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This review focuses on the most recent findings about the role of leptin in onset, development, clinical manifestations and outcomes of multiple sclerosis (MS) and applications of a possible leptin antagonist as a therapeutic agent for MS patients and strategies for designing such an antagonist. After an introduction to leptin, we will focus on its role in the immune system and autoimmunity. Afterwards we will review the literature around leptin and MS and finally will discuss the relevant potential therapeutic strategies based on the link between MS an leptin. Based on the available evidence, strategies aimed at leptin antagonism might represent a novel therapeutic platform which deserves further attention.
In 1994, leptin was discovered by Friedman and colleagues as a product encoded by the ob gene through the study of obese mice ( Zhang et al., 1994). The ob/ob or obese mouse is a mutant mouse suffering from a complex syndrome primarily characterised by excessive eating, which results in profoundly obese mice (Meier & Gressner, 2004). Leptin is a protein acting as both hormone and cytokine consisting of 167 amino acids and is an Î±-helical-bundle cytokine ( Zhang et al., 1997). The structure of leptin is highly similar to other members of this large cytokine family including growth hormone, interleukins such as interleukin-6 (IL-6), IL-11, IL-12, granulocyte colony stimulating factor (G-CSF) and leukemia inhibitory factor (LIF) (Baumann et al., 1996; Tartaglia, 1997). Leptin is predominantly produced by adipocytes and its circulating level positively correlates with white adipose tissue mass (Friedman & Halaas, 1998a). Administration of leptin to ob/ob mice increases basal metabolism and reduces food intake, leading to a markedly rapid weight loss (Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995).
Leptin interacts with leptin receptor, also known as Ob-R which is encoded by the db gene in human and has a single transmembrane-spanning domain (Tartaglia et al., 1995). Ob-R has also been designated as CD295 (cluster of differentiation 295) (Laschober et al., 2008) and is categorized under class I cytokine receptor superfamily (Chen et al., 1996). Six isoforms of leptin receptor has been discovered (Ob-Ra, b, c, d, e and f): one long (Ob-Rb), four short (Ob-Ra, c, d and f), and one secreted (Ob-Re) (Hegyi et al, 2004; Mercer et al., 1996), which are products of alternative mRNA splicing, and are different in the length of their intracellular tails although share equal extracellular-binding domains. (Sinha et al., 1996). Leptin binds to the ventromedial nucleus of the hypothalamus, which is named the "appetite center"(Saladin et al., 1995). Ob-Rb is present in a number of hypothalamic nuclei (Saladin et al., 1995). The long isoform Ob-Rb has a long intracellular domain in human and is responsible for most of the recognized properties of leptin through its complete intracellular tail, at which the signalling of four different pathways involving JAK-STAT, MAPK, PI3K and AMPK can occur (Hegyi et al., 2004). Ob-Rb is also expressed by endothelial cells, CD34+ bone marrow precursor cells, macrophages, monocytes, B- and T- lymphocytes (Ducy et al., 2000a; Friedman & Halaas, 1998b; Lord et al., 1998a; Park et al., 2001; Sanchez-Margalet et al., 2003; Sierra-Honigmann et al., 1998; Tartaglia, 1997; Tartaglia et al., 1995). db/db mice possess a deletion in the long isoform of the leptin receptor and thus are resistant to leptin ( Lee et al., 1996).
The short form (Ob-Ra) is much more widely expressed, often at higher levels compared to long form, and is expressed in different organs such as in the choroid plexus, kidney, cells of the immune system, lung and liver (Tartaglia, 1997). The short isoforms are believed to have some signalling capabilities and may also be involved in leptin transport through the blood brain barrier and maybe in other unknown functions (Peelman et al., 2006).
The cytokine structure of leptin and recent evidence has indicated that it has a pleiotropic nature (Ducy et al., 2000b). Probably the main function of leptin is to control body weight by the inhibition of food intake and to increase energy consumption by increased thermogenesis (Matarese et al., 2002). In addition, leptin appears to be part of the complex network that coordinates immune responses to various stimuli. Leptin also balances the body's energy status and thus adjusts the immune response to appropriate levels. Immune responses are an energy-demanding processes, and their inhibition during starvation may conserve energy necessary for survival of core body functions. Such interactions between energy homeostasis and the immune system appears to be bi-directional (Peelman et al, 2005).
