TNFα

TNF Bharat B. Aggarwal1,*, Ajoy Samanta1 and Marc Feldmann2 1

Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, P.O. Box 143, Houston, TX 77030, USA 2 Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK * corresponding author tel: 713-792-3503, fax: 713-794-1613, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05001.

SUMMARY The search for the cause of hemorrhagic necrosis in tumors led to the discovery of tumor necrosis factor (TNF). This cytokine is a protein that exists in both soluble (157 amino acids long) and transmembrane form (233 amino acids long), produced primarily by macrophages in response to various inflammatory stimuli. It mediates its action through two distinct receptors, a p60 form and a p80 form. Extensive research within the last 15 years has revealed that although TNF is required for protection against bacterial infection, it is involved in cell growth modulation, viral replication, immune system regulation, septic shock, autoimmune diseases, rheumatoid arthritis, inflammation, and diabetes. Agents that can block TNF action, such as thalidomide, soluble TNF receptors, and anti-TNF antibodies, have been approved for human use for autoimmunodeficiency disease syndrome (AIDS), rheumatoid arthritis, and inflammatory bowel disease, respectively.

BACKGROUND

Discovery The story of TNF began more than a century ago (1868), when the German physician Dr P. Brunes noted spontaneous regression of tumors in patients following acute bacterial infections, e.g. tuberculosis (for references see Old, 1985; Aggarwal, 1987). This observation led William B. Coley (1894) to study the effects of bacteria-free filtrate preparations from streptococci and other bacteria on tumors in humans;

in many cases the tumor volume was reduced. These preparations have been referred to in the literature as Coley's toxins. Gratia and Linz (1931) used the experimental model systems and reported on the regression of liposarcoma tumors in guinea pigs by Escherichia coli culture filtrates. By 1944 one of the major inducers of TNF, endotoxin, had been isolated as bacterial polysaccharide from a gram-negative bacterium by Murray Shear and his colleagues. This polysaccharide caused hemorrhagic necrosis of tumors. Glenn Algire and his colleagues reported in 1952 that the mechanism of this necrosis was systemic hypotension leading to collapse of tumor vasculature, resulting in tumor cell anoxia and cell death. Subsequently, O'Malley and coworkers in 1962 reported the production of a factor in the serum of normal mice challenged with endotoxin; since this serum could induce necrosis when administered to tumor-bearing animals, it was named tumor necrosis serum (TNS). When Carswell et al. (1975) confirmed the activity in serum of mice injected with endotoxin, they renamed it as tumor necrosis factor (TNF). This serum factor was found to be cytotoxic to tumor cells in culture (Helson et al., 1975) but its molecular identity was not clarified until a decade later. In the meantime, our laboratory isolated a cytotoxic factor from the human B lymphoblastoid cell line RPMI-1788 and named it lymphotoxin (LT) (later we renamed it as TNF ; Aggarwal et al., 1984, 1985a). Soon thereafter, our laboratory also reported the isolation of another cytotoxic factor from the human promyelomonocytic HL-60 cell line and named it as TNF (later we named it TNF; Aggarwal et al., 1985b). The amino acid sequences of TNF and TNF (Aggarwal et al., 1985a,b), and the isolation of

414 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann their cDNA, revealed that TNF and TNF are structurally homologous factors that can induce regression of Meth A sarcoma in mice, the assay used by Carswell et al. to identify TNF (Gray et al., 1984; Pennica et al., 1984). Shortly thereafter we demonstrated that TNF and LT share a common receptor (Aggarwal et al., 1985c). Since both forms of human TNF were isolated by using an in vitro bioassay based on murine L-929 fibrosarcoma cell line, and since the recombinant human protein was also active in mice in vivo, TNF is not species specific (Kramer et al., 1988). Though originally identified because of its antitumor activity, the pluripotent activity of TNF led to its later rediscovery and the assignment of different names in the murine system. Drs Bruce Beutler and Anthony Cerami, then from Rockefeller University, reported the isolation of a protein they named cachectin that was responsible for endotoxin-induced cachexia in mice (Beutler et al., 1985a). The isolation and partial amino acid sequence of murine cachectin revealed that it was highly homologous to the previously described human TNF. A T cell differentiation-inducing factor (DIF) discovered by Takeda et al. in 1986 was also found to have the same structure as TNF. This review will be limited to studies with TNF or TNF. For TNF , LT or LT, reader may refer to the Lymphotoxin chapter. Since its initial isolation, TNF continues to be a major topic of scientific investigation as indicated by over 13,000 citations published. These studies have indicated that TNF is a homotrimer with a subunit molecular mass of 17 kDa and that it plays a major role in growth regulation, differentiation, inflammation, viral replication, tumorigenesis, and autoimmune diseases; and in viral, bacterial, fungal, and parasitic infections (for references see monographs by Aggarwal and Vilcek, 1992; Beutler, 1992 and reviews by Tracey and Cerami, 1994; Armitage, 1994; Van Ostade et al., 1994a; Aggarwal and Natarajan, 1996).

Alternative names TNF is also referred to as TNF, cachectin, differentiation-inducing factor (DIF), and TNFSF2.

exist as a homotrimer in aqueous solution (Aggarwal et al., 1985b; Jones et al., 1992).

Main activities and pathophysiological roles Besides inducing hemorrhagic necrosis of tumors, TNF was found to be involved in tumorigenesis, tumor metastasis, viral replication, septic shock, fever, inflammation, and autoimmune diseases including Crohn's disease, and rheumatoid arthritis as well as graft-versus-host disease.

GENE AND GENE REGULATION

Accession numbers Once the amino acid sequence of the purified cytokine was identified, the cDNA for human TNF was cloned (Pennica et al., 1984; Aggarwal et al., 1985b). The gene accession number is X02910 and X02159.

Chromosome location The gene was localized on human chromosome 6 in a region between p23 and q12 (p21.1 and p21.3).

Relevant linkages Human TNF was found to be encoded by a single gene of 3.6 kb that is split by three introns (Nedwin et al., 1985). The TNF gene is located at 210 kb from the HLA-B locus of the class I gene family and is closely linked with the LT gene. Slightly more than 1000 base pairs separate the polyadenylation site of LT mRNA from the 50 end of TNF mRNA. These two genes have different tissue specificity, and their expression is regulated independently through their own distinct promoters (Nedwin et al., 1985; Nedospasov et al., 1986).

