 responses. The process is far from clear at present, but would seem to require the active participation of the host animal’s lymphoid tissue to produce regulatory T cells[162]. When transplanted tolerant lymphocytes from a donor were associated with immunization of the recipient there was also conferred an inducible and selective tolerance to the antigen used on the donor[163]. All this must mean that the donor tolerant lymphocytes home-in to the recipient’s lymphoid tissue (perhaps also the thymus), find the appropriate niche(s), be engrafted there and “infect” the surrounding recipient’s lymphoid cells with “tolerance,” which probably involves immature dendritic cells, up-regulated Foxp, ILTs and probably other factors linked to the antigen concerned and coupled with an initial proliferation stimulus, later being mature cells especially vulnerable to dissolution. If the process is anything like the hypothesis from 1968, it will involve dissolution (possibly apoptosis-like) of the donor’s tolerant lymphocytes, release of nuclear “factor(s),” which could include those transcription modulators such as NF-κB, NFAT, CRE, FoxP etc., DNA, tRNA, mRNA, chaperones and RNA-binding proteins, nuclear membrane fragments, structural proteins, sphingosine-1-phosphate and CTLA-4, whose fates and effects under the dissolution process are unclear, but could insulate, modulate or dilute agonist peptides (± other molecules) within the phagocytic endosomes[164] or at the APC-T cell synapse[165] ,[166]. (Such dissolution products, other than antigenic peptides, are rarely considered by those studying regulatory T cells. Given the immense number of specific breakdown peptides that can be expected to be released before and after the ingestion of a single cell undergoing dissolution, one has to wonder how the dendritic cell concerned can emphasize [by x 1 – 10 thousand times], and process selectively for such efficient treatment, a single one [or only a few] peptides to be important immune antigens and, in the case of the gut, transduce through two dendritic cells before presenting an antigen peptide to a lymphocyte. There would be appreciable opportunities for antigenic modulation within the endosome’s soup, as by blocking or modifying a peptide’s epitope by, say, mRNA fragments, transcription promoters or McM proteins[167] etc. or simple having a molecule like S1P received with the antigenic signal; this, with its receptors separate from the MHC-TCR union, could aid positive selection and reduce the immune responsiveness[168], features necessary for tolerance amplification. The roles of TCRγδ, lymphocyte/DC dialogue, movement of the MTOC into the Calcium flux [perhaps stimulating the Needham glucolysis pathway] and the delivery system concept are inceptive.)

The dissolution products, and plasma membrane antigens, are then taken up by (probably immature) dendritic cells[169],[170] and [CD4+ CD25(lo)-hi T cells], possibly involving Toll-like receptors[171], and affect these cells and/or are presented to the recipient’s lymphocytes[172] (CD4+ CD25hi Treg); both the membrane-derived antigen factors and the specific “tolerance” or “regulatory signal.” This latter seems to involve IL-10 in some contexts (TR1 subtype), but there are inconsistencies[173], and, in particular, a failure of antibodies to IL-10, TGF-b and CTLA-4 to block the tolerance-inducing effects in some contexts[174],[175] and an inability to characterize apoptosis or necrosis in the cell losses (Jonuleit et al. 2000, see earlier). This leaves open the possibility that one or more nuclear/cellular factors could be transferred conjointly with the membranous antigenic TCR factors, (remembering that vesicle formation may be important in some receptor activities; see earlier); both components being derived from a cellular dissolution process generally referred-to as apoptosis and that, specifically, is not well characterized in this context yet. There would have to be an amplification mechanism, such as in vivo Treg proliferation[176] with reduced apoptosis initially, to have the capacity to “infect” all the recipient’s specifically activated lymphoid progenitors, probably involving cell-to-cell contact (Jonuleit et al. 2000, see earlier) and a later propensity to dissolution.

There is the likelihood that clonal selection and “infectious tolerance” mechanisms coexist: but only when the former is appropriately suppressed (by the LF 15-0195, or its relatives[177], with a cell cycle block between S & G2 and inhibition of IKK and subsequently suppressed NF-κB activity[178]), or bypassed (and induced, as in EAE recovery[179]), is the Foxp control permitted and identifiable. Then the “infectious tolerance” can be seen to be operating. The induction of Tregs and their ability to inhibit effector functions is reviewed[180].