Leptin and the immune system
Leptin and its receptors are independently regulated gene products: the ob gene encodes for leptin, while the db gene encodes for the leptin receptor. Mice with homozygous mutations in the leptin gene are designated ob/ob and mice homozygous for mutations in the leptin receptor gene are designated as db/db. The diverse roles for leptin in mammalians are shown by the complex syndromes displayed by leptin-deficient (ob/ob) mice and deficient leptin receptor mice (db/db). These mice are not only obese, but also show abnormal reproductive function, bone structure, wound repair, hormone levels, and immune function (Chehab et al, 1996; Ducy et al., 2000a; Fleet, 2000; Frank et al, 2000; Howard et al., 1999; Ring et al., 2000). In addition, both ob/ob and db/db mice suffer from thymic atrophy and have reduced numbers of circulating lymphocytes (Chandra, 1980; Faggioni et al., 2000a; Mandel & Mahmoud, 1978). Impaired T cell immunity in these mice points towards a direct effect of leptin on T lymphocytes (Howard et al., 1999), which may reflect CD4+ and CD8+ T cells express functional leptin receptor(s) (Lord et al., 1998b; Martin-Romero et al., 2000). Leptin concentrations lowered by starvation appear to correlate with impaired immune responses in mice (Ozata et al, 2000). Since administration of leptin to ob/ob but not db/db mice prevented immune dysfunction, a central role for leptin as an immune system regulator has been proposed (Faggioni et al., 2000; Howard et al., 1999).
Several authors have reviewed recent findings on leptins relationship with the immune system and autoimmune diseases (De Rosa et al., 2007; Farooqi et al., 2002; Iorio et al., 2006; La Cava et al, 2004; La Cava et al, 2003; Matarese & La Cava, 2004; Matarese & Lechler, 2004; Matarese et al, 2007; Matarese et al, 2005; Matarese et al, 2002; Papathanassoglou et al., 2006). Leptins' effects on adaptive immune responses have been more extensively investigated compared to innate immunity. In vitro studies have shown that leptin enhances proliferation of circulating blood T lymphocytes in a dose-dependent manner (Lord et al., 1998b; Martin-Romero et al., 2000). Adding physiological concentrations of leptin to a Mixed Lymphocytes Reaction (MLR) induces a dose-dependent increase of the proliferation of CD4+ T cell (Lord et al., 1998a). Considering that congenital deficiency of leptin increases the frequency of infections and related mortality (Ozata et al, 1999), it was hypothesized that a low concentration of serum leptin may promote increased susceptibility to infection by reducing T helper cell priming and by affecting thymic function (Howard et al., 1999; Lord et al., 1998a). Leptin appears to affect the T helper (Th) subsets, shifting the balance towards the T helper one (Th1) subtype by stimulating production of the Th1 pro-inflammatory cytokines such as, IL-2, interferon gamma (IFN-Î³), tumour necrosis factor alpha (TNF-Î±), and IL-18, and decreases production of the Th2 cytokines: IL-4, IL-5 and IL-10 (Lord et al., 1998b; Martin-Romero et al., 2000). These effects are not observed in T lymphocytes from db/db mice, supporting the concept that this effect is directly mediated by leptin receptors, expressed on T lymphocytes (La Cava et al., 2003).
