INTRODUCTION

Signaling molecules of immune system are cytokine that may either stimulate or suppress the responses of various cells involved in host immune mechanisms and interact or convey the essential messages for the proper functioning of defense systems against numerous pathogens or disease conditions (Chauhan and Dhama, 2008; Dhama et al., 2011). They trigger signal-transduction pathways by binding to receptors on a target cell (that ultimately alter gene expression in these cells). They are involved in both innate and adaptive immunity play important role as adjunctive immunomodulators in a variety of infectious diseases (Darnell Jr., 1997; Bach et al., 1997; Chang et al., 2004; Tizard, 2004). Tumor Necrosis Factor (TNF) is one among the most studied and central pro-inflammatory cytokines (Gillett et al., 2010). It was first identified in 1984, in which the two forms, TNF-β and LT-α (otherwise called lymphotoxin or TNF-α), were isolated; TNF-β from activated macrophages and LT-α from T cells. These proteins have now become representatives of a distinctive superfamily of cytokine ligands (TNF ligand superfamily) along with their corresponding receptors (TNF receptor superfamily); altogether constituting the TNF Superfamily (Croft et al., 2012). Most of the members are expressed by immune cells or can target immune cells and thereby exert a wide range of actions including the production of inflammatory chemokines and cytokines, promoting cellular growth, differentiation and survival etc. TNF-β induces the expression of selectin which bind to mucins expressed by circulating neutrophils and this binding mediates the attachment or wandering neutrophils to the vascular endothelium (Gabay and Kushner, 1999; Dhama and Chauhan, 2008). Cytokine like TNF-β also induces coagulation; increases the vascular permeability and induces increased expression of adhesion molecules on vascular endothelial cells along with playing a central role in chronic inflammation as well as autoimmunity and malignant diseases like cancer. For maintaining the homeostasis of the immune system; inflammation as well as host defence, TNF plays a major role but there exists a dark side to this powerful cytokine (especially clear in middle aged and old aged people) (Balkwill, 2006; Dhama et al., 2008).

Tumor Necrosis Factor (TNF) which was described as a key cytokine for inflicting necrosis of tumors has now been recognized as a key regulator of the inflammatory responses. Many scientific researches have concluded that TNFSF ligand-receptor interactions and signaling pathways are active in mediating several of the inflammatory and autoimmune diseases (Watkins et al., 1995). TNF ligands were identified to have central role in a variety of inflammatory conditions viz., rheumatoid arthritis, inflammatory bowel disease, ankylosing spondylitis, psoriasis, graves’ disease, SLE, multiple sclerosis, diabetes, asthma etc. (Croft et al., 2012). Moreover various genetic polymorphisms in the proteins of this superfamily have been associated with the susceptibility to developing disease (Haritunians et al., 2010; Jung et al., 2010).

As the involvement of the TNF superfamily ligand and receptor molecules in the incidence of the above pathological conditions has been established, it is clear that the inhibition of these communications can prevent the downstream signaling pathways, leading to the suppression of inflammatory immune cells and thereby diminishing the pathology of autoimmune and inflammatory diseases (Locksley et al., 2001). Several TNF blocking drugs have been presented with therapeutic success and based on the success of these therapies, attention is put for further developments (Thompson et al., 2011; Dhama et al., 2013).

TNF SUPER FAMILY

The Tumor Necrosis Factor (TNF) superfamily consists of approximately 50 membrane-bound and soluble proteins. About 350-450 million years ago there was evolution of ligand and receptor molecules along with adaptive immune system (Croft, 2009). These are highly conserved proteins, found in all mammals (Croft, 2003a). Important ligand members of this family include TNFα, LT-α, LT-β, OX40L, TRAIL, CD40L, CD27L, CD30L, FASL, 4-1BBL, OPGL, LIGHT, APRIL, DR3, DR4, DR5, RANK and TALL-1 etc. (Ashkenazi and Dixit, 1998; Shu et al., 1999; Croft, 2003b; Kodama et al., 2005). With the exception of LT-α all ligand members are type-II membrane proteins forming the hallmark of ligand family with C-terminal extracellular domain having conserved region of 150-amino acids that folds into homotrimers formed by β-pleated sheet. It can generate a soluble functional form when released by proteiolytic degradation. But unlike other protein families of notable homology, these proteins display only about 20±25% homology at the protein level (Gillett et al., 2010; Croft et al., 2012).