Structure

Regulatory sites and corresponding transcription factors

Human TNF is a type II transmembrane protein consisting of 233 amino acid residues. Mature soluble human TNF is a 157-amino-acid-long nonglycoprotein containing a single disulfide bridge and known to

Human TNF mRNA consists of about 1.7 kb and TNF gene expression has been found to be cell type specific. The 50 flanking region of the TNF gene contains, besides a TATA box and a GC box,

TNF consensus binding sites for various transcription factors including NFB, PU.1 (purine-rich box), a cyclic AMP response element (CRE), ATF-2, c-jun/ AP-I, AP-2, SP-1, Krox-24 and NF-AT (nuclear factor-activated T cells). Studies have shown that a CRE-binding site in the human TNF gene promoter binds to ATF2/Jun proteins and that this CRE site is critical to the regulation of the gene in multiple cell types stimulated by a variety of cellular stimuli, including TNF itself (Tsai et al., 1996a,b and references therein). In macrophages, TNF production has been shown to be dependent on NFB activation (Foxwell et al., 1998). The 30 untranslated region (UTR) contains an AU-rich element (ARE), the AUUUA pentanucleotide, which is known to control posttranscriptional regulation of TNF gene expression by destablizing the mRNA and interfering with translation (Carballo et al., 1998). Agents such as tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of TNF mRNA (Lai et al., 1999). This is a common phenomenon present in many cytokines. Certain agents such as LPS have been shown to induce TNF expression by inducing factors that bind to other regions in the TNF promoter (Myokai et al., 1999 and references therein). The regulation of the TNF gene is cell type specific, and different response elements in the TNF promoter are activated by different stimuli (Tsai et al., 1996a; Foxwell et al., 1998). Besides transcription factors, various kinases are known to regulate the production of TNF, including protein kinase C, Cot kinase, p38 MAP kinase, and p42 MAP kinase. Table 1 is a list of agents that can either upregulate or downregulate TNF expression by their action at the transcriptional, translational, or posttranslational level.

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Table 1 Inducers and suppressors of TNF production TNF inducers Bacterial products:

Endotoxin/lipopolysaccharide Lipid A Muramyl peptides Toxic shock syndrome toxin 1 (TSST-1) Listeria monocytogenes Corynebacterium parvum Staphylococcal exotoxins Mycoplasma orale Pseudomonas aeruginosa Candida albicans Mycobacterium avium Lipoteichoic acids

Viruses:

Human immunodeficiency viruses (HIV) Influenza A virus Sendai virus Respiratory syncytial virus Reovirus (UV-inactivated) Newcastle disease virus Neurotropic virus

Parasite products:

Malaria exoantigens Entamoeba histolytica

Cytokines:

Tumor necrosis factor IFN IL-1 IL-2 GM-CSF M-CSF

Cells and tissues that express the gene TNF was originally isolated from the human myeloid cell line HL-60. It is clear now, however, that besides HL-60 almost all myeloid cell lines, including U-937, THP-1, ML-1a, can produce TNF. Besides myeloid lines, B lymphoblastoid cell lines (Raji, Daudi, RPMI-1788) and T cell lines (Jurkat, HuT-78) produce TNF. In addition, a wide variety of tumor cell lines of neither lymphoid nor myeloid origin can produce TNF, including melanoma, breast, ovarian, glioma, and renal cell carcinoma (for references see Aggarwal and Vilcek, 1992). Among the normal cells, macrophages, T lymphocytes, NK cells, dendritic

Drugs:

Inducers of protein kinase C (phorbol esters) Inhibitors of phosphatases (okadaic acid and calyculin) Lithium chloride Cyclooxygenase/5-lipoxygenase inhibitors Flavone-8 acetic acid (FAA) Sodium periodate Benzodiazepine Complement (C5a)

Others:

Tumor cells Hyperthermia

416 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Table 1 (Continued )

Table 1 (Continued ) X-ray radiation

Ethanol

UV radiation

Novel oxamide derivatives

Cyclic GMP

Adenosine and carbocyclic analogs

Substance P

Heat shock

Trace elements, e.g. zinc

CC-305 (water-soluble Thalidomide)

Myelin P2 basic protein TNF suppressors Cytokines:

IFN and IFN TGF IL-4 IL-6 IL-10 IL-11

cells, endothelial cells, peripheral blood leukocytes, osteoblasts, astrocytes, mast cells, Kupffer cells, and smooth muscle cells can produce TNF (see Table 2). Monocytes/macrophages are probably the major producers of TNF in most conditions.

IL-13 G-CSF Viruses:

Epstein-Barr virus Adenoviral proteins

Drugs:

Phosphodiesterase inhibitors, e.g. pentoxifylline, rolipram Intracellular inducers of cyclic AMP Metalloprotease inhibitors

PROTEIN

Accession numbers The protein sequence accession number is the same as that for the gene (see Gene accession numbers). The crystal structure of TNF is described by Jones et al. (1989) and Eck and Sprang (1989).

Lipooxygenase inhibitors Inhibitors of NFB activation Thalidomide Cyclosporin A Lipid IVA Norepinephrine Dexamethasone cis-Urocanic acid Chlorpromazine Linomide Estradiol and progesterone Prostaglandin E2 Vitamin D3 Dietary n-3 fatty acid Gangliosides Vasoactive intestinal peptide Lactoferrin Lisofylline Amiloride Histamine

Sequence Mature human TNF is a 157-amino-acid-long protein containing one disulfide bridge and no carbohydrates. It also lacks methionine. The N-terminal sequence of the soluble natural TNF starts with VRSSSRTP (Aggarwal et al., 1985b). There are reports, however, of heterogeneous N-termini (see references in Aggarwal and Vilcek, 1992). The first 5±10 residues of the N-terminus of the protein appear to be less critical for its biological activity than the rest of them.

Description of protein The molecular mass of TNF protein under denaturing conditions is about 17 kDa. Under native conditions, however, TNF is a trimer with an approximate molecular mass of 50 kDa. It is an acidic protein with an isoelectric point around 5.6 (Aggarwal et al., 1985b). Circular dichroism studies have revealed that TNF is entirely the -sheet class of proteins (Narhi et al., 1996).

TNF

Discussion of crystal structure The active form of TNF appears to be a homotrimer. The crystal structure of TNF revealed that each monomer consists of two antiparallel -pleated sheets with a jelly roll topology that interact with each other in a head-to-tail fashion to form a heterotrimeric structure (Eck and Sprang, 1989; Jones et al., 1989).

Important homologies In mammalian species the amino acid sequence of TNF is highly conserved. Human and murine TNF have 79% homology at the amino acid level, and both TNFs have two cysteine residues that form an internal disulfide bridge. The genes for rat, rabbit, bovine, cat, dog, porcine, equine, and ovine TNF have been cloned (for references see Van Ostade et al., 1994a). The amino acid sequence of human TNF also displays similarity to human lymphotoxin, being 22% identical and 51% homologous (Pennica et al., 1984; Aggarwal et al., 1985b). The TNF sequence is 15±25% homologous to other members of the TNF superfamily which include FasL, LT, RANKL, THANK, LIGHT, CD40L, CD27L, CD30L, VEGI, APRIL, TRAIL, LT, LT , Ox40L, and 4-1BBL (for references see Gruss and Dower, 1995; Mukhopadhyay et al., 1999).