Leptin also influences other immune cells. Peritoneal macrophages from ob/ob mice display a lower phagocytic activity, compared to macrophages from normal mice, and when leptin was administered, the phagocytic activity was restored (Loffreda et al., 1998). Furthermore, the production of granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) (Gainsford et al., 1996) and the pro-inflammatory cytokines such as, TNF-Î±, IL-6 and IL-12 (Loffreda et al., 1998) by murine macrophages is enhanced after treatment with leptin. It has also been shown that leptin induces TNF-Î±, IL-6 and IFN-Î³ production by resting human peripheral blood mononuclear cells (PBMCs) and enhances the release of these cytokines from stimulated PBMCs (Zarkesh-Esfahani et al., 2001). In human neutrophils, leptin appears to mediate its effects indirectly, probably involving the release of TNF-Î± from monocytes (Zarkesh-Esfahani et al., 2004) which activates chemotaxis of lymphocytes and monocytes (Taub et al., 1993), to sites of inflammation (Mach et al., 1999; Taub et al., 1993). Moreover, in ob/ob mice, numbers of intraepithelial lymphocytes (IELs) are reduced and these IELs exhibit decreased IFN-Î³ secretion, while the lamina propria mononuclear cells of these mice show increased apoptosis(Siegmund et al, 2002).
Leptin also appears to be a regulator of natural killer (NK) cells development and activation. The db/db mice show decreased numbers of NK cells in the liver, spleen, lung and peripheral blood, and in normal mice leptin administration increases the basal or induced lysis of splenocytes, not seen in db/db mice (Tian et al, 2002).
Leptin and Autoimmunity
Leptin, plays a signficant role in CD4+ T cell-mediated immune responses, promoting a pro-inflammatory Th1 response. The Th1 enhancing properties of leptin have been shown to increase the susceptibility of mice to develop experimentally induced autoimmune diseases such as type 1 diabetes melitus (T1D), antigen-induced arthritis (AIA) and experimental autoimmune encephalomyelitis (EAE), an immune-mediated model of human multiple sclerosis (Ozata et al, 1999). Accumulating evidence suggests that leptin also plays a pivotal role in the development of CD4+ T cell mediated autoimmune diseases in human including Crohn's disease (Sartor, 2005), rheumatoid arthritis (RA)(Fraser et al, 1999), multiple sclerosis (Sanna et al., 2003) and type I diabetes mellitus (T1D)(Matarese et al, 2002a). ob/ob mice resist induction of several experimental models of inflammatory and autoimmune diseases, such as experimental arthritis (Busso et al., 2002), T cell-mediated hepatitis (Faggioni et al., 2000b) and acute and chronic intestinal inflammation (Mykoniatis et al., 2003).
In experimental mouse model systems of inflammatory bowel disease (Crohn's disease with acute and chronic colitis), leptin-deficient ob/ob mice showed a significant (72%) lower colitis disease severity with a concurrent decrease in pro-inflammatory cytokines (IFN-Î³, TNF-Î±, IL-1Î², IL-18 and IL-6) in colon cell culture supernatants, compared to wild type mice (Siegmund et al., 2002). Administration of leptin to ob/ob mice eliminates resistance to experimentally induced Clostridium difficile (CD) colitis (Siegmund et al., 2002). In this model, CD toxin A caused a severe colitis in wild type mice; ob/ob as well as db/db mice appeared to be partially protected against CD toxin A-induced gut inflammation (Mykoniatis et al., 2003). In this case, leptin administration in ob/ob, but not in db/db mice reversed this effect (Mykoniatis et al., 2003), which is consistent with dependency on leptin receptor signalling.
The organ-specific autoimmune condition termed chronic idiopathic thrombocytopenic purpura (ITP) is characterized by production of antibodies against platelet membrane antigens which causes their enhanced disintegration by macrophages (Cines & Blanchette, 2002). Leptin enhances in vitro secretion of IgG anti-platelet antibodies by splenocytes and PBMCs from patients with chronic ITP (Zhan et al, 2004). After depletion of CD4+ T cells, this phenomenon was no longer observed (Erikci et al, 2006). Further studies showed that leptin could increase platelet reactive T cells (Kuwana et al, 2002). These findings indicate that leptin may in some way be related to the pathogenesis of chronic ITP and may represent a target for therapy (Ren et al., 2006).
