Journal of Cytokine Biology
Open Access

Chemokines; Synovial hyperplasia; Inflammation

Introduction

The micropathophysiology of the gouty attack in the human joint is well-known; and the specifics of this cascade of events are very well documented [1-9]. It is assumed that the reader is well-versed in the basics; and the author refers the reader to those excellent references. Certainly, as one studies both the historical research of gouty inflammatory arthropathy, and is involved in the treatment of patients.The role of colchicine becomes clear. One of the best articles, in my opinion was written by Dalbeth, et al. [9]. In this excellent review, “Mechanism of Action of Colchicine in the Treatment of Gout” the three pathways associated with the initiation and amplification of the acute MSU-induced gout attack are described in great detail (Figure 1).

Figure 1: Mechanism of Action of Colchicine in the Treatment of Gout.

This important review can be cross-referenced and data-mined for review of many analogous actions of Lithium. This was the starting point of my search for the potential mechanisms and actions of colchicine that may be analogous to those of Lithium. Of course, we know that colchicine disrupts the microtubules of the pseudopodia that are critical for the migration of the WBCs and macrophages from the endothelium into the synovium [9]. In my early research into Lithium’s actions: I came across an article describing its ability to affect the motility of sperm flagellae [10]. Understanding that this locomotion was also directed by microtubular activity; I began to look closer at the similarities and differences between colchicine and lithium. The main action of colchicine is to bind tubulin, and prevent further assembly and subsequent locomotion [11]. Lithium affects locomotion differently, and prevents assembly of microtubules by inhibiting GSK-3b. Many of Lithium’s actions are due to its ability to replace magnesium in the enzyme pocket of glycogen synthetase kinase 3-b [12]. As a very reactive alkali metal, Li+ ion readily substitutes for the slightly larger Mg++ ion. Because of its ability to block GSK-3b and other kinases, Lithium affects many pathways [13]. Lithium is known to be neuroprotective and to increase survival of neural and glial cells [14-17]. The action and effects of Lithium far exceed those of colchicine. My observations are tabulated (Table 1).

Table 1: Observations comparing the actions of colchicine and lithium.

The table shown above begins with the relative similarities between colchicine and lithium; and one clearly sees the numerous effects of lithium within the CNS and spine.

Methods

The myriad actions of lithium within the CNS may have implications for pain management in the future; and may be also have both scientific and clinical application to the neural cells and pain pathways within peripheral nervous system. It stands to reason, that topical application of lithium may produce a local response; with only a fraction of the applied dose reaching the serum. In 3 patients tested who were applying 2% Lithium Carbonate in witch hazel to the foot; no detectable concentration was seen in the 8-1.2m Eq/L lab range expected to be therapeutic. Taken together, I feel that the reduction of nerve pain and gouty inflammation seen in patients treated off-label; and reduction in pain, swelling, and KC-GRO (IL-8) seen in the MSU rat model, may be attributed to the 2% Lithium Carbonate applied topically and delivered to the desired target tissues as Li+ ion after passage through the epidermis, without any measurable uptake in serum. The study of Lithium Carbonate topical Pharmaco Kinetics (PK) was beyond the designated scope of this proof-of-concept study (Figures 2-7).

Figure 2: (Normal Joint) there are no inflammatory cells in joint (blue arrow) Normal synovial lining (green arrow).

Figure 3: @20x note a single layer of synovial fibroblasts (green arrow) and collapsed vessel (blue arrow) which indicates lack of inflammation in the normal physiologic state of the Sprague Dawley Rat.

Figure 4: (MSU Gout Attack) Notice the edema within the joint; and take note of the raft of inflammatory cells that have infiltrated and occupy the joint (green arrow) and hyperplasia of the synovium (blue arrow).

Figure 5: @20X Neutrophils and fibrin are free within the joint cavity (blue arrow). The synoviocytes are proliferative (green arrows) and the capillary endothelium is hypertrophied (black arrows).

Figure 6: (DMSO Treated) the synovium is focally thickened (blue arrow), but there is less acute inflammation within the joint cavity (green arrow). Presumptive Mono Sodium Urate (MSU) (black arrow) is present.