Posttranslational modifications The mRNA encodes for the TNF precursor protein of 233 amino acids (Pennica et al., 1984). The amino acid sequence of the natural TNF (Aggarwal et al., 1985b) demonstrated that the mature protein of 157 amino acids is preceded by a 76 amino acid signal sequence involved in protein secretion. It is expressed as a type II transmembrane precursor protein of 26 kDa that is converted to the mature human TNF of 17 kDa. This conversion is a highly regulated process and occurs by proteolytic cleavage of the 76 amino acid signal peptide from the C- terminal end of the membrane-bound protein by a membrane-bound matrix metalloproteinase called TNF-converting enzyme (TACE) (for references see Black and White, 1998). It has been suggested that this cleavage is an essential step before release of the cytokine. The 80 kDa enzyme cleaves pro-TNF between the Gln-Ala and Val-Arg sequences (Black et al., 1997). Interestingly, the sequence of human TACE cDNA revealed that it is a member of the ADAM (a disintegrin and metalloproteinase) family

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of metalloproteases. More than 20 members of the ADAM family with extensive homology to the snake venom metalloproteases have been identified. The homozygous deletion of the TACE gene from mice was found to be lethal, and most mice died between day 17.5 and the first day after birth (Peschon et al., 1998). The inhibitors which block TACE activity have been found to block the shedding of the TNF receptor and the processing of TNF (Crowe et al., 1995). There are reports, however, which indicate that the transmembrane form of TNF binds preferentially to the p75 receptor (Grell et al., 1995) and is able to kill target cells by cell-to-cell contact and provide costimulatory signal for B cells. The human TNF is not glycosylated although murine TNF has a potential Nlinked glycosylation site.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Although first isolated from HL-60, a macrophagelike cell line, it is now clear that TNF is produced by wide variety of different cell types (Table 2). It includes, besides macrophages and monocytes, T cells, B cells, astrocytes, fibroblasts, basophils, mast cells, NK cells, Kupffer cells, smooth muscle cells, epidermal cells, breast tumor cells, ovarian tumor cells, glioblastoma, melanoma, leukemia, prostate tumors, and pancreatic cancers (for references see Aggarwal and Vilcek, 1992). Indeed, extensive research within the last decade has revealed that the ability of most cells to produce TNF is regulated by the 30 AU-rich elements (ARE) region of the gene. This region is responsible for TNF mRNA destabilization and translational repression. Indeed, transgenic mice lacking TNF ARE from the genome produce abnormal levels of TNF (Kontoyiannis et al., 1999).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Although LPS and phorbol esters (PMA) were initially found to be potent inducers of TNF production (Pennica et al., 1984; Aggarwal et al., 1985b), several additional inducers have subsequently been found (see Table 1). These include lipid A (derived from LPS), Ca2‡ ionophores, and immunepotentiating cytokines such as IL-1, IL-2, GM-CSF,

418 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Table 2 A list of TNF-producing cell lines and cell types Immune system:

Macrophages (e.g. HL-60, U-937, THP-1, ML-1a) Natural killer cells T lymphocytes (e.g. Jurkat, HuT-78) Normal B cells and B lymphoblastoid cells (Raji, Daudi, RPMI-1788) Polymorphonuclear cells

Tumor cells:

Breast adenocarcinoma (e.g. MCF-7) Renal cell carcinoma Ovarian sarcoma Fibrosarcoma (TNF-resistant L929 cells) Epidermal cells (A431, KB) Glioblastomas (e.g. U-251) Hairy cell leukemia B cell chronic lymphocytic leukemia Melanoma

Other cells:

Mast cells Kupffer cells Granulosa cells Retinal pigment epithelial cells Spermatogenic cells Fibroblasts Smooth muscle cells Astrocytes Osteoblasts

IFN, IFN , and IFN . Interestingly TNF can also upregulate its own synthesis through the activation of NFB. In addition, viruses, fungi, parasites, protozoa, immune complexes, and tumor cells stimulate TNF release. Indeed almost all stressful and inflammatory stimuli have been shown to induce TNF release, including UV light and X-rays. Several agents downregulate TNF expression, including inhibitors of prostaglandin synthesis (salicylate), phosphodiesterase inhibitors, cyclosporin A, and immunosuppressive cytokines (IL-4, IL-6, IL-10, IL-11, IL-13, TGF ). Because TNF transcription is dependent, in part, on NFB activation (Foxwell et al., 1998) and the latter is regulated by reactive oxygen intermediates, most inhibitors of NFB activation (e.g. IL-10, IL-4, IL-11, IL-13, -melanocyte stimulating hormone, leflunomide, -lapachone) and antioxidants (PDTC, curcumin, capsaicin, sanguinarine, superoxide dismutase, -glutamyl cysteine

synthetase) have potential in downregulating TNF expression. Overexpression of cells with IB, the inhibitory subunit of NFB, has been shown to block TNF production (Bondeson et al., 1999). Endogenous activators of TNF include GM-CSF, G-CSF, M-CSF, IL-1, IL-3, IL-2, IL-12, IL-15, and the IFNs. Endogenous suppressors include IL-4, IL6, IL-10, IL-11, IL-13, and TGF . (For references see Aggarwal and Natarajan, 1996). In general, cytokines produced by TH2 cells such as IL-4, IL-10, and IL-13 downregulate the expression of TNF produced by TH1 cells. Most inflammatory stimuli, infection, or stress lead to TNF production.

RECEPTOR UTILIZATION For details, see the TNF receptor chapter. Briefly, TNF binds to two different receptors referred to as p60 (also called p55, or type I or CD120a) and p80 (also called p75 or type II or CD120b) based on their molecular weight. These two receptors are homologous in their extracellular domains but distinct in their intracellular domains (for references see Armitage, 1994; Darnay and Aggarwal, 1997). The p60 receptor is expressed on all cell types, whereas the p80 form is expressed chiefly on cells of the hematopoietic and immune system and on endothelial cells. Most cells express 1000±5000 receptor sites with an affinity ranging from 0.1 to 1 nM. The binding of TNF to its receptor can be displaced by LT, suggesting they have a common receptor (Aggarwal et al., 1985c). TNF muteins have been made by site-specific mutagenesis; these bind to either the p60 (mutated at R32W and S86T) or to the p80 (mutated at D143N and A145R) receptor (Loetscher et al., 1993; Van Ostade et al., 1994b; Haridas et al., 1998).

IN VITRO ACTIVITIES

In vitro findings TNF is a multipotential cytokine that exhibits different activities in different cell types. Some of these activities are outlined in Table 3.