Such a basic mechanism seems likely to be involved in the Lithium-induced tolerance to EAE antigens as shown by Levine & Saltzman (1991- see earlier), but it must extend beyond mere tolerance, or else cancers would be expected to lose immune restraints when patients were treated with Lithium in this way; and in the case studied (see elsewhere on the website), that was certainly not the case; rather, the reverse. There must be more to it – a duality of immune modulation.

By way of an hypothesis, Lithium may, after a modest bolus dose, precipitate a wave of dissolution (apoptosis ?) of the most vulnerable thymocytes (Péret-Cruet & Dancey 1977, see earlier), probably before any selection stage in development, as in late DN (Prockop et al. 2004, see earlier) and empty the cell niches. Thymocyte dissolution (apoptosis ?) transfers the (?)nuclear/cellular “antigen factor(s)” (see earlier) to the dendritic cells and/or the early thymocyte progenitors before the more mature thymocytes are released, in order to have the specific Treg status augmented and confirmed by exposure to the peripheral antigen concerned[181]. There may be produced a tolerance to the self antigens involved or cell dissolution of the chosen cells (as for double negative T cells[182]) at the time (prematurely) but, by terminating the middle-order thymocyte pathway, the Lithium also may abort the clonal selection process, which may be more relevant for more primitive antigens, such as tumour stem-cell antigens. The Lithium bolus would still be enough to trigger the cascades of gene transcription factors, such as AP-1, CRE and Foxp etc., which may be augmented. A day without a Lithium bolus and a low basal level may provide a recovery time for parts of this machinery, such as the proliferating thymocyte progenitors, which will be ready for another release of (?)nuclear/cellular “antigen factor(s)” after ~48 h. Similar effects may occur in the peripheral lymphoid[183] tissue, but at a lower level and slower. There is the potential for the selection of different bolus-to-bolus intervals to achieve different effects.

In order to clarify thinking on clinical experiences, there is a need to classify the observed responses in relation to time scales (as loosely defined earlier): -

Conditions under consideration

Condition                     Effect-time      Effect                            Other effect

Multiple sclerosis

Hyperacute

Abort relapse

Little apparent progression

Sarcoidosis

Acute

Pain/symptom relief

 

Lupus erythematosus

Acute

Chronic

Pain relief

Prophylaxis

 

Reduce steroid dose

Hepatitis C

Acute

Serum ALT fall

T3 thyrotoxicosis + ALT↑

Paranoid ideation

Rheumatoid arthritis

Subacute

ESR/inflammation ↓

Prophylaxis for 5-6 week

Cancer (NSCLC)

Chronic

Reduce size

Serum marker↓

Felt better (relative’s

account)

 

The mechanism of Lithium treatment on Hepatitis C and Cancer/neoplasia poses problems because one of the important features of these diseases is the (what appears to be) an inappropriately active Treg population. Some clarification may come from classifying conditions according to the perception as to the origin of the immune mechanisms and of the location of the original observable lesion  –

Conditions and relevant immune sites

Condition                      Site of immune activation                    Site of initial lesions seen

Cancer

Local, with sentinel node

Local (T1)

Hepatitis C

Local (liver), with sentinel node(s)?

Liver

EAE

Local (site of injection) to systemic [swamping of sentinel node(s) irrelevant to the brain]

Brain (multiple sites)

Multiple sclerosis

? Systemic [no sentinel node(s)]

Brain (multiple sites)

Rheumatoid arthritis

Multiple effector sites (systemic)

Polyarthritis

Lupus erythematosus

Multiple effector sites (systemic)

Multi-organ

Sarcoidosis

? Multiple effector sites (? systemic)

Multiple nodes/organs

Green = Local; White = ? systemic effect; pink = Systemic

The immune assault for EAE is clearly peripheral (by injection) initially, and outside the brain. There is no relevant sentinel node system for the brain, meaning that the immune stimulation is effectively systemic and against self antigen(s) globally within the neural structures. The status of multiple sclerosis is, by analogy (questionably), probably similar; meaning that there may be an aberration of the systemic immune mechanisms outside and remote from the brain, acting as the genesis for the neural lesions, which are multiple, scattered globally and against self antigen(s). There are no sentinel nodes to act as buffers.

