Tumour necrosis factor and lymphotoxin: Molecular ...

Immunology and Cell Biology (1996) 74, 465-472

Tumour necrosis factor and lymphotoxin: Molecular aspects and role in tissue-specific autoimmunity HEINRICH KORNER and JONATHON D SEDGWICK Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital. Camperdown. New South Wales. Australia Summary Tumour necrosis factor (TNF) is a highly potent, proinflammator>' cytokine with broad-ranging functions from the regulation of endothelial cell adhesion molecules to facilitate entr\' of leucocytes into tissues, to direct induction of cellular cytotoxicity. This diversity of function potentially attributable to TNF in the genesis of inflammatory disorders place TNF as a primary candidate for clinical targeting and considerable success in this regard has been achieved, particularly in rheumatoid arthritis (RA). In this article we provide a short overview of TNF and its homologue lymphotoxin (LT) a and p. Particular emphasis is placed on recent discoveries regarding the cell surface expression ofthese cytokines and the role of TNF/LT in experimental autoimmune encephalomyelitis (EAE). an animal model of the human demyelinating disease, multiple sclerosis (MS). Key words: apoptosis, autoimmune encephalomyelitis, autoimmunity, cytokines, lymphotoxin, tumour necrosis factor.

Tumour necrosis factor The phenomenon of induction of haemorrhagic necrosis of tumours during some bacterial infections has been long recognized. The first person to attempt to exploit this well-known phenomenon for the treatment of inoperable tumours was William Coley (1862-1936), a New York surgeon, who used preparations of Gram-positive and Gram-negative bacteria for cancer therapy and claimed success in some cases.' As expected, the side effects of his treatment were intolerable and these early attempts at therapy had to be stopped. Much later LPS was isolated as the active ingredient in comparable bacterial preparations to those used by Coley-'"^ and the presence of a tumour necrotizing agent in serum of Bacillc Calmette-Guerin (BCG)-infected and LPS-challenged animals was demonstrated by transfer experiments."^'' This substance, which induced necrosis of the meth-A sarcoma in mice, also killed transformed cells in vitro, including WEHI 164 cells.'" This otherwise undefined and unpuritied substance was termed tumour necrosis factor (TNF). In a parallel line of research. Cerami ('/ al, attempted to identify the underlying cause of wasting in chronic infectious disease." Rabbits infected with Trypanosoma brucei developed severe cachexia losing, despite low parasitic load, up to 50% of their live weight. Paradoxically, the final stage of wasting was marked by a profound accumulation of triglycerides in the scrum. A systemic deficiency in the activity of the enzyme, lipoprotein lipase, was shown to be responsible for this hypertriglyceridaemia.'' The factor responsible for metabolic suppression of this Correspondence: J Sedgwiek, Centenary Institute of Caneer Medicine and Cell Biology, Building 93, Royal Prince Alfred Hospital, Missenden Road, Camperdown. NSW 2050, Australia. Received 23 April 1996; accepted 23 April 1996.

enzyme also was induced in certain endotoxin-sensitive mouse strains (C3H/HeN) by LPS and the activity was transferable to LPS-resistant strains (C3H/HeJ).'- The fact that this substance could completely block lipase activity was exploited in development of a sensitive bioassay'•' fora molecule subsequently called cachectin.'"* '^ Endotoxin-treated macrophages were found to be a major source of the TNF and 'cachectin' activities'^'"' and cachectin exhibited a strong cytotoxic activity for WEHI 164 cells in vitro indicating a high degree of homology."* Identity of the two proteins as the same molecule was finally established by molecular cloning and expression.'''-*' Murine TNF is located on chromosome 17, closely linked to the structurally-related LTa"' and LTp-- molecules and 70 kb proximal to the D region (H2D) of the MHC complex and at a distance of only about 1 Mb to the I (MHC class II; IA and IE) and the H2K regions.-^ Human TNF is located on chromosome 6-'' and closely linked to HLA-B.-^ This close genetic linkage to the MHC complex supports the possible involvement of TNE in those inflammatory autoimmune diseases where a strong association with the use of certain HLA types, such as in ankylosing spondylitis (HLA-B27), can be demonstrated.^^ About 70% of the amino acid sequence of TNF was found to be conserved when TNF from nine mammalian species were compared. TNF is expressed as a 26 kDa cell-associated pre-peptide which is cleaved and released as a 17 kDa mature protein. Both the secreted 17 kDa protein and the 26 kDa propeptide spontaneously trimerize and form the bioactive ligand.-' The threedimensional structure of the trimer has been determined-** and a model of ligand-receptor interaction deduced.-**