There are also data supporting a role for leptin in the development of rheumatoid arthritis (RA). Injection of methylated bovine serum albumin (BSA) into the knees of mice results in the development of antigen-induced arthritis. Ob/ob and db/db mice developed less severe arthritis (compared to wild type mice), with lower IL-1Î² and TNF-Î± present in articular synovial fluid in the knee and decreased levels of circulating methylated BSA antibody. Furthermore, decreased antigen-specific T cell proliferation, lower IFN-Î³ and a higher IL-10 secretion, indicate a shift towards an anti-inflammatory Th2 phenotype (Busso et al., 2002). Reducing leptin levels in RA patients by fasting ameliorate the clinical signs of the disease (La Cava & Matarese, 2004).
In non-obese diabetic mice (NOD) model, used to study type 1 diabetes (an autoimmune pancreatic inflammatory disease, which destroys Î²-cells), there is a pro-dromal increase in serum leptin levels prior to development diabetes (in females). Injection of leptin also accelerates autoimmune mediated lysis of Î²-cells and increases IFN-Î³ production by peripheral T cells. These events support leptin as promoting the development of type 1 diabetes through activation of Th1 responses (Matarese et al, 2002b). It has been found that natural leptin receptor mutants of the NOD/LtJ strain of mice (named NOD/LtJ-db5J) display reduced susceptibility to T1D ( Lee et al , 2005). In general, women have higher circulating leptin levels than men (Elbers et al., 1997) which may help explain their greater to propensity to develop autoimmune diseases (Cooper & Stroehla, 2003), leads to the conclusion that sexually dimorphic leptin concentrations constitute the basis of higher rates of autoimmunity in females ( Matarese et al, 2002).
It has been shown that leptin can affect the survival and proliferation of autoreactive CD4+ T cells in EAE through the nutrient/energy-sensing AKT/mammalian target of rapamycin signaling pathway which can help to explain a link between chronic inflammation and autoimmune T cell reactivity (Galgani et al, 2010).
Leptin and multiple sclerosis
MS, an autoimmune neurodegenerative disorder most often affects in younger (and female) adults. While the exact causes of MS remain unknown, MS pathophysiology is thought to involve complex interactions among genetic, environmental and immunologic factors (Ewing & Bernard, 1998). Relapsing-remitting MS, the most common form of MS, is more common in females(Rosati, 2001). MS therefore is an example of an autoimmune disease whose progression and severity depends on increased levels of many cytokines and chemokines.
It is well appreciated that myelin targeted Th1 CD4+ cells might participate in pathogenesis of MS and it has been shown that Th1 cytokines are increased in the inflammatory lesions of EAE (Steinman & Zamvil, 2003). In contrast, elevation of Th2 cytokines are typically associated with recovery from EAE as well as protection from MS (Williams et al, 1994). As mentioned before, leptin is known to shift immune responses towards the Th1 polarity. One of the most convincing findings demonstrating the critical role of leptin in the induction of EAE was presented by Matarese et al (Matarese et al, 2001). A surge in serum leptin levels has been shown to precede the clinical onset of EAE manifestations . Genetically ob/ob mice resist disease induction in both active and adoptively transfer models of EAE. This resistance vanishes by leptin administration and is associated with the conversion of T cells from Th2 to Th1 programming, as well as increased IgG2a secretion over IgG1. Similarly, in wild-type C57BL/6J which are highly vulnerable to development of EAE, leptin increases disease severity by releasing IFN-Î³ and by augmenting production of IgG2a. These findings suggest that leptin is both required for development of EAE, and likely also human MS.