Type I transmembrane proteins constituting a variety of cysteine-rich motifs in their extracellular domains (disulphide bridge-based structural modules) are the members of receptor superfamily. There are two distinct types of TNF receptors; type I and type II with molecular sizes 55 kD and 75, respectively. A pre-ligand assembly domain (PLAD) has been described recently for a subset of receptors, including Fas, TNF-receptor I (TNF-RI) and CD40 which is thought to facilitate homotypic association between monomeric receptor subunits, by which the receptor complexes are capable of responding rapidly to environmental cues. With the emergence of adaptive immunity, an extended family gives rise to this core family of ligand-receptors through en-bloc gene duplication events (partially from single ancestral genes). They include the TNF superfamily homologue Eiger and the TNF-RSF homologue Wengen (Clark et al., 2005). Regarding the expression, different molecules of TNF ligand-receptor family are not ubiquitously expressed. The expression of several of these molecules is increased following immune-cell activation and this suggests that they have a central role in modulating immune responses. Studies of TNFRs expressed by conventional T cells and their ligands expressed by antigen-presenting cells (ApCs) has led to the hypothesis that antigen recognition by T cells engages and bidirectionally activates TNF-TNFR pair that contribute to the immune cells (including T lymphocytes) effector responses (Croft, 2003a, 2009; Fang et al., 2008).

Cytokine binding to some TNF receptor family members, such as TNF-RI, TNF-RII and CD40 favour the recruitment of TNF receptor-associated factors (TRAFs) to the cytoplasmic domains of the receptors. The TRAFs activate transcription factors like nuclear factor KB (NF-KB) and activation protein-I (API). Cytokine binding to other family members, such as TNF-RI, leads to recruitment of an adapter protein that activates caspases and triggers apoptosis. Thus, different members of the TNF receptor family can induce gene expression and cell death and some can do both. TNFRI knockout mice show more impaired host defense than do TNF-RII knockout mice, suggesting that TNF-RI is more important for the function of the cytokine. Then within 10-20 min, on ligation with TNF-β, TNFR I get trimerized and recruits various adapter molecules. This will activate NF-κB, inducing several anti-apoptotic genes (Complex I formation) and survival signal (Lee et al., 2002). This is followed by (more than 2-3 h) by an endocytosis of receptor complex resulting in the dissociation of certain adapter proteins (TRAF-2, RIP) and recruitment of Fas-associated Death Domain (FADD) and procasepase-8 to form Death-inducing Signaling Complex (DISC). In the DISC, caspase-8 is activated and released into the cytoplasm where it activates effector caspases to induce apoptosis (Croft, 2003a).

ACTION AS MEDIATORS OF INFLAMMATORY PROCESS

TNF ligands interacts with the either of the two receptors, TNFR1 and TNFR2, that are differentially expressed on cells and initiate varied signalling cascades, leading to diverse cellular responses viz., cell death, proliferation, differentiation and migration (Bradley, 2008). Various pro-inflammatory changes are the outcomes of TNF responses of vascular endothelial cells that lead to adhesion and trans-endothelial migration of leukocytes along with vascular leak. TNF also induces the expression of certain pro-coagulant proteins and down-regulate anticoagulant protein, thereby inducing intravascular thrombosis (Mark et al., 2001; Croft, 2003a). Another major role of TNF is as a modulator of host defense mechanism against infectious agents; especially gram negative bacterial organisms. This role has been confirmed by studies conducted in mice deficient in TNF receptor. Those mice had severely reduced clearance of the bacterium Listeria monocytogenes and immediately yielded to this infection (Pfeffer et al., 1993; Rothe et al., 1993; Bradley, 2008). There are also evidences that increased serum TNF levels were found in the cases of uncomplicated Plasmodium falciparum malaria and markedly increased in cases of fatal cerebral malaria (Bradley, 2008). This indicates that TNF is physiologically an important factor for the normal defense against infections, but can be harmful if the production is inappropriate or extreme. In hepatic diseases there will be reduced TNF production and accompanying incapacity to fight infection (Gillett et al., 2010).