Based on the proposed classification set-out above (EAE & MS regarded as Systemic), the same conditions in relation to the type of regulatory cells that probably would be most involved in the early stages of the disease process can be presented :

Conditions and their regulatory classification

Condition          Site            Ag#    Regulatory T     Li+ effect (proposed)

Cancer

Local

Non-

self

Induced Treg/TR

(Node)

Delete induced Treg/TR1, allow immune

 rejection

Hepatitis C

Local

Non-self

Induced Treg/TR

(Node)

Delete induced Treg/TR1, allow immune

rejection

EAE

Systemic

Self

Natural Treg

 (Thymus*)

Delete natural Treg, permit new setting for Treg at a lower threshold

Multiple sclerosis

Systemic

Self

Natural Treg

 (Thymus*)

Delete natural Treg, permit new setting for Treg at a lower threshold

Rheumatoid arthritis

Systemic

Self

Natural Treg

 (Thymus*)

Delete natural Treg, permit new setting for Treg at a lower threshold

Lupus erythematosus

Systemic

Self

Natural Treg

 (Thymus*)

Delete natural Treg, permit new setting for Treg at a lower threshold

Sarcoidosis

Systemic

Self?

Natural Treg

 (Thymus*)

Delete natural Treg, permit new setting for Treg at a lower threshold

*The thymus is probably the main site, but this function may also be applicable for thymus-derived Treg cell proliferation in lymph nodes generally.

             #Ag = antigen (main types involved)

 

By contrast, Type 1 diabetes mellitus (T1DM, studied in mice)[184],[185] and transplantation (also in mice, being CTLA-4 & IL-10 dependent)[186] could, in relation to Lithium treatment, have the following features:

T1DM

Local

Self

Induced Treg/TR

(Node)

May make worse – a need to increase specific Treg/TR*.  

Transplant

Local

Non-self

Induced Treg/TR

(Node)

May make worse – a need to increase specific Treg/TR*.  

*As may be done with Vitamin D analogues[187] - remember that these can be used to treat Psoriasis, considered a disease involving CD4 Th1 lymphocytes[188], possibly of bone marrow origin[189], where the skin may be regarded as acting like a lymph node (the CD4 cells bypassing the thymus to the skin, except for some processed through the thymus, that are then unable to regulate reaction to the self antigens of arthritis etc., although there may be some doubts[190]), whereas Lithium can make Psoriasis worse; agreement with the table.

Whilst some patients with chronic Hepatitis C (an RNA virus) with evidence of hypersensitivity phenomena can have decreased levels of regulatory T cells[191] and heightened sensitivity to cytokine effects, these features are uncommon and late. In the common, more typical cases, the Treg suppress specific HCV CD8+ responses independently of TGF-b and INF-γ, and suppress their maturation[192],[193]. However chronic HCV patients demonstrate a relative increase in Foxp3 lymphocytes in the portal tracts, and also scattered through the lobules: the serum ALT level being correlated with the number of CD8+ T cells in the portal tracts[194]. The case with HCV (see Submission to the NH&MRC on the website): review of the patient’s liver biopsy pre-Lithium treatment confirmed mild cirrhosis and steatosis, mild-moderate monocytic portal tract infiltrate and mild numbers of small mononuclear cells within the lobules.) He had two treatment courses with Lithium and had initial serum ALT levels above those in the studies quoted. Since CD4+ CD25hi Foxp3+ INFγ- T cells seemed up-regulated/activated by HCV[195], an abnormally accentuated Treg action may be an indirect target of Lithium treatment, which may simply inhibit the IL-2 receptor mechanism, possibly at the G-protein level, but would have to be able to exert an effect after only about 2-3 h of inhibition (unlikely), or prevent IL-2 production by blocking NF-κB (or other) promoter (or both/more). In which case, there may be precipitated auto-immune phenomena associated with treatment, which may have occurred. His serum ALT levels fell following treatment with Lithium on each occasion. These responses may indicate a reduction in the number/activity of CD8 T cells, based upon the studies of patients in relatively steady-states. He developed a short-lived antibody negative T3 thyrotoxicosis about 10 days after the start of treatment, associated with a minor ALT rise and followed by an episode of paranoid ideation. These could be indicators of virus-affected cell destruction (liver and thyroid) or, in the case of the liver, simply an effect of the thyrotoxicosis upon it. Also, the paranoia may simply reflect a post-T3 thyrotoxicosis effect.  At this stage, we may wonder if this was due to a block on the Treg suppressive function, allowing an overshoot inflammatory cytokine production (or some other effect upon the chronic viral load. The claimed inhibition of the DNA Herpes simplex viral mRNA transcription using an in vitro concentration of 30 mmol/L of Lithium chloride[196] is unlikely to have clinical significance. The clinical observations of reductions in the incidence and/or severity of Herpes simplex infections[197],[198] must involve other mechanisms, so that the observations with Hepatitis C virus could be relevant).