Investigators have also examined links between leptin and MS (Matarese et al., 2005; Matarese et al, 2008). Leptin is elevated up to 6.5-fold higher in acute/active MS compared to chronic, silent MS (Steinman, 1999). In acute phases of MS, leptin secretion and CSF production of IFN-Î³ were increased (Matarese et al, 2005). In acute MS, leptin secretion was increased in samples of both CSF and serum of MS patients; however this did not track closely with body mass. Leptin elevation in the CSF was higher than that in serum, reflecting latent, secondary sites of leptin synthesis within the CNS and/or the enhanced transport of leptin across the blood vascular barrier following its systemic production (Matarese et al., 2008). . Increased secretion of leptin into the serum has also been detected before MS relapses (even during treatment of MS with IFN-Î²), and leptin has the capacity to enhance the secretion of TNF-Î±, IL-6, and IL-10 from peripheral blood mononuclear cells of patients with MS in vitro during the acute phase of the disease; this does not occur in patients with stable disease (Batocchi et al., 2003). It has been reported that leptin secretion is increased in both serum and cerebrospinal fluid (CSF) of treatment-naive MS patients; CSF leptin levels parallel the secretion of IFN-Î³ in the CSF and are reciprocally related to the abundance of circulating regulatory T cells (Treg). These tolerogenic cells essential for maintenance of anergy are comparatively reduced in patients with MS. Importantly in MS patients, peripheral Treg cells are suppressed by high serum levels of leptin (Matarese et al, 2005). T cells from MS patients which are autoreactive to human myelin basic protein (hMBP) -specific were observed to secrete leptin as well as to increase their expression of leptin receptor (Matarese et al, 2005; Matarese et al., 2005; Sanna et al., 2003) . Up-regulation of the Ob-R in mononuclear cells is seen in relapsing-remitting MS (RRMS) patients during relapse, but is not seen in remission or in controls (Frisullo et al., 2007). This finding suggests that Ob-R may play a role in the pathogenesis of MS by up-regulating the immune response in the acute phase of the disease (Frisullo et al., 2007).
These data strongly suggest a central role for leptin in the pathogenesis of CNS inflammation in both EAE and MS. Therefore, leptin antagonism may offer a new treatment option for MS patients.
It has been shown that blocking leptin signalling with either anti-leptin antibodies or with a recombinant mouse leptin receptor decoy, prior to or following the initiation of EAE, reduced evidence of clinical disease, with reduced disease progression, fewer relapses, less evidence of proteolipid protein 139-151 myelin peptide-induced T cell proliferation, and increased conversion to a Th2 cytokine secretion profile (De Rosa et al., 2006). CD4+ T cells recovered from mice which had been injected with leptin blockers showed lowered responses to PLP139-151 peptide, (measured as the accumulation of intracellular cyclin-dependent kinase inhibitor p27 (p27Kip-1)). Diminished responses induced by leptin blockade were associated with a reduction in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, suggesting that ERK1/2 activity regulates the etiology of EAE and perhaps also human MS (Chen et al, 1999).
Both anti-leptin and anti-leptin receptor blocking antibodies diminished the proliferative responses of the hMBP-sensitized T cell lines to antigenic stimulation, indicating the possibility of using leptin-based interventions to terminate this autocrine loop and block autoreactivity (Matarese et al, 2005). .
Pharmacological inhibition of leptin using several classes of receptors antagonists reduces clinical initiation, progression, and subsequent relapses in both primary or passively transferred EAE (De Rosa et al., 2006). These reactions were correlated with a significant inhibition of delayed-type hypersensitivity reaction against PLP139-151 peptide, reduced CD4+ T cell activation, and an elevated IL-4 and IL-10 production in response to challenge with myelin antigens. Foxp3, a marker for Treg cells and a key regulator of immune tolerance, is more intensely expressed by CD4+ T cells from mice in which leptin function had been neutralized, suggesting that they had switched to the Treg phenotype. Lower T cell responsiveness might represent maintenance of p27Kip-1, (a pro-anergy factor) or reduced phosphorylation of regulatory tyrosine residues on ERK1/2 and STAT6. These finding provide a mechanistic basis allowing for clinical intervention in EAE (and possibly in human MS), which would exploit leptin signaling in the design of therapeutic agents to treat MS (and possibly other chronic inflammatory states) (De Rosa et al., 2006). Leptin neutralization profoundly alters intracellular signalling of myelin-reactive T cells, increasing the number of regulatory T cells which improve the course of EAE (Matarese, 2006).