In order to develop and modulate immune system, majority of members of ligand family play important roles as lymphoid-enriched tissues are their main expression sites (Khare et al., 2000). TNF, which is predominantly from the macrophages, is a key mediator of inflammatory responses and defenses (Tracey and Cerami, 1994; Khare et al., 2000). Fas ligand mainly synthesized by activated T cells, modulates thymocyte apoptosis mediated by T cell receptor (Castro et al., 1996). CD40L, which is also expressed by activated T cells, signals for B cell survival and proliferation as well as immunoglobulin isotype switching (Khare et al., 2000; Gillett et al., 2010).

Many members of the TNFSF were recognized to modulate some non-lymphoid cells also. TNF which was initially described as a circulating factor, causing necrosis of tumour, has since been identified as a key regulator of the inflammatory responses. TNF ligands were identified to have central role in a variety of inflammatory conditions viz., rheumatoid arthritis, inflammatory bowel disease, ankylosing spondylitis, psoriasis, graves’ disease, SLE, multiple sclerosis, diabetes, asthma etc (Croft et al., 2012).

In approximately 1% of population Rheumatoid Arthritis (RA) develops most commonly. The condition is characterized by persistent synovitis resulting in progressive damage, erosion of adjacent bone and cartilage and systemic inflammation, leading to chronic disability. Synovial hyperplasia accompanied by angiogenesis is a prominent feature of the condition (Geiler et al., 2011). Predisposing causes to the condition include genetic causes, autoantibodies and environmental factors etc. Several pro-inflammatory cytokines viz., TNF, IL-1, GM-CSF, IL-6, are produced within the inflamed joint cavity. Because of their predicament in the inflammatory reaction cascade, tumor necrosis factor ligands proven to be the most important cytokines involved in rheumatoid arthritis (Geiler et al., 2011).

Crohn’s disease-like inflammatory bowel disease and ulcerative colitis are inflammatory conditions inflicting the lamina propria of intestine and an over-expression and increased immunoreactivity of various TNF family members have been demonstrated to be involved with the development of these inflammatory conditions (Sands et al., 2004). Patients affected with Crohn’s disease are more prone to another autoimmune condition called Ankylosing spondylitis, which is an inflammatory arthritis, particularly affecting the spine along with sacroiliac joints. In those patients with this condition, elevated serum levels of various TNF proteins were identified that correlates the role of TNF in the disease incidence (Lange et al., 2000; Francois et al., 2006).

Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which there will be production of autoantibodies that are specific for the DNA, RNA or various proteins associated with nucleic acids. This will lead to the formation of immune complexes in small blood vessels, especially in the kidneys (Yang et al., 2009). The affected patients with SLE usually found to have abnormal B- and T-cell function (Croft, 2009; Croft et al., 2012). Psoriasis, an autoimmune and inflammatory disorder affecting the skin, is characterized by the presence of psoriatic plaques, formed by inflammatory cell infiltration and hyperkeratotic lesions. A study conducted in patients with psoriasis has shown an upregulated expression of TNF receptors in blood vessels associated with dermal layers (Chaudhari et al., 2001; Gottlieb et al., 2004; Bradley, 2008).

TNF exerts its action in the central nervous system also, where its main sources are microglia and astrocytes, in response to infections, inflammations, ischaemic as well as traumatic injuries. But it has been shown to have both detrimental and favorable effects in brain, depending upon the conditions (Arnett et al., 2001; Bradley, 2008). In murine model, blockage of TNF during ischaemic injury to the brain resulted in amelioration of the condition (Nawashiro et al., 1997) but in case of experimental autoimmune encephalomyelitis, those with lack of TNF were highly susceptible to the condition and treatment using TNF supplementation reduced the condition. SLE is a multiple organ system related autoimmune disease with exceptionally diverse clinical manifestations. Various studies have shown that TNF ligand-receptor pair has been implicated in this autoimmune condition also (Croft, 2009).