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The main activities of TNE in vivo are proinflammatory, including up-regulation of adhesion molecules enabling inflammatory leucocytes to enter the tissue^°-^' and host defence against intra-cellular bacteria! infection.-'-"^'^

Lymphotoxin Soluble cytotoxic factors responsible for so-called 'bystander' killing^^ and potential mediators of tissue damage in delayed-type hypersensitivity^^"^^ were studied in the late 1960s. One factor, produced by T lymphocytes after antigenic or mitogenic stimulation, was studied in detail and given the name lymphotoxin (LT). The relationship of LT with TNF/cachectin was only determined following complete sequencing of the molecule.-'^•^^ In hindsight, it appears that the biological activity attributable to T cell LT was probably derived mostly from TNF. It only became apparent subsequently that T cells, in addition to macrophages, produced TNF.""^ Lymphotoxin and TNF are only about 30% homologous in their primary amino acid sequence,-*^ but of greater significance is the observation that the regions of major sequence homology indicated similarity in the tertiary and quaternary structure of TNF and LT.-" Lymphotoxin, like TNF, forms a trimeric structure'"^ and is sufliciently structurally conserved to bind the same receptor as TNE with similar aflinity,"*' but the cellular response to this binding appears different.'*^ In vilro. LT duplicated the activities of TNF (e.g. WEHI 164 cell lysis) and consequently was considered as functionally redundant to the more prevalent TNF, which apparently differed only in its site of production. Following the cloning of both molecules,-^-^'-^^-'*^ T cells (and indeed almost all cells after appropriate activation) were found to be capable of producing substantial amounts of TNF. while B lymphocytes also produced LT.''^ Thus, some cells produced both TNF and LTa implying an evolutionary pressure for maintenance of both and so possibly a unique role for both cytokines. Moreover, despite a similar binding aflinity to the TNF receptor molecules, the stability of the trimeric structure and duration of binding to the TNF receptors, and different physiological reactions, suggested unique roles for the two cytokines in i'/vt>.''Recent studies ofthe in vivo function of LTa employing gene targeting technology unexpectedly uncovered the central involvement of LT in the formation of secondary lymphoid organs during development.'*-^ LTa-deficient mice almost completely lacked lymph nodes and Peyer's patches and exhibited disturbed splenic architecture. More recently it was shown that the germinal centre reaction in the spleen after immunization also depended on the presence of LTa and TNF receptor (R) p55-'^ to which secreted LTa bind. The latter result implicating TNFR p55 in germinal centre formation did not, however, exclude the possibility that TNE itself may be involved. This possibility will soon be investigated using TNE gene knockout mice.

