Eur. J. Biochem. 202, 3- 14 (1991) (c) FEBS 1991

Review The molecular action of tumor necrosis factor-a Giovanni CAMUSSI’, Emanuele ALBANO’, Ciro TETTA3 and Federico BUSSOLIN04 Dipartimento di Biochimica e Biofisica, la Facolta di Medicina, Universita di Napoli, Italy Laboratorio di Immunopatologia and Dipartimento di Genetica, Biologia e Chimica Medica, Universiti di Torino, Italy

’ Dipartimento di Medicina e Oncologia Sperimentale, (Received April 22,1991)

-

EJB 91 0524

Tumor necrosis factor-a (TNF-a) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-a may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-a may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNFa are triggered by the binding to specific cell surface receptors. The formation of TNF-a-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-bindingregulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-a. The full understanding of the molecular mechanisms of TNF-a will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.

Tumor necrosis factor/cachectin (TNF-a) is a polypeptide hormone with a wide range of biologic activities [l]. It was initially described in serum of endotoxin-treated mice as the mediator of the necrosis of some transplantable tumors [2]. It was subsequently reported that TNF-a is cytocidal or cytostatic for some tumor cells in tissue culture [3]. Furthermore, recent studies indicate that TNF-a may act either as a mediator in beneficial processes of host defense, immunity and tissue homeostasis or in the pathogenesis of infection, tissue injury and inflammation [4]. General properties of the molecule Molecular structure

Concomitant studies on the molecular basis of cachexia led to characterization of cachectin, a protein secreted by Correspondence to P. G. Camussi, Laboratorio di Immunopatologia, Corso Polonia 14, 1-10126 Torino, Italy Abbreviations. TNF-c(, tumor necrosis factor-cl; MHC, major histocompatibility complex; LPS, lipopolysaccharide; IL-1, interleukin-I ; IFN, interferon; PAF, platelet-activating factor; PMN, polymorphonuclear neutrophils; PG12, prostacyclin; LFA-1, lymphocyte-function-associated antigen-I ; ELAM-I , endothelial leukocyte adhesion molecule-I ; ICAMs, intracellular leukocyte adhesion molecules; INCAM-I 10, inducible cell adhesion molecule110; VCAM-1, vascular cell adhesion molecule; TGF, transforming growth factor; GM-CSF, granulocyte, monocyte colony-stimulating factor; PDGF, platelet-derived growth factor.

macrophages in response to endotoxin [5]. Cachectin suppresses the lipoprotein lipase activity of adipocytes and causes hypertriglyceridemia and wasting [ 5 ] .Amino acid sequencing of cachectin and cloning of the cDNA for the genes of cachectin [6] and TNF-a [7] demonstrated that the two molecules are identical. The human TNF-a subunit is a 157amino-acid peptide (molecular mass 17 kDa) produced as a propeptide of 233 amino acids and activated by cleavage of a 76-amino-acid signal peptide. It was suggested that the cleavage of the molecule at the cell surface may be essential for the secretion of TNF-a [8]. In humans, at variance with other species, the molecule is nonglycosylated. Each subunit consists mainly of a /I-pleated sheet structure [9] and three subunits combine noncovalently to form the active hormone [lo]. Human TNF-a shows 76% identity with murine TNF-cr and 30% with lymphotoxin, a protein with cytotoxic activity secreted by lymphoid cells [Ill. TNF-a and lymphotoxin bind to the same receptor [12] and share several biological activities. In humans, these cytokines are encoded by separate genes on the short arm of chromosome 6, suggesting an ancestral tandem duplication event [13]. The TNF-a and lymphotoxin genes are separated by only about 1100 pairs of bases. Both genes reside within the major histocompatibility complex (MHC) and contain three introns, one of which interrupts the sequence encoding the mature polypeptide. A coordinated control of the expression of both genes is suggested by the consistent similarity in the 5’-untranslated region of the TNF-a and lymphotoxin [131.