Regulatory molecules: Inhibitors and enhancers The activity of TNF in most instances is potentiated by the presence of IFN, IFN , and IFN (Aggarwal and Eessalu, 1987). The cytotoxic activity of TNF is

TNF Table 3 A partial list of in vitro effects of TNF Hematopoietic system Induction:

Functional and morphological maturation of myeloid leukemia cells T cell colony formation Superoxide production by EBV-transformed B lymphocytes Invasiveness of T cell hybridomas and cytotoxic T cell clones

Suppression:

Normal and leukemia late-stage erythropoiesis Competence signal delivered by HIV to normal B cells G-CSF receptor expression on myeloid leukemia cells and PMNs Alters red blood cell kinetics and induces anemia c-kit proto-oncogene product expression in normal and myeloid leukemia CD34‡ cells

Osteoblastic cells Bone metabolism Induction of proliferation Production of IL-1 and IL-6 Inhibits proliferation and alkaline phosphatase activity Endothelial cells Induction:

Nitric oxide synthase IL-1 G-CSF GM-CSF IL-3 receptor Permeability of albumin Cell surface antigens Urokinase-type plasminogen activator Plasminogen activator inhibitor Release of platelet-activating factor Prostacyclin synthesis Eosinophil oxidant production and toxicity towards endothelium Galactosyltransferase activity and verocytotoxin receptors Fibrin deposition P-selectin ICAM-1 VCAM-1 E-selectin (ELAM-1)

Suppression:

Proliferation Protein S secretion Thrombomodulin Expression of vitronectin receptor (integrin B30) Regrowth of ionizing radiation-treated cells Stability of B-actin mRNA Glutathione synthesis

419

420 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Table 3 (Continued ) Macrophages Induction:

Expression of MHC antigens (class I more than class II) Stimulation of metabolism IL-1 and prostaglandin E2 GM-CSF M-CSF and IL-1 Triggers granulocytes to internalize complement-coated virus particles Cytosolic calcium levels

Suppression:

Proliferation of granulocytes and macrophage colonies Monocyte proliferation

Others:

Involved in the T cell-independent pathway of macrophage activation in SCID mice Serves as an effector molecule for tumor cell killing Binding and transmigration of lymphocytes across endothelial cells

Fibroblasts Induction:

Proliferation Membrane-associated IL-1 Interferon 2 IL-6 Leukemia-inhibitory factor Matrix metalloproteinases

Suppression:

Respiratory activity Collagen gene transcription and collagen synthesis Tissue inhibitor of metalloproteinases production

Others:

Affects LTR-controlled oncogene expression

Neutrophils Induction:

Activation Superoxide anion generation Release of leukotriene B4 and platelet-activating factor Calcium oscillation and calcium-activated chloride current Asbestos-induced production of reactive oxygen metabolites

Suppression:

Chemotaxis to N-formyl-L-leucyl-L-phenylalanine. Cell surface expression of sialophorin CD43 Bone marrow and lymphocytes

Others:

Dose- and time-dependent transmigration Sequestration in lungs Increased adherence to extracellular matrix

Adipocytes Suppresses the synthesis of lipids Eliminates binding of nuclear factor- and an octamer-binding protein to the lipoprotein lipase promoter Regulation of collagen gene expression in 3T3-L1 cells

TNF

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Table 3 (Continued ) Endocrine system Inhibits the effect of FSH on Sertoli cells Inhibits growth hormone secretion from cultured pituitary cells Inhibits gonadotropin action in granulosa cells Inhibits gonadotropin-supported ovarian androgen biosynthesis Enhances inhibitory effects of IL-1 on Leydig cell steroidogenesis Enhances human chorionic gonadotropin in choriocarcinoma cells Smooth muscle cells Induction of nitric oxide synthase Other cell types Induction of plasminogen activator activity in human pulmonary epithelial cells (A549) Causes a depression of liver drug metabolism and increase in plasma fibrinogen levels Affects the hepatic development of malaria parasites via IL-6 secretion Inhibits malaria parasites in vivo and in vitro Decreases the expression of intestinal IgG-binding site by H29-N2 colonic cancer cells Increases GTPase activity of the Gi protein in HL-60 and L929 cells Enhances radio-antibody uptake in colon carcinoma xenografts Suppresses the production of HIV mRNA and core protein p24 Amplifies measles virus-mediated Ia induction on astrocytes Serves as a paracrine and autocrine growth factor Shedding of HLA antigens expressed by melanoma cells Depresses cytochrome P450-dependent microsomal drug metabolism in mice In vivo biological responses Inhibition of growth of rat trophoblasts Decreases thyroid hormone levels in the serum in mice Protection of mice upon passive immunization from endotoxin Mediation of anorexia in rat Sepsis-induced apoptosis of the thymocytes in mice. Stimulates leukotriene production in rats Plays a protective role in experimental murine cutaneous leishmaniasis Inhibits albumin gene expression in nude mice Decreases catalase activity in rat liver Negatively regulates hepatitis B virus gene expression in transgenic mice. Stimulates nitric oxide formation in cultured hepatocytes

also enhanced by inhibitors of protein transcription or translation (Kramer and Carver, 1986). The activity of TNF is inhibited by various cytokines including IL-4, IL-10, IL-13, and TGF and by various antioxidants. In addition, inhibitors of serine

proteases and caspases are also known to downregulate TNF activity (Suffys et al., 1988; Tewari et al., 1995). Various members of the Bcl-2 family of proteins are also known to block the apoptotic activity of TNF (Johnson and Boise, 1999, and

422 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann references therein). Interestingly, in some cells, TNF induces the expression of the members of the Bcl-2 family members (Tamatani et al., 1999), which may explain how cells develop resistance to TNF.

Table 4 A partial list of the in vivo effects of TNF Decreases thyroid hormone levels in the serum in mice Protects mice upon passive immunization from endotoxin

Bioassays used

Mediates anorexia in rat

Perhaps the most frequently used assay for TNF involves its cytotoxic effects against murine fibroblast cell line L929 and WEHI 164. The L929 assay was used initially to isolate TNF (Aggarwal and Kohr, 1985). It is described in more detail in the chapter on Cytokine Assays. Besides murine cell lines, human rhabdomyosarcoma cell line KYM-1 has also been employed to assay for human TNF (Meager, 1991).

Stimulates leukotriene production in rats

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The true normal physiological role of TNF in vivo is unclear. In general, it is believed that TNF is required for protection against bacterial, fungal, parasitic, and perhaps even viral infections and other stressful stimuli. Besides hemorrhagic necrosis, whether TNF is able to block tumorigenesis and metastasis in vivo is still not fully understood (Lejeune et al., 1998). There is also evidence that TNF is involved in tumorigenesis and metastasis (Orosz et al., 1993). A recent report indicates that mice deficient in TNF are resistant to skin carcinogenesis (Moore et al., 1999). Some of the in vivo roles of TNF are outlined in Table 4.

Species differences Human TNF is known to transduce responses in murine cells and vice versa. Experience over the years has, however, shown that TNF have species preferences rather than species specificity. This is further supported by the fact that the p60 receptor is not species-specific whereas the p80 receptor is. Furthermore, murine TNF is more toxic to mouse in vivo than human TNF (Kramer et al., 1988), suggesting the synergistic role of the p60 and p80 receptors in inducing toxic effects.