Receptors and signalling Tumour necrosis factor and LTa induce a plethora of overlapping and seemingly redundant cellular responses. To understand this further it is important to identify the molecular mechanisms involved and particularly the receptors that bind these molecules and deliver the signal intracellularly. Cytokines normally signal through high affinity receptors on the cell surface. These signals are channelled to cytoplasm or nucleus initiating changes in the metabolic or transcriptional state of a receptive cell. TNF (and soluble LTa) signal through two receptors"''''*** which are expressed on almost all cell types."*^ The receptor molecules, with molecular weights of about 55 and 75 kDa, are named accordingly TNFR I p55 and TNER II p75 (sometimes termed p60 and p80). The binding site for the receptor on the trimeric ligand is the interface between two TNF (or LTa) molecules and signalling is triggered by receptor oligomerization. Both receptor molecules signal independently and with distinct pathways.^" TNER I has been found to be responsible for almost all TNF-mediated functions in viiro and in vivo including cellular cytotoxicity. host defence (antiviral and antibacterial). MHC molecule induction and so on. In only a few cases has independent activity of TNFR p75 been demonstrated, the most important being the induction of thymocyte proliferation.-''' Explanations for this puzzling inactivity of the p75 receptor were: (i) that p75 provided only a supportive or modulating function for the p55 receptor in cellular activation; (ii) that the secreted form of TNFR p75 functioned as a buffer for soluble TNF and stabilized the bioactive form;^- or (iii) that the main function of TNER p75 was so-called 'ligand-passing', involving the transfer of bound TNF to TNER p55."-5-* Very recently, however, two distinct intracellular TNER-associated proteins were characterized^^ indicating two separated pathways of signalling. This implied that rather than a subservient role for TNFR p75 the main cellular responses to independent engagement of TNFR p75 simply had not yet been unravelled, or that the role of TNFR p75 in the collaboration with TNFR p55 was more substantial than assumed. TNF and LTa/p as cell surface molecules Largely for historical and technical reasons, active TNE and LTa were (and still are by some) considered to act mostly as (secreted) soluble (sol) proteins (Fig. I). This view has now changed. While TNF has no transmembrane sequence, a hydrophobic stretch in the signal peptide was found to be sufficient to target the molecule to the membrane (Fig. 1) and serve as membrane anchor. It was shown that human TNE (murine TNE behaves similarly) retained a 12 amino acid target sequence situated extracellularly and that specific protease cleavage released the 17 kDa mature protein from the cell surface (Fig. 1).^^ Cell-membrane TNF (mTNF) displays a highly dynamic expression pattern. T cells activated in vitro exhibit a peak of mTNF expression after only 8 h. Eorty-eight hours after activation, surface TNF is again virtually

TNF molecules and autoimmunity

minor and m.i|tir hetprotrimenc forms ot ml.Ta/|)

homotnmenc. soluble LTa

mTNF

transient cell surface expression

Figure I Cell-surface and soluble forms of three 'TNF-family' members: TNF. LT and FasL. ^-^' The proteinase responsible for this efficient cleavage is a highly specific cell surface-associated matrix-metalloproteinase (Fig. 1; reviewed*^*). Interestingly, treatment of mice with an enzyme inhibitor rescued the animals injected with a lethal dose of LPS, from endotoxie shock-'''' implying that it was the free and not mTNF that was the major participant in this pathological process. By 1988. non-TNE-secrctable mutant cell lines were described and this form of TNF proved to be as active as the soluble form in terms of cytotoxicity and anti-tumour activity.^^-^^ A recent study showed that the engagement ofthe p55 and the p75 receptors by surface TNF under cefl-cell contact conditions was able to trigger much more dramatic and perhaps qualitatively different effects.*"' Taking these findings together, it seems likely that solTNF is an exception physiologically, triggered under unusual circumstances such as in massive bacteraemia, while mTNF acts in small doses and is carefully regulated transcriptionally, translationally and via regulated secretion. The view of LTa has changed similarly in recent years. LTa has no transmembrane sequence and no hydrophobic signal sequence. Despite this, membrane LTa was detected on the surface of activated B and T cells as well asT ceil hybridomas, but in association with a 33 kDa glycoprotein.'*'*•*'- This associated protein was identified subsequently as a new memberofthe TNF family, termed LTp. and shown to serve as the membrane anchor for LTa in a LTa/p heterotrimer (Fig. 1, mLT--) LTp, in contrast to LTa, is a type II membrane protein not unlike TNF. The LT membrane complex is not as dynamic as surface TNF after in vitro induction, reaching its peak expression after 24 h and remaining at the surface for a considerable time. The fact that LTp is constitutively expressed, whereas LTa is highly inducible^^ probably underlies the regulation of this surface complex. It is presumed that LTa is 'secreted' when the available cytoplasmic p chain is saturated, p chain homotrimers are not detectable on the cell surface.-The observation that hcterotrimeric mLT did not bind to TNER I or II led the way to the discovery of yet another receptor, the TNFR-related protein (TNFRrp) or LTpR.*"* In vivo, no biological function has been ascribed to this