4 Induction and production

TNF-a is synthesized by a number of cells, including monocytes/macrophages, lymphocytes, natural killer cells, glomerular mesangial cells, astrocytes and microglial cells of the brain, and Kupffer’s cells of the liver [4]. Beside endotoxin/ lipopolysaccharide (LPS), that represents the main stimulus, virus, fungal and parasital antigens, enterotoxin, mycobacterial cord factor, C5a anaphylatoxin, immune complexes, interleukin-1 (IL-1) and, in an autocrine manner, TNF-a itself, may trigger the synthesis of TNF-a. Lipid A also induces production of TNF-a but is less effective than wild-type LPS [14]. The ability of lipid A to induce TNF-a synthesis seems closely connected with the conditions in which it is at least hexacylated and/or contains hydroxylated fatty acids [14]. TNF-a does not exist in a stored form but is synthesized de n o w following cell activation. The biosynthesis of TNF-a is tightly regulated by transcriptional and post-transcriptional mechanisms [15]. Glucocorticoid hormones inhibit TNF-a biosynthesis provided that they are administered before the cells have been exposed to LPS [15]. Beutler et al. [15] have demonstrated that macrophages contain a pool of TNF-a mRNA that is not expressed as protein. After LPS stimulation, the cells [16] and tissues [17] markedly increase their content of TNF-a mRNA that is immobilized for translation with production of the bioactive peptide [15, 161. While gene transcription is accelerated approximately threefold, TNF-a mRNA levels rise within the cell by a factor of 50- 100, and the secretion of mature hormone rises by a factor of about 10000 [ I 5,161. Glucocorticoids inhibit both gene transcription and mRNA translation [15], thus preventing the synthesis of TNF-a. However, once translation of TNF-a mRNA is in progress, glucocorticoids are unable to suppress the production of TNF-a [15].In contrast, interferon y (IFN-y) exerts a permissive effect on the translation of TNF-a mRNA that enhances the biosynthesis of TNF-a [18]. It was recently suggested that K B-type enhancers are involved in LPS-mediated transcriptional activation of the TNF-a gene in macrophages and that factors interacting with an MHC class-11-like ‘Y box’ can additionally modulate the activity of the gene [19]. Collart et al. [20] demonstrated that regulation of TNF-a transcription in macrophages involves four K B-like motifs and constitutive as well as inducible forms of nuclear factor (NF) K B. In addition, the biosynthesis of TNF-a is largely regulated at a post-transcriptional level. The 3’-untranslated TTATTTAT element present in several cytokines and protooncogenes is capable of suppressing the translation of mRNA molecules in which it is expressed. Han et al. [21], using constructs in which the chloramphenicol acetyltransferase (CAT) coding sequence is followed by varying segments of the TNFa 3’-untranslated region, demonstrated that downstream sequences present in TNF-a mRNA are sufficient to mediate induction of CAT synthesis in response to activation by LPS. The induction of CAT activity was correlated not with variation in the concentration of cytoplasmic ,“nRNA but to a marked enhancement to translation efficiency [6].On the other hand, the mRNA transcript of TNF-a and of several others cytokines contain a 33-nucleotide 3‘-untranslated sequence composed of repeated and overlapping copies of the consensus octamer UUAUUUAU [6]. It would seem that the sequence reduces mRNA half-life, thus preventing inappropriate persistent production of a large amounts of biologically active polypeptides [6]. At 0.26-nm resolution by X-ray crystallography, the 17kDa TNF-a monomer forms an elongated, antiparallel p-

pleated sheet sandwich with a ‘jelly roll’ topology. Three monomers associate intimately about a threefold axis of symmetry to form a compact bell-shaped trimer, revealing a striking structural similarity to several viral coat proteins [22]. The analysis of structural and functional residues observed between TNF-a and lymphotoxin [23] suggests that lymphotoxin also binds to the same receptor as a trimer and that the ‘base’ of the trimer is the site of interaction with the receptor [22]. In addition to the soluble secreted TNF-a molecule, a cell-associated form of TNF-a has been described [8]. Cellassociated TNF-a, which in macrophages is responsible for cell-mediated cytotoxicity against TNF-a-sensitive target, exists in two forms: a molecule attached to its receptor [24] and a 26-kDa transmembrane protein [25]. The 26-kDa integral protein is a precursor of the 17-kDa secretory molecule, but may also play a role as a paracrine mediator [25]. Further understanding of structure/function relationship was recently obtained using human/mouse chimeric TNF-a proteins [26] or TNF-a analogs produced by making in vitro deletions of the coding sequence of the human TNF-a gene [27]. It was found that the carboxy-terminal amino acids of TNF-a are essential for the biological activity and are part of an epitope recognized by neutralizing monoclonal antibodies [27]. Biological activities