Diverse actions of leptin discussed earlier on many organ systems and immune functions suggest that attempts to block leptin signalling in vivo should be carefully evaluated as it may cause undesirable, off-target effects. The main concern in the development of leptin-based therapeutic strategies for autoimmune diseases, like MS remains that complete leptin/leptin receptor blockage might interfere with leptins' hypothalamic body mass regulating role. Indeed, treatment of mice with the S120A/T121A leptin mutant (which acts as leptin antagonist) induces significant weight gain by affecting satiety. Weight gain in S120A/T121A treated mice also indirectly implies that the mutant also functions centrally and is actively transported across the blood-brain-barrier (Peelman et al., 2004).
There are different rationales for the design of leptin antagonists. Non-specific agents which block leptin signal pathways which overlap with other systems, such as JAK-STAT, may result in detrimental off-target effects. So far, there is no approved commercially available specific leptin antagonist that can be used for clinical studies with human subjects. The recent development of leptin mutant mice and proteins that interfere with leptin activity or signalling suggest the eventual possibility of leptin modulation in clinical therapy of inflammatory states (Gertler, 2006). A monoclonal antibody against human leptin receptors which has a leptin antagonist effect has been previously described (Fazeli et al., 2006). This antibody inhibits pro-inflammatory actions of leptin by blocking peripheral immune actions of leptin and leptin-induced induction of TNF-Î± by human monocytes, and T cell proliferation (Fazeli et al., 2006). The DNA sequence encoding this antibody has been cloned, and different forms of blocking antibody (Fab and ScFv) produced with similar blocking efficacy as the whole antibody, a first step towards a therapeutic antibody. The greatest advantage of recombinant antibody (rAb) technology is that rAbs can be manipulated genetically to yield specific properties (e.g. humanized conjugated with other molecular motifs, etc) and more importantly producing bifunctional molecules which can simultaneously bind to at least two different ligands, one of which is cell-/tissue-specific, permitting blockade of leptin receptors on a specific target tissues.
The adipose tissue and neuroendocrine system also secretes factors which like leptin also regulate caloric intake and metabolism also affect influence immune status. These mediators include adiponectin, visfatin, neuropeptide Y (NPY), and ghrelin (Tilg & Moschen, 2006). Ghrelin, a hormone stimulated by NPY and Agouti-related peptide (AgRP), (a neuropeptide produced in the brain), is secreted mainly by the stomach and also by the small intestine, pancreas and thyroid (Gil-Campos et al, 2006). Ghrelin is secreted when blood levels of leptin and glucose drop, and stimulates appetite. It is usually increased before meals, decreased after food intake (Shiiya et al., 2002) stimulates the anterior pituitary gland to secrete growth hormone, and is a biological antagonistic to leptin. also has a potent ability to suppress leptin-mediated Th1 cytokine production (Dixit et al., 2007). In humans, ghrelin blocks leptin-induced secretion of Th1 cytokines by T cells (Dixit et al., 2004), and in mice, suppresses EAE through reduction of mRNA levels of TNF-Î±, IL-1Î², and IL-6 in spinal cord cellular infiltrates and microglia (Theil et al., 2009). Therefore, ghrelin may also represent an endogenous antagonist of leptin, and thus find use in the treatment of MS.
Adequate nutrition is a prerequisite for generating appropriate immune responses against invading pathogens. Conversely, sufficient energy stores may be one of the factors required for long-term, detrimental immune reactions, like those observed in autoimmune diseases. Therefore, leptin can be considered as a link between the immune tolerance, metabolic state, and autoimmunity. Leptin, as an inflammatory cytokine, may be responsible for balancing immune responses between immunosuppression and autoimmunity, (Fig. 1) with higher circulating leptin levels predisposing individuals to autoimmune diseases, while low serum leptin reducing autoimmunity, but increase susceptibility to infection (Matarese et al., 2002). As early leptin research has primarily focused on the roles of leptin on body weight regulation, less attention has yet been given to the development of leptin antagonists specifically designed for its peripheral immune-regulating effects. Based on the available evidence in the literature, leptin receptor antagonism might represent an important and novel therapeutic approach for treating autoimmune diseases, including MS. Development of monoclonal antibodies against the leptin receptors that block leptin signalling in specific tissues or organs could be a promising future tool for many immune mediated chronic inflammatory conditions (Fazeli et al., 2006).