TNF has its role in cardiovascular diseases also, in which the disease pathology has been found to be associated with the exacerbated level of various TNF cytokines. Variety of pathological conditions of cardiovascular system viz. atherosclerosis and myocardial infarction; myocarditis, heart failure and cardiac allograft rejection and vascular endothelial cell responses have been implicated with TNF. Several studies point out that certain chronic inflammatory and autoimmune conditions such as rheumatoid arthritis have predisposition towards cardiovascular diseases. This predisposition of increased cardiovascular risk has been found to be in association with elevated serum level of inflammatory mediators, mainly TNF and there also evidences for amelioration of these conditions on anti-TNF therapy (Wolfe and Michaud, 2004; Croft, 2009).

The key role of TNF is connected with the incidence of several inflammatory conditions in respiratory system also and some of those conditions include asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, chronic bronchitis etc (Mukhopadhyay et al., 2006). In the case of asthma, leukocytes collected from bronchiolar lavage of the patients were to have augmented release of TNF and it was shown to play role in bronchial hyper-responsiveness by inflicting inflammation and remodeling. TNF is associated with the pathogenesis of various renal diseases like glomerulonephritis, renal transplant rejection, renal ischaemia etc. (Al-Lamki et al., 2005). It has been found that in case of diseases associated with renal inflammation, difference in the signalling of TNF receptor subtypes leads to difference in the efficacy and adverse effects of various blocking agents (Al-Lamki et al., 2010).

DIFFERENT TNF LIGAND RECEPTOR INTERACTIONS IN MEDIATING INFLAMMATORY AND AUTOIMMUNE DISEASES

OX40-OX40L: One among the TNF receptor-ligand pair is OX40-OX40L which is proinflammatory in function. Various knockout studies have proven that they play major role in the development of autoimmune disease like Rheumatoid Arthritis (RA), inflammatory colitis, diabetes, Multiple Sclerosis (MS), asthma and atherosclerosis in murine models (Croft, 2009; 2010). Compared to healthy individuals, in case of individuals with mild asthma there is upregulation of expressed OX40 and OX40L in the submucosa of bronchi (Siddiqui et al., 2010). Mice deficient in OX40L suffer from polymicrobial sepsis showing improved survivality and reduction in production of cytokine and damage of organ. Ocular inflammation is also found to have diminished in murine autoimmune uveitis thereby widening the scope of disease control by OX40 and OX40L interaction (Zhang et al., 2010). In a model of coxsackie virus B3 driven myocarditis, heart inflammation shown to be ameliorated by the blockade of OX40L, thereby leading to increased survivability (Fousteri et al., 2011). This indicates the connection of this ligand-recceptor interaction with atherosclerosis development (Croft, 2009; 2010). This interaction has also been proven in correlation with the risk of development of Systemic Lupus Erythematosus (SLE). OX40 has been found to be associated with lupus nephritis condition by demonstrating its increased expression on Th17 cells which infiltrate kidney parenchyma (Dolff et al., 2010).

CD30: CD30, known for its increased expression on malignant tumors like Hodgkin’s lymphoma, has been proven for its role in autoimmune diabetes and asthma. Blockage of CD30-CD30L interaction was found to reduce the development of these diseasevin the NOD mouse models (Oflazoglu et al., 2009). In case of colitis, the blocking of CD30L interactions lead to the suppression of trinitrobenzene sulfonic acid (TNBS)-induced colitis (Sun et al., 2008; 2010). CD40-CD40L interaction, which was described to modulate B cell activity and isotype switching has now been identified to promote various inflammatory and autoimmune conditions in murine models. These conditions include Graves’ disease, SLE, RA, MS, diabetes, asthma, psoriasis, inflammatory bowel disease etc. (Peters et al., 2009). CD40L is also expressed by platelets which indicate that it may be involved in the development of atherosclerosis (Lievens et al., 2010). This is supported by the finding that platelets from CD40L-deficient mice were fail to adhere on vascular endothelium in vivo and unable to form aggregates of platelet and leukocyte atherosclerotic lesions (Lievens et al., 2010; Croft et al., 2012).