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receptor. It is possible that the signals necessary for formation of lymph nodes are signalled through this molecule, but no direct evidence is yet available for this. The existence of other TNF and LT receptor molecules is possible and until a genetically-targeted mouse strain lacking the LTp-receptor is available that exhibits the same phenotype as LTa-deficient mice, the primacy of this receptor for mLT will remain uncertain. TNF in autoimmunit} A plethora of cytokines are detectable in inflamed tissues involved in tissue-specific autoimmunity. probably most produced by inflammatory' cells but some by resident cells in response to inflammatory events. While it is still unclear precisely which of these cytokines are involved in tissue damage linked to autoimmunity. considerable evidence now supports a role for TNF (and LTa has not been excluded) in pathogenesis. First. TNF particularly but also LT are present at the site of inflammation, and second, and more significantly, neutralizing antagonists of these cytokines exhibit potent in vivo functions in terms of disease suppression. Studies arc most advanced in rheumatoid arthritis (RA), an inflammatory, degenerative disease of the joints and clinical, double-blind placebocontrolled trials using anti-TNF antibodies have revealed a clear clinical benefit to this therapy.''^ '** Additionally, patients in one study showed a rapid but transient increase in the number of peripheral lymphocytes and a decrease in circulating mononuelear cells following treatment, and a reduction in serum levels of ILl and other inflammatory' markers.^^ In related work, mice with collagen-induced arthritis also responded well to treatment with an anti-TNF antibody, and in this animal model a broad anti-inflammatory capacity was exhibited by anti-TNF mAb, even when mice received treatment after the onset of clinical disease.^^ Nevertheless, it is not well understood how TNF is involved in the initial generation of the inflammatory process. Confusion regarding the mechanismsof action of TNF is due in large part to the considerable range of physiological effects potentially mediated by TNF. On the one hand. TNF can exert cytotoxic effects on different cell types while, on the other hand. TNF has different modulating activities including the induction of other cytokines. and in particular the regulation of vascular adhesion and MHC molecules. Using two other models of tissue-specific autoimmunity, experimental autoimmune encephalomyelitis (EAE), and experimental autoimmune uveoretinitis (EAU). it has been possible to pursue these issues further. EAE, an inflammator>' autoimmune disease ofthe central nervous system (CNS), normally studied in rat or mouse, is initiated and probably largely mediated by autoantigen-specific T cells.*''* EAE displays some features ofthe human CNS demyelinating disease multiple sclerosis (MS). Only a low percentage of CNS-infiltrating cells are actually antigen-specific T cells.'" These encephalitogenic cells produce TNF and LT but whether pathogenicity of these cells is directly related to the amount of produced cytokine^' is controversial.'- Nevertheless, blocking of

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TNF (or LT) in vivo using neutralizing antibodies or receptor molecules effectively inhibits or ameliorates EAE.'^-" The mechanism of action of administered TNE antagonists is unclear but anti-TNF mAb were shown not to interfere with the inductive phase ofthe disease.'^''*'^ It was suggested more recently, that it was the lack of TNF-dependent adhesion molecule up-regulation following TNF blockade in vivo that was responsible for disease inhibition, by preventing effector cell access to the CNS.^° To shed more light on the role of TNF/LT in the pathogenesis of EAE a soluble human TNFp55 receptor fused to human IgGl (TNF-Rp55-huIgG) was used to treat rats with a passively acquired form ofthe disease.'* In this case, however. CD45 congenic T cell donor and recipient animals were used that enabled tracking of the antigen (myelin basic protein, MBP)-specific donor T cells to the CNS using RT7 (CD45) allotype-specific mAb to label the donor T cells (Fig. 2). Injection of 5 x 10^ T ceUs in con180