Figure 7: (Lithium Formulation-Treated) The synovium is less thickened (green arrow) when compared to the Acute MSU attack, and also is less thickened than the DMSO treated ankle. There is also less acute inflammation and WBCs within the left ankle joint cavity (blue arrow) when compared to DMSO treatment alone.

also is less thickened than the DMSO treated ankle. There is also less acute inflammation and WBCs within the left ankle joint cavity (blue arrow) when compared to DMSO treatment alone.

Results and Discussion

The IL-8 seen in the human is not expressed in the murine model. The murine homologue is called KC-GRO [18-20]. The cytokine CXCL1 that binds the human IL-8, and its receptor CXCL-2 are essential for the development of the acute gouty neutrophilic response to urate crystals in the murine gout subcutaneous air pouch model. In a resting state, neutrophils are rare in synovial fluid. The CXC chemokine IL-8 in one study accounted for more than 90% of the neutrophil chemotactic activity seen [21]. IL-8 is considered an important marker for gout. The IL-8 acts on CXCL1/CXCL2 to recruit neutrophils out of the endothelium [22]. This excellent article by Girbl, et al. illustrates the effects of chemokine signaling on neutrophil recruitment from the endothelium. The 3-d illustrations and animated real-time imaging is remarkable (Figure 8).

Figure 8: 3-d illustrations and animated real-time imaging is remarkable.

In the author’s current study of MSU-induced gout, several cytokines IL-1b, IL-6, KC/GRO, and TNF-a were seen in synovial fluid analysis. The elevation of these cytokines would be expected as a normal baseline inflammatory response to MSU injected into the rat ankle. However, the application of topical lithium carbonate caused an increase in the cytokines IL-1b, IL-6, and TNF-a in synovial fluid. The increase in these cytokines, although somewhat unexpected; may be seen as an indication that Lithium Ions actually affected the joint. Lithium is known to have both anti-inflammatory and neuroprotective effects [14-17, 23]. Lithium acts to modulate TNF and IL-1 induction early in the signaling pathway [24]. The inhibition of GSK3b by LiCl increases the TNF-a protein synthesis by greater than a 3-fold margin in neutrophils [25]. Lithium led to a consistent increase of IL-1b, IL-6 and TNF-a in the serum of 30 subjects tested [26]. There is a large body of data which indicates that under certain experimental conditions lithium also exhibits pro-inflammatory properties e.g., induction of IL-4, IL-6 and other pro-inflammatory cytokines’ synthesis [27]. It may be reasonable to attribute the “priming” of the inflammatory response, and induction of cytokines IL-1b, IL-6, and TNF-a in the BRT model to the topical Lithium Carbonate applied.

Attenuation of cytokine KC/GRO was seen in synovial fluid with application of a topical Lithium Carbonate as the test ingredient in the BRT rat MSU model. This was a surprising and also an unexpected finding in light of the increase in the other cytokines that were tested. An NIH-funded study in 1994 established the fact that KC is the murine homologue of human GRO-a; and the KC receptor is also an IL-8 receptor homologue capable of binding both KC and the macrophage inflammatory protein-2 with high affinity [18]. The interaction of KC/GRO (as it binds the IL-8 receptor) triggers neutrophil activity. IL-8 is abundant in the synovial fluid in both acute gout and pseudogout [28,29]. Rapid release of IL-8 and binding to CXCL2 stimulate the adhesion and diapedesis of neutrophils out of the endothelium into the synovium. The CXCL1/CXCL2 interaction is also known to regulate and activate the NLRP3 inflammasome in macrophages [30]. The neuronal inflammatory cytokines CXCL1/ CXCL2 are regulated by GSK3 signaling [31] (Table-2). Lithium is well-known to block GSK3; and its effect on synovial tissues by reducing KC/GRO may be used to treat and prevent gout/pseudogout via IL-8 (Figure 9).

Table 2: High Levels of IL-6, TNF-a, and IL-1b seen in MSU gout model amplified by topical lithium carbonate

Figure 9: KC-GRO (murine IL-8) seen in MSU Gout Model IL-8 in Synovium is unexpectedly reduced by topical lithium carbonate.