4-1BB: 4-1BB, another important member of this superfamily, was identified as a stimulatory molecule of activated T cells, has now been shown to express on activated antigen presenting cells and other cell types. Various cancer studies conducted in murine tumor models have shown that they contributes for the augmented activity of T cytotoxic cells and NK cells (Tansey and Szymkowski, 2009). 4-1BB may also control sepsis and biliary cirrhosis. This ligand has shown to exert its effect on sepsis in which the ligand deficient mice exhibited reduced mortality in a sepsis model. In case of primary biliary cirrhosis, the expression of 4-1BBL has shown a positive correlation with the disease markers of serum viz., interleukin (IL)-18, bilirubin, glutamyltransferase etc. (Nguyen et al., 2009; Croft et al., 2012).

CD70-CD27 l: CD70-CD27 ligand-receptor interaction has been found to be between a range of immune cells and also between immune and some nonimmune cell types. This interaction is implicated in several autoimmune diseases especially in the case of Experimental Autoimmune Encephalomyelitis (EAE), ie mouse models of multiple sclerosis and also in SLE of humans and murine models (Nolte et al., 2009; Croft et al., 2012). Recent studies with collagen-induced murine models of rheumatoid arthritis shown to had diminished disease symptoms upon anti-CD70 antibody therapy. Expression of CD70 is reported to be Th1 cell-specific which correlates with the finding that therapies involving CD70 blockade had its ameliorating effects on Th1-driven hypersensitivity reactions and contact hypersensitivity reactions, however no effect on the extent of pathology of Th2-type disease models as in the case of Th2-driven asthma and experimental allergic conjunctivitis (Sumi et al., 2008; Behrendt and Hansen, 2010).

DR3-TL1A: Death receptor 3 (DR3) and TNF-like factor 1A (TL1A) interactions are associated with Th1 co-stimulation and these molecules are now been implicated in the pathology of gut inflammatory conditions (Croft, 2009) such as IBD, ulcerative colitis and Crohn’s disease (Tremelling et al., 2008; Haritunians et al., 2010). There is also evidence of increased TL1A expression in connection with the development of T cell-dependent inflammatory small bowel pathology. In addition to Th1 cells the expression of DR3 is found on Th17 cells (Meylan et al., 2011; Croft et al., 2012). The TL1A-DR3 interaction is not restricted only to those diseases which are regulated by Th1- or Th17 cells, but also by Th2 cells. In anti-TL1A therapy to Th2-driven murine models of asthma, it displayed impaired expression of Th2 cytokines, leading to the suppression of airway inflammation and mucus production (Zhang et al., 2009). Glucocorticoid-induced TNF receptor-related protein (GITR) and its ligand (GITRL): The GITR-GITRL interaction pathway has reported to be active in the development of type 1 diabetes and pancreatitis. This has been proven by the study in NOD mice using agonist anti-GITR antibody therapy, leading to a higher incidence of diabetes (Galuppo et al., 2011; Croft et al., 2012).

THERAPEUTIC TARGETING OF TNF

It has been established that the appropriate regulation of TNF ligand and receptor interactions and functions are crucial for the proper immune system activity. Excessive production of various TNF cytokines has been attributed with the development of an array of autoimmune as well as inflammatory conditions, some of which are discussed above. In order to suppress the immunopathology and disease progression of these conditions, therapies should be aimed mainly against the responses of immune cells like T cells, APCs, NK cells and NKT cells. It is ideal that this approach be accompanied by the maintenance of Treg cells, thereby facilitating the control of disease for long-term (Croft, 2009). As per these findings several successful endeavors have been made to target these ligand-receptor interactions for the therapeutic management of inflammatory/autoimmune conditions (Gillett et al., 2010). Cytokine therapy is an emerging and promising treatment regimen as are the other ones like monoclonal antibodies, avian egg antibodies, gene silencing, gene therapy, apoptins, herbal and panchgavya elements (Mahima et al., 2012; Deb et al., 2013; Dhama et al., 2005a, b; 2013).