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Figure 2 TNFR-IgG prevents passively-transferred EAE in rats without impeding MBP-specitic T cell movement into the CNS. Histogram. PVG-strain CD45-congenic rats received 5x10'^ MBP-spccihc CD4' T cell blasts i.v. (dO). the T cells derived from MBP-immunized CD45 allotype-disparate PVG rats. Recipient rats were injected with 200 ng TNFR-IgG (—•— and hatched bars) or huIgG (—O— and open bars) daily i.p. as indicated (!)• Bodyweights are represented by lines and clinical scores by bars. Letters A-C correspond to histological plates. Histology. In parallel experiments. CNS tissue was removed from animals on day 5. Shown are PVG CD45^ • MBP-specific T cells stained in the medulla (A and B) and spinal cord (C) of huIgG (A) or TNFR-IgG-treated (B and C) PVG CD45«^ host rats. V, vessel. Arrows indicate host inflammatory cells negative for the donor T cell CD45 allotype. (A and B) Transmitted light interference contrast. (C) Bright-field illumination. Bars=100|im. Reproduced from Proc, Natl Acad. Sa, USA 1995; 92: 11 066-70 by copyright permission ofthe National Academy of Sciences.

trol recipient rats led to a reliable monophasic course of disease with symptoms of mild paralysis at day 5 and a rapid recovery after day 6 whereas TNF-Rp55-huIgGtreated rats exhibited no clinical signs of disease. Row cytometric analysis of CNS cells recovered from both treated and control groups showed that a substantial number of donor encephalitogenic T cells were present in the clinically healthy TNF-R-treated recipient rats, indicating that the T cells indeed entered the CNS. The major detectable difference was the absence of non-specific leucocytes at an early stage of disease in the treated animals although such cells did accumulate later. The locality of MBPspecific T cells in the CNS of treated versus control rats was the same (Fig. 2).

TNF molecules and autoimmunity

Comparable findings have emerged from studies in actively-induced EAE using Lewis rats immunized with MBP and adjuvant (H Korner, G Chaudri and JD Sedgwiek unpubl. data) where the treated animals remained free of clinical signs of EAE despite a substantial number of leucocytes in the CNS. Flow cytometric phenotyping of this infiltrate revealed that the inflammatory T cells in treated (and still healthy) rats, were highly activated but non-specific inflammatory macrophages and resident microglia were apparently less activated, at least as defined by MHC class II and CDl Ib/c expression. These simple but unambiguous experiments demonstrated that inhibition of TNF/LT function could prevent EAE without simply inhibiting movement of effector T cells into the CNS. The data placed emphasis on a direct TNF/LT effector (i.e. tissue damaging) function in the pathogenesis of EAE. As a result of these findings, we speculated that if T cell effector function has been blocked, specific target tissues should remain intact despite the presence of inflammatory cells within the tissues. To resolve this issue, we turned to experimental autoimmune uveoretinitis (EAU), an established model of human posterior uveitis. In EAU, unlike EAE, both leucocytic infiltration ofthe tissue (the retina) and destruction of a specific target tissue (rod outer segments (ROS) of the retina) can be readily identified and graded semiquantitatively by light microscopy. These studies''* demonstrated that: (i) there was quantitatively normal movement of highly activated (CD25*MRC OX40*MHC class II*) T cells to the retina of TNFR-lg-treated rats (relative to controls); and (ii) despite this, the ROS and other neuronal elements within the retina were relatively preserved. The results were consistent with the previous studies in EAE regarding the uninterrupted movement of autoreactive T cells to the CNS after TNFR-IgG treatment, but demonstrated additionally that a specifically targeted tissue was protected. Figure 3 illustrates potential points of TNF function in CNS inflammation. It is known that blockade of TNF function (at least with TNFR-Ig) is not sufficient to prevent activated T cell adhesion and extravasation (point 1) but is probably preventing disease (EAE/EAU) by interfering with TNF-mediated tissue damage (point 2). We speculate that mTNF is the most relevant molecule in this regard and consistent with this is the capacity of TNFRIgG to bind to the 26 kDa cell-surface form of TNF on rat T cells (H Korner, G Chaudri and JD Sedgwiek unpubl. data).