Knockout mouse phenotypes The deletion of the TNF gene in mice has shown that these mice develop normally. Thymus is normal, but

Mediates sepsis-induced apoptosis of thymocytes in mice Plays a protective role in experimental murine cutaneous leishmaniasis Inhibits albumin gene expression in nude mice Decreases catalase activity in rat liver Negatively regulates hepatitis B virus gene expression in transgenic mice Inhibits growth of rat trophoblasts Stimulates nitric oxide formation in cultured hepatocytes

spleen architecture is not. However, their ability to fight infection is compromised. Both follicular dendritic cell clusters and germinal centers are absent from the spleen of immunized TNF gene-deleted mice. In addition, TNF knockout mice show low toxicity to TNF, are resistant to the lethality of minute doses of LPS and exhibit increased susceptibility to candida challenge (Marino et al., 1997). These mice also show an anomalous late response to heat-killed Corynebacterium parvum. Consistent with TNF's known role in obesity-induced insulin resistance in mice, the TNF knockout mice were protected from such resistance (Uysal et al., 1997).

Transgenic overexpression Extensive studies have been carried out on the pathological effects of TNF transgene upregulated by the lack of the 30 untranslated region in mice (for references see Probert et al., 1996). Depending on the organ in which the transgene is expressed and whether soluble or transmembrane TNF is expressed, the effects may vary (Table 5). The TNF transgene induces several effects in mice, including localized tissue toxicity, chronic inflammatory arthritis, and spontaneous and inflammatory demyelinating disease. The overexpression of wild-type murine or human TNF transgenes by resident astrocytes or neurons is sufficient to trigger a neurological disorder characterized by ataxia, seizures, and paresis, with histopathological features of chronic CNS inflammation and white matter degeneration. Furthermore, it was shown that transmembrane human TNF is sufficient to trigger CNS inflammation and degeneration when

TNF

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Table 5 Effect of TNF transgenes on the phenotype Promoter

Gene

Cell type

Abnormality

Wild-type and 30 -modified

TNF

Spleen, kidney, brain

Arthritis

Insulin

TNF

Pancreas

Insulitis No diabetes

CD2

TNF

T cell

Wasting syndrome Ischemia Lymphoid abnormalities

TNF

CNS

Chronic inflammatory demyelinating disease

For references see Aggarwal and Natarajan (1996).

overexpressed by astrocytes, but not when it was overexpressed by neurons, indicating that target cells mediating the neuroinflammatory activities of TNF localize in the vicinity of astrocytes rather than neurons (Alexopoulou et al., 1997). Thus both soluble and transmembrane forms of TNF can play critical roles in vivo in the pathogenesis of CNS inflammation and demyelination. In addition, when the TNF transgene was expressed in T cells, it led to wasting, cachexia, and lymphoid abnormalities in mice.

Pharmacological effects TNF is a pleiotropic cytokine that produces varying immunologic and inflammatory host defense responses. Administration of TNF to various animals leads to hemorrhage, necrosis, local inflammation, shock, and death. The lethal dose of human TNF in mouse has been found to be in excess of 1 mg/kg body weight, whereas murine TNF causes death in mice at doses as low as 10 mg/kg (Broukaert et al., 1992). These differences are related to the species-specific affinity of the receptor. Subcutaneous and intradermal injection of TNF in mice or rabbits rapidly leads to (within 8 hours) neutrophil margination and edema formation (Dunn et al., 1989; Rampart et al., 1989). When injected subcutaneously daily over 5±7 days, TNF causes skin necrosis and intense neutrophil infiltration (Sheehan et al., 1995). Subcutaneous administration of TNF in baboons also led to hemorrhage, necrosis, and dense aggregates of both macrophages and neutrophils (Van Zee et al., 1994). Systemic administration of TNF in baboons caused acute hypotension, tachycardia, increased plasma lactate, and organ dysfunction (Van Zee et al., 1994). Administration of TNF to dogs also causes

irreversible tissue damage and death (Tracey et al., 1987). Furthermore TNF that binds p80 receptor was found to be less toxic than that which binds p60 receptor in baboons. Because TNF is not toxic when administered to mice lacking p60 receptor (Rothe et al., 1993), most of the systemic toxicity has been associated with the presence of p60 receptor. Cachexia Prolonged exposure to TNF causes anorexia, loss of body weight, dehydration, and loss of body proteins and lipids. This condition, called cachexia, may occur during chronic parasitic, bacterial, and viral infection. The symptoms also appear in cancer patients. The mediator involved in this process was named cachectin, later recognized to be the same as TNF. The cytokine is known to regulate the synthesis of several metabolic enzymes, e.g. lipoprotein lipase, to increase the level of cytokine-induced synthesis of acute phase proteins, and accelerate amino acid uptake, energy expenditure, lipolysis, and protein turnover rate. These reactions are identical to those in chronic infection and cancer. In severe diseases like AIDS, the TNF level is significantly increased when the signs and symptoms of cachexia develop (Benyoucef et al., 1996 and references therein). Rabbits infected with Trypanosoma brucei develop severe cachexia associated with hypertriglyceridemia having lipid clearance defect (Nakamura, 1998). Inflammation TNF is an inflammatory cytokine. It is chemotactic to monocytes and neutrophils. Stimulation of these cells with TNF induces phagocytosis, adherence of these cells to endothelial cells, and generation of free radicals of oxygen-superoxide anion, and hydrogen

424 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann peroxide. Stimulation of cultured human endothelial cells with TNF induces procoagulant activity. TNFinduced endothelial cell activation leads to the structural reorganization of the endothelium, resulting in vascular leakiness which is partly due to its capacity to upregulate VEGF, also known as vascular permeability factor (Giraudo et al., 1998). TNF increases the expression of ICAM-1 and endothelial leukocyte adhesion molecule 1 (ELAM-1). Both molecules can bind neutrophils and monocytes. The cytokine also induces synthesis of an inflammatory cytokine, IL-8, and other chemotactic cytokines that regulate the migration, degranulation, and respiratory burst response of neutrophils. IL-8 also stimulates the interaction of ICAM-1 and neutrophils, a very important interaction during transvenule migration of the cells. TNF induces the synthesis of other chemokines, such as MCP-1, that promotes accumulation of monocytes at the site of inflammation. In several inflammatory diseases like rheumatoid arthritis and related autoimmune diseases TNF is produced in the inflammatory sites (for references see Feldmann et al., 1995), suggesting its role in tissue degradation as well as in augmentation of inflammatory response. Endotoxin-Mediated Shock LPS is one of the major inducers of TNF production both in vitro and in vivo. Administration of high doses of LPS or infection with gram-negative bacteria leads to septic shock. TNF is considered a major mediator of septic shock syndrome because it is overproduced during sepsis, it causes shock and tissue injury identical to the sepsis, and anti-TNF antibodies given early protect animals from septic shock. In addition, transgenic mice with the TNF p60 receptor mutation were protected from the septic shock. However, so far anti-TNF antibody therapy has not been successful in clinical trials of sepsis. Bone Resorption and Tissue Remodeling TNF is involved in remodeling of connective tissue. From fibroblasts and synovial cells TNF induces the release of collagenase and other matrix metalloproteases enzymes. It inhibits collagen synthesis in bone; in explanted cartilage it reduces the content of alkaline phosphatase and induces resorption of proteoglycan.