Certain catabolic effects on fat cells and whole animals are exerted by TNF-β and have been proven by TNF-α messenger RNA expression profiling in rodent models (obese fa/fa rats) wherein an increase in the uptake of glucose in response to insulin peripherally has been observed significantly (Hotamisligil et al., 1993). As TNF-α bears a neuro-inflammatory domain in the nervous system it plays a pivotal role in drug development in the treatment of neuropathic pain originating both centrally and peripherally. It has been proven by various studies conducted in animal models of neuropathic pains based on various types of nerve injuries viz. peripheral versus spinal nerve as well as ligation versus chronic constrictive injury (Leung and Cahill, 2010).

Preclinical studies have analyzed the activity of neutralizing antibodies that are targeting specifically for TNF ligand proteins, or of Fc fusion proteins with a TNFR that binds to the ligand and thereby blocks the endogenous interaction. The effects of blocking the TNF ligand-receptor interactions discuss have been assessed in models of inflammatory disease (viz., allergy, bronchial asthma, organ transplantation, graft-versus-host disease (GvHD) and atherosclerosis) and autoimmune disease (including experimental autoimmune encephalomyelitis (EAE), diabetes, colitis, adjuvant- or collagen-induced arthritis and systemic lupus erythematosus (Taylor et al., 2002). These studies have shown that neutralizing any one of these TNF-TNFR interactions can result in overpowering of conditions, which in most of the cases, is precisely linked to decline in CD4+ or CD8+T cells activity, or in other cases results in the impairment of NK- and NKT-cell function (Zimmerer et al., 2012).

TNF-β is a tumor promoter helping to produce the toxic effects involved in conventional cancer therapy viz. cytokine release syndrome as well as nephrotoxicity induced by cisplastin. TNF-β antagonists have been developed effectively against rheumatoid arthritis and inflammatory bowel disease due to the central role played by TNF-β in inflammation (Szlosarek and Balkwill, 2003). Inhibition of TNF has also been proven to be an effective therapy for inflammatory diseases viz. psoriasis and psoriatic arthritis; ankylosing spondylitis but the efficacy of preventing septic shock and acquired immunodeficiency syndrome (AIDS) have been questioned. Certolizumab pegol is a novel TNF inhibitor (Esposito and Cuzzocrea, 2009). Different therapeutic agents are now in market under the label of TNF blockers. This includes monoclonal antibodies to TNF and TNF receptor fusion proteins. Drugs named Infliximab and Adalumimab are among the monoclonal antibodies to TNF whereas Etanercept and Lenercept are the TNFR fusion proteins (Gillett et al., 2010; Croft, 2009; Croft et al., 2012). Etanercept comes under a human recombinant protein which is soluble in nature. It is a fusion protein in which TNFR2 has been coupled with the Fc portion of immunoglobulin G. Infliximab is a chimera of human and murine immunoglobulin G1 monoclonal anti-TNF antibody. Adalibumab is produced by the phage display and is a human anti-human TNF antibody (Bradley, 2008; Gillett et al., 2010). In case of rheumatoid arthritis condition all the above drugs have shown to be effective. For patients with Crohn’s disease, infliximab is shown to be effective in inducing remission but not etanercept (Tracey et al., 2008; Bradley, 2008). In ankylosing spondylitis condition, both etanercept and infliximab are proven to be effective. Various clinical trials have shown that infliximab, etanercept and adalimumab are operative for treating all the manifestations in case of psoriasis. In case of patients affected with refractory asthma has shown evidence for beneficial effects of the drug etanercept on markers of asthma to control the condition (Tyring et al., 2007; Tracey et al., 2008; Bradley, 2008; Gillett et al., 2010; Croft et al., 2012).