Conclusion

This study demonstrates for the first time, the ability of topicallyapplied 2% Lithium Carbonate to decrease pain, swelling and inflammation of MSU-induced gout in the ankles of male Sprague Dawley rats.

This study also measured for the first time, the reduction of KCGRO (murine IL-8) in the synovial fluid of gouty ankles of male Sprague Dawley rats induced by the topical application of 2% Lithium Carbonate.

The histopathology of this study validates the ability of topical 2% Lithium Carbonate to favorably affect the MSU-induced gouty ankles of male Sprague Dawley rats. The topical application of 2% Lithium Carbonate reduced KC/GRO (IL-8) in the rat synovial fluid; but the histopathology confirmed that topically-applied Lithium reduced inflammation, synovial hyperplasia and cellular infiltration of WBC’s into the joint . This has implications far beyond gout, and also pseudogout in my opinion. It is my opinion, that our study validates the ability of topical Lithium Carbonate to reduce the amount of KCGRO in the synovium; and may prove effective as blocking the critical amplification of the CXCL1/CXCL2 cascade seen in the crystalline inflammatory arthropathies of gout and pseudogout. The recent literature and our research, have demonstrated the importance of KCGRO and CXCL1/CXCL2 in gout, pseudogout, OA, and RA. The indications for the use of Topical Lithium Carbonate might be extended to the inclusion of other forms of chronic neuroinflammatory pain, and the chronic pain associated with degenerative arthritis. This attenuation of inflammation may be achievable by blocking a complex cascade of events in the tissues around/within the synovium.

Disclaimer

The following research and discussion do not in any way represent the view or opinions of the Department of Veterans Affairs or the US Government. This research was not supported, funded, or conducted with their resources or guidance.