Interactions with cytokine network TNF synergizes with IFN in vivo for its antitumor effects (Lienard et al., 1992) and for antiparasitic

effects (Liew et al., 1990). Although this activity has not yet been fully demonstrated in vivo, in vitro TNF induces the production of IL-1, IL-6, IL-8, G-CSF, GM-CSF, and M-CSF. In vivo, after LPS challenge, TNF, IL-1, and IL-6 were produced. Similarly IL-6 and various CSFs regulate the production of TNF in vitro. IL-1 can induce TNF in vitro, but this does not seem to occur in rheumatoid joint tissue cultures (Butler et al., 1995).

Endogenous inhibitors and enhancers Perhaps the most potent inhibitor of TNF is the soluble form of the TNF receptor, which when shed from the cell surface is effective in blocking the activity of TNF. Based on in vitro studies, TGF , IL-4, IL-6, IL-10, IL-11, and IL-13 can also inhibit either TNF production or block TNF action. The major augmenter of the effects of TNF in vivo is IFN .

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects TNF is not detected in the serum of normal human subjects. Low levels of TNF in the serum have, however, been detected in patients with gram-negative bacterial infection, fever, or cancer.

Role in experiments of nature and disease states TNF has been shown to play a role in a wide variety of diseases, and some of these are listed in Table 6. In human rheumatoid arthritis, TNF protein has been observed in the synovial fluid, and TNF mRNA in the synovial cells, including monocytes and macrophages (Yocum et al., 1989; Macnaul et al., 1990). Since TNF has also been detected in cancer patients, especially those with Hodgkin's disease, what cells produce this TNF is not clear. Perhaps the most likely source is tumor cells themselves. Based on in vitro experiments, the role of TNF in tumorigenesis and metastasis has been demonstrated.

TNF Table 6 A partial list of TNF-mediated diseases Bacterial

Table 6 (Continued ) Tumor allograft rejection

Bacteria-induced preterm pregnancy loss

Gynecologic malignancy Lymphoproliferative disease

Human decidua (infection-induced preterm labor)

Murine mesocestoides-corti infection

Pneumocystis carinii pneumonia

Inflammatory hyperalgesia

Cryptogenic fibrosing alveolitis

Head injury

Bacterial meningitis

Fever

Mycobacterium tuberculosis

Down's syndrome

Nocardia brasiliensis Shock

Candida septic shock syndrome T cell-mediated lethal shock Septic shock

Autoimmune disease

425

For references see Aggarwal and Vilcek (1992), Beutler (1992), Aggarwal and Natarajan (1996).

Rheumatoid arthritis Crohn's disease Glomerulonephritis (renal disease) Guillain-Barre syndrome Systemic lupus erythematosus Multiple sclerosis Childhood chronic inflammatory bowel disease Diabetes

Viral diseases

HIV-related lung disease Advanced HIV infection Viral meningitis Murine retrovirus infection

Parasitic diseases

Histoplasmosis in mice Plasmodium vivax malaria Dysentery (Shigella dysenteriae) Toxoplasma gondii Pertussis in children

Other diseases

Diabetes Pulmonary fibrosis Chronic osteomyelitis Pulmonary artery occlusion reperfusion Langerhans cell migration Graves' disease T cell leukemia with hypercalcemia Hairy cell leukemia Chronic lymphocytic leukemia Allergic asthma Graft-versus-host disease Allograft rejection

The standard bioassay for TNF involves its cytotoxic effects against L929 cells. This assay has been described in the chapter on Cytokine Assays.

IN THERAPY

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. Antibodies against TNF have been used in animal model systems to block endotoxin-induced septic shock and other inflammatory diseases. Antibodies have also been used in human subjects to treat rheumatoid arthritis, Crohn's disease, and septic shock. In addition, thalidomide, which inhibits TNF production, has been approved for use in AIDS patients (Klausner et al., 1996). Anti-TNF Therapy TNF is one of the most rapidly produced cytokines in vivo or in vitro in response to a wide variety of insults, and together with its potentially pathogenic effects this has led to anti-TNF being tested in a wide spectrum of animal models of diseases, and a smaller number of human clinical trials. The first was in animal models of sepsis where it was effective in mice as well as larger animals such as monkeys (Beutler et al., 1985b; Hinshaw et al., 1990; Walsh et al., 1992). However, in all the experimental models, which involved injection with large numbers of gram-negative bacteria, the timing of anti-TNF therapy was critical. Protection was only seen if the

426 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann anti-TNF antibody was administered before or at the same time as the infection. A few hours' delay resulted in loss of protection. This was echoed in the results obtained in the human clinical trials of sepsis, which have been uniformly disappointing. Subsequently, anti-TNF therapy was tried in animal models of rheumatoid arthritis. It was found to be protective in animal models such as collagen-induced arthritis, adjuvant arthritis, as well as streptococcal cell wall arthritis (Thorbecke et al., 1992; Williams et al., 1992), as illustrated in Figure 1. The results have been echoed in the success of anti-TNF in rheumatoid arthritis. Animal models of inflammatory bowel disease have also been analyzed. It was found at first that in the IL-10-knockout mice there was little benefit, but subsequent studies have showed clear benefit. The same is true in the T cell transfer model, and is echoed by the success of anti-TNF therapy in Crohn's disease (Powrie et al., 1994; Neurath et al., 1997). Interesting results were obtained in experimental allergic encephalomyelitis. Treatment of mice upon transfer of sensitized T cells was effective (Ruddle et al., 1990), and treatment of mice with chronic relapsing encephalomyelitis revealed a beneficial effect of anti-TNF administered i.p. It was of interest, however, that 10±100 times less antibody was needed if the mice were injected intracerebrally (Baker et al., 1994). This suggests that the active beneficial component of anti-TNF therapy in this model of EAE was that which entered the brain. In this context it is interesting that the only anti-TNF biological which has been tried in multiple sclerosis, TNFRp55:Fc fusion protein (lenercept) was not beneficial in its primary outcome measure and made

patients worse, as judged by relapses. The simplest mechanism to explain this difference might be the inability of the TNFRp55:Fc to cross the blood-brain barrier (The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS/ MRI Analysis Group, 1999). Anti-TNF Therapy in Sepsis The first disease to be treated with anti-TNF antibody was sepsis. The results, however, have not been clinically useful, although it is likely that they will teach us things about clinical trial design. Several distinct antibodies have been used in sepsis trials. The ones most extensively used have been mouse monoclonal antibodies, in clinical studies initiated before the advent of chimeric or fully humanized monoclonals. The encouraging early results led to larger clinical studies. It is not clear why the clinical trials were unsuccessful, but a likely reason is the fact that sepsis patients, unlike the animal models, are treated rather late in the course of the disease. Another reason is the fact that other serious diseases, such as diabetes or cancer, tend to be underlying the sepsis. Moreover, in order to get the huge trials done in a realistic period of time, hundreds of trial sites were used, with consequent heterogeneity of protocols for concurrent therapy, misdiagnoses, etc. One of the puzzling aspects has been that there have been marked variability in the results: in some trials the results were neutral (no worsening, no benefit), while the use of soluble TNFR:Fc fusion protein in one relatively small trial led to a dose-dependent worsening in clinical outcome (Abraham et al., 1995; Fisher et al., 1996).