Non small cell lung cancer (as, probably, was the Lithium-treated patient studied) is associated with an increased expression of regulatory T cells CD4+ CD25+ CTLA4+ CD45RO+ which showed specificity for autologous T cells suppression, and produced TGFb[199] (with undetectable IL-10), indicating an induced/nodal type. A study of breast and pancreatic ductal carcinomas differed, in that the Treg produced IL-10[200], being more typical for induced/nodal type. (The distinction between CD25+ and CD25hi in the flow cytometry studies of 2002-3 was often not clear.) Induced TR1­ in a patient with colon cancer could inhibit proliferation by production of TGF-b, without cell-to-cell contact, but CTL activity was not assessed[201].

The cytolytic action of sensitized CD8+ lymphocytes against cancers may be inhibited by the CD+ CD25+ Treg producing TGF-b and suppressing the CD8+ cytotoxicity via their TGF-bR[202]. The Tregs have been shown to have a significant role in the growth and spread of ovarian cancer[203], where the Treg are found less in the sentinel nodes, but more in close association with the tumour mass and ascites, the chemokine CCL22 derived from the tumour and macrophages being implicated; being negatively associated with survival. Similarly, for hepatocellular carcinoma, there is infiltration of the tumour with Tregs rich with TGF-b, with decreased perforin and Granzyme B in the CD8+ component and vascular invasion linked to Foxp3 Treg cells[204]; the suppression induced in the CD4+ CD25- not relaxed by removal of the Tregs[205].  The simplest explanation then for Lithium to have an anticancer effect may be by blocking the TGF-bR pathway, as by inhibiting the G-protein/PKC machinery, quite apart from other potential roles. However, this seems too simple and, considering the short interval that Lithium may exert its inhibitory role on the G-proteins after a bolus dose, lacks plausibility.

In the hypothesis of 1968, elaborated in 1970, note was made that “. . . the spontaneous regression of tumours is lessened, in those periods when the cortex is predominant” (ie when active tolerance, chiefly in the thymus, but also in the periphery, was predominant). This implied that tolerance-inducing lymphocytes were able to inhibit tumour rejection.  More recently that, generally, has been the findings with the Treg cells in a murine model, where the presence of Treg cells in the proximity of tumours (a fibrosarcoma), inhibits T8+ cytotoxicity, and rejection[206] (this reference also containing a summary of the recent literature). There was an appreciable up-regulation of Foxp3 in the tumour-related lymphocytes, and both TGF-b and IL-10 seemed to be involved. The overall pattern is reminiscent of the work done in 1989[207], in which Vinblastine was used to eliminate specifically tumour-sensitized suppressor (Treg/TR1) cells at about day 15 after a lymphoma is introduced into mice and, more recently, when Gemcitabine was used to reduce the activity of myeloid-derived suppressor cells, in conjunction with anti-cancer immunization and modulation[208]. The resulting tumour remission was attributable to the reactivation of CTL T8 cells induced into tumour-related dormancy by the time of the Vinblastine treatment. Lithium, a much safer, cheaper and less toxic drug, may do much the same, but only when administered in the appropriate protocol.

 

CONCLUSION

Lithium, an old medication dug out of the ground, works in mysterious ways. Perhaps my musings may stimulate some interest in this simple, yet complex material. The enigma of its dual ability to act as an immune activator and as an immune suppressor, depending on circumstances, has been addressed and some conclusions proffered. No hypothesis has value unless it leads somewhere – perhaps some may be inspired to trial its clinical use in novel ways, as in bolus doses, for patients whose outlook otherwise is very bleak. Since its cellular modes of action are still unknown, any and all treatment regimes must be empirical and finely tuned. With care, it is safe, cheap and easily monitored.

 

 

Copyright © MA Traill January 28th 2008


 

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