1. Garrod AB (1859) The Nature and Treatment of Gout and Rheumatic Gout Walton and Maberly, London
2. Ure A (1841) On Gouty Concretions, with a New Method of Treatment. Med Chir Trans 24: 30–5.
3. Edison, Thomas A (1890) An Account of Some Experiments of Electrical Endosmose to the Treatment of Gouty Concretion Telegraphic Journal.
4. Peterson, Frederick (1893) Tetra-Ethyl-Ammonium A New Solvent for Uric Acid Discovered at the Edison Laboratory. The New York Medic J Weekly Review of Medicine: 320-322.
5. Abramowitsch D, Neoussikine B (1946) Treatment by Ion Transfer (Iontophoresis). Physical Therapy:285
6. Busso N, So A (2010) Gout Mechanisms of Inflammation in Gout. Arthritis Res Ther 12:206.
7. Chen J, Wu M, Yang J, Wang J, Qiao Y,et al. (2017) The Immunological Basis in the Pathogenesis of Gout Iran. J Immunol: 90-98.
8. Cronstein BN, Terkeltaub R (2006) The Inflammatory Process of Gout and its Treatment. Arthritis Res Ther.
9. Dalbeth N, Lauterio TJ, Wolfe HR (2014) Mechanism of Action of Colchicine in the Treatment of Gout. Clin Ther: 1465-79.
10. Gibbons BH, Gibbons IR (1984) Lithium Reversibly Inhibits Microtubule-Based Motility in Sperm Flagella. Nature: 560-2.
11. Bhattacharyya B, Wolff J (1976) Stabilization of Microtubules by Lithium Ion. Biochem and Biophys Rese Communic: 383-390.
12. Freland L, Beaulieu JM (2012) Inhibition of GSK3 by Lithium, from single Molecules to Signaling Networks. Front Mol Neurosci 5:14.
13. Jakobsson E, Chiu SW, Fazal Z, Kruczek J, Pritchet L, et al. (2017) Towards a Unified Understanding of Lithium Action in Basic Biology and Its Significance for Applied Biology. J Membr Biol: 587-604.
14. Lovestone S, Davis DR, Webster MT, Kaech S, Brion JP, et al. (1999) Lithium Reduces Tau Phosphorylation: Effects in Living Cells and Neurons at Therapeutic Concentrations. Biol Psychiatry 15:995-1003.
15. Makoukji J, Belle M, Meffre D, Stassart R, Grenier J, et al. (2012) Lithium Enhances Remyelination of Peripheral Nerves. Proc Natl Acad Sci U S A 109:3973-8.
16. Muñoz-Montaño JR, Moreno FJ, Avila J, Diaz-Nido J (1997) Lithium Inhibits Alzheimer’s Disease-Like Protein Phosphorylation in Neurons. FEBS Lett 411:183-8.
17. Nonaka S, Hough CJ, Chuang DM (1998) Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium influx. Proc Natl Acad Sci U S A 95:2642-7.
18. Bozic CR, Gerard NP, Kolakowski LF, Conklyn MJ, Breslow R, et al. (1994). The Murine Interleukin 8 Type B Receptor Homologue and Its Ligands. J Biol Chem 269:29355-8.
19. Fan X, Patera AC, Pong-Kennedy A, Deno G, Gonsiorek W, et al. (2007) Murine CXCR1 is a Functional Receptor for GCP-2/CXCL6 and Interleukin-8/CXCL. J Biol Chem 282:11658-66.
20. Fujiwara K, Ohkawara S, Takagi K, Yoshinaga M, Matsukawa A (2002) Involvement of CXC Chemokine Growth-Related Oncogene-a in Monosodium Urate Crystal-Induced Arthritis in Rabbits. Lab Invest 82:1297-304.
21. Terkeltaub R, Baird S, Sears P, Santiago R, Boisvert W (1998) The Murine Homolog of the Interleukin-8 Receptor CXCR-2 is Essential for the Occurrence of Neutrophilic Inflammation in the Air Pouch Model of Acute Urate Crystal-Induced Gouty Synovitis. Arthritis Rheum 41:900-9.
22. Girbl T, Lenn T, Perez L, Rolas L, Barkaway A, et al. (2018) Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis. Immunity 49:1062-1076.
23. Bloomfield FJ, Young MM (1982) Influence of Lithium and Fluoride on Degranulation from Human Neutrophils in Vitro. Inflammation 6:257-67.
24. Beyaert R, Osthoff KS, Roy FV, Fiers W (1991) Lithium Chloride Potentiates Tumor Necrosis Factor-Induced and Interleukin 1-Induced Cytokine and Cytokine Receptor Expression. Cytokine 284-291.
25. Giambelluca MS, Bertheau‐Mailhot G, Laflamme C, Servant MJ, Pouliot M, et al. (2014) TNF-a Expression in Neutrophils and its Regulation by Glycogen Synthetase Kinase-3: A Potentiating Role for Lithium. The FASEB Journal 3679-3690.
26. Petersein C, Sack U, Merg R, Schönherr J, Schmidt FM, et al. (2014) Impact of Lithium Alone and in combination with Antidepressants on Cytokine Production in Vitro. J Neural Transm (Vienna) 122:109-22.
27. Nassar A, Azab AN (2014) Effects of Lithium on Inflammation.ACS Chemical Neuroscience: 451–458.
29. Becker MD, O'Rourke LM, Blackman WS, Planck SR, Rosenbaum JT (2000) Reduced Leukocyte Migration, but Normal Rolling and Arrest, in Interleukin-8 Receptor Homologue Knockout Mice. Invest Ophthalmol Vis Sci 41:1812-7.
30. Jeong JH, Jung JH, Lee JS, Oh JS, Kim YG, et al. (2019) Prominent Inflammatory Features of Monocytes/Macrophages in Acute Calcium Pyrophosphate Crystal Arthritis: a Comparison with Acute Gouty. Arthritis Immune Netw.
31. Boro M, Balaji KN (2017) CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2. J Immunol: 1660-1671.
32. Chai HH, Fu XC, Ma L, Sun HT, Chen GZ, et al. (2019) The Cytokine CXCL1 and Its Receptor CXCR2 Contribute to Chronic Stress-Induced Depression in Mice. FASEB J 33(8):8853-8864.