Figure 1 Serum C-reactive protein and IL-6 levels reduced after treatment with Infliximab. From Williams et al. (1992) with permission.

TNF Anti-TNF Therapy in Rheumatoid Arthritis (RA)

Statistically significant reductions in joint swelling were noted within 2 weeks, and plateaued at 70% reduction at 4±8 weeks. However, all 20 patients who had markedly improved, subsequently relapsed, the longest response being 6 months (Elliott et al., 1993). The success of this trial prompted a randomized, double-blind placebo-controlled trial, in which a single dose of 1 mg/kg (44% responders) or 10 mg/kg (79% responders) proved significantly better than placebo (8% responders), with the criterion of response being the composite Paulus 20 score. The results are illustrated in Figure 2, and the serum and other samples from these patients have been an invaluable resource for elucidating the mechanism of action of anti-TNF therapy in RA (Elliott et al., 1994). Subsequent studies have included a larger phase II study to evaluate the effects of longer term treatment, of 3 months continuous therapy, evaluated up to 6 months (Maini et al., 1998), and a phase III study, evaluating therapy at 4±8 week intervals up to 2 years, although this has only been decoded to 54 weeks as yet (Maini et al., 1999). The results of combination therapy of anti-TNF with methotrexate (MTX) were interesting. At a low dose of 1 mg/kg there was transient benefit, which was not sustained despite repeated injection. However at that dose, in the presence of low-dose methotrexate

The most extensive series of studies of TNF blockade have been performed in this disease. So far, six different anti-TNF biological agents have been used clinically, with reports of success with all of them. This topic has been reviewed by Feldmann et al. (1995, 1997). The first antibody to be used in rheumatoid arthritis, and which proved the principle, was cA2, a chimeric human IgG1, K, mouse Fv anti-TNF antibody. It was first used in an open-label trial of patients who had long-standing active RA and had failed existing therapy. To verify that the effect was due to anti-TNF, the patients who were taking existing potential disease-modifying rheumatic drugs, known as `DMARDS', were taken off these drugs but were permitted to remain on a stable dose of nonsteroidal anti-inflammatory drugs, as well as a stable dose of corticosteroids. The antibody dose used was comparable to that found to be effective in the mouse model of collageninduced arthritis (Williams et al., 1992), namely 20 mg/ kg over a 2-week period. The results were dramatic. There was symptomatic benefit, including reduction in pain and morning stiffness, as well as signs such as joint swelling, beginning within hours. Relief of fatigue and lethargy was particularly rapid within hours.

Figure 2 Single infusion Infliximab. (a) Therapeutic response: primary end point Paulus 20% responses at week 4. (b) Change in tender joint count over 4 weeks. (c) Change in serum CRP over 4 weeks. From Elliott et al. (1994) with permission. (b) Tender joint count (0– 60)

Placebo 8%

1 mg/ kg

Change in tender joint count over 4 weeks

30

20

10

0 0

44%

1

10 mg/ kg

p < 0.0001

79%

p = 0.0083

Responders

(c)

7.0

CRP mg/ dL

(a)

427

2 Weeks

3

4

Change in serum CRP over 4 weeks

5.0

3.0

Non-Responders

Normal range

1.0 0 0

1

2 Weeks

3

4

428 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann (7.5 mg/week) there was a significant clinical effect, which persisted to week 26. At higher repeated doses of 3 mg/kg and 10 mg/kg there was good clinical benefit, which was augmented by MTX as illustrated in Figure 3. The mechanism of the MTX benefit is not clearly established, one component is the reduced immunogenicity of the antibody in the presence of MTX, clear at the 1 mg/kg, but less so at the other doses (Maini et al., 1998). It is likely that other mechanisms are operating, and is probably analogous with the synergy of anti-T cell agents with anti-TNF in animal models (Williams et al., 1994). As MTX reduce TNF production and promotes T cell apoptosis, the mechanism of synergy is likely to be a similar to that occurring in collagen-induced arthritis. Longer term treatment in phase 3 has verified the clinical benefit of 3 and 10 mg/kg in the presence of MTX, but most importantly has shown that there is significant joint protection with anti-TNF therapy, as was previously demonstrated in the mouse models (Williams et al., 1992) (unpublished data reported in Wall Street Journal and presented at the American College of Rheumatology Meeting, November 1999) (P. Lipsky et al., submitted 2000). The degree of joint protection was remarkable; joint destruction, as assessed by a modified Larsen X-ray score, was halted by 6 months in all four dose groups of RemicadeTM (Centocor) in combination with MTX. Half of the treated patients had evidence of improvements in X-ray modified Larsen score at one year. The mechanism of action of anti-TNF therapy has been studied in these clinical trials. Several mechanisms of actions were detected. The first mechanism anticipated from the in vitro studies with human rheumatoid arthritis synovium involved downregulation of the cytokine cascade. This was most easily

shown by evaluation of serum IL-6 levels, but reductions in serum IL-1 have also been noted, as well as VEGF and multiple chemokines (Lorenz et al., 1996; Paleolog et al., 1998; Charles et al., 1999). The second important mechanism was a reduction in synovial cellularity, which is in part due to reduced leukocyte trafficking. Adhesion molecule expression (e.g. E-selectin, ICAM-1) was diminished, as was that of chemokines (Paleolog et al., 1996; Tak et al., 1996), and reduced trafficking was verified by a direct study using radiolabeled (Tc99) granulocytes (Taylor et al., 1998). There are suggestions of increased apoptosis in joints after anti-TNF therapy but this has not yet been quantified. A third mechanism being evaluated is the reduction in VEGF production and angiogenesis after anti-TNF (Paleolog et al., 1998). The success with cA2 first reported at a meeting in September 1992 and published in 1993 (Elliott et al., 1993) has led other groups to enter the field, and the first confirmation came with a humanized anti-TNF antibody, CDP 571, used by Celltech/Bayer (Rankin et al., 1994). This was reported to be effective, but the results were less clear cut than that with cA2 (RemicadeTM). The results with the most recent entrant to the field, D2E7, a `human' antibody, generated from phage display libraries, has been effective in its early studies, and is now entering phase 3 studies later in 1999. The efficacy appears to be good, comparable with EnbrelTM and RemicadeTM (Rau et al., 1998). The TNFRp55:IgG Fc chimeric fusion protein (known as lenercept) was effective in clinical trials in rheumatoid arthritis but of variable efficacy, perhaps due to problems with production and immunogenicity, and for these reasons has not been continued in trials (Hasler et al., 1996).