TNF in apoptosis The Fas ligand (FasL) molecule, so clearly important in delivering apoptotic signals via Fas (CD95) to a range of cells (reviewed^'^-^') is also illustrated in Fig. 1. In contrast to TNF, FasL was generally viewed as a surface molecule (Fig. I) but it is now known that, like TNF, FasL may also be cleaved from the cell surface and released (Fig. 1).^Also like FasL, the cytotoxic activity of TNF is based on its ability to induce programmed cell death (apoptosis). This can be achieved via binding to the TNFR I which

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T-T-mediated Apoptosis

M0-induced T cell Apoptosis Figure 3 Multiple potential sites of interaction for TNF in induction and resolution of autoimmune inflammation. Shown here is a schematic version of possible TNF-mediated events in the CNS. (I), Leucoeyte-endothelial cell adhesion; (2), mTNF and solTNF-mediated tissue damage (macrophage (MO) or T cell-mediated); (3), mTNF-mediated T cell apoptosis. T cells are known to die by apoptosis in the CNS although the role of TNF in this process is purely speculative at this stage. (MG. mieroglia; AST. astrocyte).

contains an intracellular 'death-domain' homologous to the Fas cytoplasmic tail death-domain*^ or by signalling through both TNF receptors in parallel using a synergistic combination of both, distinct, signalling pathways.^' Interestingly, the intracellular death-domain of TNFR I and Fas tend to self-association and support the receptor clustering which would lead to cell death. This means there has to be intracellular factors normally preventing this, thus introducing another level of regulation.'^'* To date, relatively little is known about the functional significance of TNF-mediated apoptosis, at least within the context of TNF-TNFR cell-cell interactions. There are different spheres of action conceivable: First, the autoregulation of mature, peripheral T cells (T cell receptordependent, activation-induced cell death) via TNF has been demonstrated. In this one /// vitro case, CD8 * T cells seemed more susceptible to TNF-dependent apoptosis while CD4 * T cells were more susceptible to the action of FasL.*^ A second possibility is the clearance of inflammatory post-activated T cells from tissues (Fig. 3). There is evidence that apoptosis is a major mechanism of elimination of T cells from inflammatory lesions in the CNS**^ and antigen-reactive T cells are preferentially removed. Our own studies indicate that the lethal hit could be mediated by resident tissue macrophages (microglia; A Ford and JD Sedgwiek unpubl. data). Another study^' has shown that M-CSF-differentiated human blood monocytes induce antigen-dependent apoptotic CD4* T cell death in what appears to be a FasL-independent mechanism. Whether mTNF is involved in apoptosis in microglial cell-T cell interactions is currently being investigated in our laboratory. Finally, a tumoricidal activity of mTNF may be of some importance. Many transformed cell lines

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are resistant to TNF-induced cell death but a recent study showed that the simultaneous engagement of TNFR I and II by membrane TNF rendered a variety of tumour cell lines susceptible to apoptotic death.*' In summary, the general and long-held impression amongst workers in the TNF/LT field that the membrane forms of these molecules were the most physiologically significant, is now receiving increased experimental support.

Conclusion TheTNFfamilyof ligands and their receptors continue to grow at a considerable pace and now comprises 11 members. These members are trimeric type II membrane proteins which also have an active secreted form and function by oligomerization of their receptors. The only exception known is LTa which is secreted exclusively. However, the discovery ofthe function of LTp as a membrane anchor has abrogated this anomaly. All members of the TNF family are involved in regulation of homeostasis, some more in a role as costimulatory molecules supporting leucocyte differentiation and proliferation, others in regulation or induction of cell death. TNF, Fas L, soluble LTa and the newly discovered TRAIL*'*' are all able to induce programmed cell death and do so in an orderly and highly regulated manner. The close analysis of new mouse strains deficient for one or more of the TNF family of molecules will undoubtedly reveal much about the physiologically important collaborative interactions between members of this molecular family.

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