Figure 3 Paulus 20% responses to Infliximab with and without methotrexate. From Maini et al. (1998) with permission.

et al.

Arthritis Rheum

TNF

429

Figure 4 Etanercept in rheumatoid arthritis: ACR20, 50, and 70 responses at 6 months. From Moreland et al. (1999) with permission.

n n n

The clinical development of the TNFRp75:IgG Fc chimeric fusion protein (known as etanercept or EnbrelTM) has been very successful, and despite starting years after cA2 (RemicadeTM) it was licensed for use in the US in rheumatoid arthritis before RemicadeTM. Recent results with etanercept are illustrated in Figure 4. Etanercept is given subcutaneously twice per week, in contrast to RemicadeTM which is given by slow intravenous infusion every 4±8 weeks (Weinblatt et al., 1999). The clinical effects are comparable to those of RemicadeTM (Moreland et al., 1996, 1997) and there are reports that it has been successful in protecting joints, as assessed by reduction in X-ray progression (Wall Street Journal ). Potentially, the mechanism of action of EnbrelTM is different from that of RemicadeTM, as it is given in lower and repeated doses, so that there may not be a `wash out' of TNF, as with the high, intermittent doses of RemicadeTM. Another difference is that in contrast to RemicadeTM, EnbrelTM also recognizes lymphotoxin, and this may account for Enbrel's efficacy in juvenile chronic arthritis. Anti-TNF Therapy in Crohn's Disease Of the two common forms of inflammatory bowel disease, the results with Crohn's disease have been very convincing, while those with ulcerative colitis have not. This has led to further trials of cA2 (RemicadeTM) in Crohn's disease, and the licensing of this drug for severe Crohn's disease, including that with fistulas. One of the first trials used single doses of 5, 10, 20 mg/kg, which were found to induce significant improvement in 50±80% of patients who were doing poorly on existing therapy involving high-dose

corticosteroids. The efficacy was judged by a 70point improvement in the Crohn's disease activity index at 4 weeks, without changes in concomitant medication or surgery. Benefit continued for over 12 weeks (Van Dullemen et al., 1995; Targan et al., 1997). The pivotal trial for gaining approval was in 101 patients with one or more fistulas. There was closure of over 50% of fistulas for at least one month in over 60% of patients, with 46% closing all fistulas, with median duration of response being 84±99 days (depending on dose) (Present et al., 1999). There has been confirmation of the benefit of anti-TNF therapy in Crohn's disease using the CDP571 humanized antibody (Stack et al., 1997). As in rheumatoid arthritis, this antibody appeared to be less effective at equivalent dosage than cA2 (RemicadeTM). Anti-TNF Therapy in Multiple Sclerosis In contrast to a number of results with anti-TNF in experimental models of MS, experimental allergic encephalomyelitis (EAE), for example (Ruddle et al., 1990; Baker et al., 1994), the only anti-TNF biological that has been tested in multiple sclerosis (MS) was not successful (The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group, 1999). The trial involved a number of different doses, and there was no effect on the primary end point, namely the number of capillary leakage sites, as judged by gadoliniumenhanced MRI. However, this lack of effect was compounded by clinical worsening as judged by the number of clinical relapses lasting 24 hours or more. The effect was dose-dependent, so appears to be credible. The reason for this worsening, in contrast to the benefit seen in animal models, is not known.

430 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann A hypothesis is that it is due to the incapacity of the fusion protein to enter the CNS, where the benefit would be exerted, as suggested by the intracerebral anti-TNF injection in mouse studies (Baker et al., 1994). There is also evidence that TNF blockade can augment immune responses (Cope et al., 1994), and this effect in the periphery might account for the augmented number of relapses. Anti-TNF Therapy in Congestive Heart Failure There have been some heart studies with the TNFRp75:Fc (Enbrel) in patients with congestive heart failure. The effects reported so far in a small number of patients were modest, but positive (Deswal et al., 1999), and there are further clinical studies in progress, which will clarify whether the benefit is reproducible.

Pharmacokinetics In one of the first clinical studies, the pharmacokinetics, toxicity, and biological activity of i.v. and i.m. administered recombinant human TNF in patients with metastatic cancer were examined (Blick et al., 1987). Recombinant TNF in doses ranging from 1 to 200 mg/m2 by alternating i.m. and i.v. bolus injections was administered with a minimal intervening period of 72 hours. rTNF had a half-life of 14±18 minutes in the serum. After i.m. administration, serum concentrations of TNF peaked within 2 hours. rTNF was well tolerated clinically in this dose range. In another study, 19 patients with advanced cancer were given TNF to determine the pharmacokinetics profile, safety, and maximal tolerated dose (MTD) (Mittelman et al., 1992). TNF was administered by continuous infusion for 24 hours followed by pharmacokinetics and a 120-hour infusion repeated every 3 weeks. The initial dose was 40 mg/m2 and was ultimately escalated to 200 mg/m2. Toxic reactions in this trial included fever, chills, rigors, hypertension, headaches, seizures, lethargy, weight loss, and malaise. At all dose levels, but more significantly at the highest doses, hematological toxicity was observed, and grade 3 neurotoxicity (headache and confusion) and hypotension were noted. Two patients died during the study, and this was felt to be related to septic episodes. Because of the severe toxicity, 160 mg/m2 was defined as the MTD. At 160 mg/m2 peak serum levels occurred within 5±20 minutes of initiation and were not detectable 1 hour later. No measurable plasma levels of TNF were observed with the administration of doses of 80 mg/m2.

Toxicity See section on Pharmacokinetics.

Clinical results Several clinical studies have been carried out with TNF, most of them in cancer patients. These studies have recently been reviewed (Lejeune et al., 1998). Perhaps the most encouraging studies thus far show that high doses of TNF along with chemotherapy, with or without IFN , can be safely administered regionally through isolated limb perfusion. This procedure produced between 70% and 80% complete remission in cases of in-transit melanoma metastases and between 25% and 36% complete remission in cases of inextirpable soft tissue sarcomas. Dual targeting is involved; TNF and IFN induce apoptosis of angiogenic endothelium, while melphalan induces apoptosis of tumor cells.

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LICENSED PRODUCTS Anti-TNF antibody ± RemicadeTM by Centocor, Inc. TNFRp75:Fc dimeric fusion protein (EnbrelTM by Immunex/AHP, Seattle, WA, USA) Most companies that sell cytokines supply TNF and antibodies against TNF. These include: R & D Systems (Minneapolis, MN, USA) and Calbiochem (La Jolla, CA, USA).