TNF-a has pleiotropic effects on a large number of different cell types. The net biological effect of this regulatory cytokine may be ultimately beneficial or detrimental for the host depending on its relative concentration, duration of cell exposure and presence of other mediators that may act in synergy with TNF-a [4, 28, 291. Cachexia. Persisting TNF-a production may provoke cachexia. In fact, TNF-a regulates the biosynthesis of several metabolic enzymes. Adipocytes and skeletal myocytes incubated with TNF-a become catabolic and lipolysis [30] and glycogenolysis [31] are markedly stimulated. In hepatocytes, TNF-a increases the production of acute-phase proteins and accelerates glucagon-mediated amino acid uptake [32]. The increased rates of whole-body energy expenditure, lipolysis, protein turnover as well as the anorexia, the anemia and the loss of body mass induced by TNF-a [33] are similar to the metabolic alterations associated with chronic infections or critical illnesses. Raised serum levels of TNF-a have been observed in cachectic patients with cancer [34], acquired immunodeficiency syndrome [35], parasitic infections [36, 371 and severe heart failure [38]. Endotoxiclseptic shock. The acute systemic release of TNFa is involved in the pathogenesis of septic shock and endotoxin-induced tissue injury [4]. TNF-a production temporally correlates with the onset of fever, mialgia, rigors, nausea and headache occurring in humans after intravenous injection of endotoxin [39]. The administration of recombinant TNF-a in primates produces clinical and metabolical alterations superimposable to those of endotoxic or septic shock [40]. The lethal effects of endotoxin injection are prevented by administration of monoclonal anti-TNF-a antibody F(ab)t fragment [41]. Raised serum levels of TNF-a have been also reported to be associated with a high rate of mortality for meningococcal infection [42], cerebral malaria [36] and Gramnegative purpura fulminans [43]. The lethal toxicity of TNFa is synergistically influenced by other molecules produced after endotoxin stimulation such as IL-1 and IFN-y and largely depends on production of secondary mediators including other regulatory peptides and eicosanoids. Leukotrienes

5 [44] and platelet activating factor (PAF) [45] have been implicated in the pathogenesis of endotoxic or septic shock. When administered intravenously to the experimental animal, PAF reproduces several aspects of the endotoxin or TNF-a-induced shock and PAF-receptor antagonists inhibit or reverse endotoxin-induced hypotension and prevent mortality [45,46]. Indeed, TNF-a stimulates macrophages, polymorphonuclear neutrophils (PMN) and vascular endothelial cells to produce and release PAF, suggesting that PAF may act as a secondary mediator [47]. InJlammation. Recent reports indicate that TNF-a is a mediator involved in inflammatory reactions other than those induced by endotoxin. TNF-a is chemotactic for monocytes and PMN, stimulates phagocytosis, adherence to endothelium and superoxide production by these cells and induces procoagulant activities in cultured human endothelial cells (for review, see [29]). In particular, TNF-a as well as IL-1, another 17-kDa cytokine that is structurally different but overlaps in biological function with TNF-a, causes endothelial cells to synthesize proteins that confer new functional capacities on the endothelium in a process defined as ‘endothelial activation’ [48]. These cytokines produce vascular leakiness by causing a structural reorganization of the endothelial cell layer dependent on rearrangement of endothelial cell cytoskeleton [49]. In vivo these cytokines initiate coagulation on the endothelial surface [50], leading to generation of agonists such as thrombin and fibrinogen peptides that cause endothelial cell contraction 14481. The ability of TNF-a t o stimulate synthesis of prostacyclin (PG12) [51], endothelial-derived relaxing factor [48] and of PAF [47] may also be involved in the early and in the sustained vasodilation, increased leukocyte delivery and increased vascular leak. In addition to directly increasing vascular permeability, PAF may induce an early (30 min) adhesion of PMN to the endothelium [52] and may favor subsequent activation and p2-integrin-dependent transmigration of PMN [53]. Despite the fact that TNF-a does not directly injure endothelium, this cytokine renders endothelial cells more susceptible to leukocyte-mediated injury [54]. In fact, TNF-a increases expression of intracellular-leukocyte adhesion molecules (ICAMs) [55] and the de novo synthesis and expression of endothelial leukocyte adhesion molecule-1 (ELAM-l), a 110-kDa cell-surface glycoprotein that binds PMN and monocytes [56]. TNF-a also causes the cell to synthesize and to secrete IL-8, a low-molecular-mass cytokine [57] that stimulates PMN movement, chemotaxis, degranulation and respiratory burst. By causing ‘molecular activation’ of /?2-integrins,such as lymphocyte-function-associated antigen-1 (LFA-l), IL-8 can enhance neutrophil interaction with ICAM-1, a molecule that is crucial in transmigration through the venule walls of marginated PMN [48]. TNF-a-activated endothelium expresses an increased amount of ICAM-1 [55]. In addition, TNF-a causes endothelial synthesis of monocyte chemotactic protein (MCP-1) [58] that may favor the delayed accumulation of monocyte in the inflamed tissue. The lymphocyte and monocyte adhesion to endothelium is also promoted by the TNF-a, IL-1 and IFN-y-increased expression of inducible cell adhesion molecule (INCAM-1 10) or vascular cell adhesion molecule (VCAM-1) [59] that occurs in the late phase of the inflammatory reaction (12-48 h) concomitantly with diminished ELAM-I [48]. Furthermore, by promoting the expression of MHC [60,61] antigen in endothelial cells, TNFCI may convert these cells into antigen-presenting cells and targets for sensitized T-cells. Finally, TNF-a acting in concert with IL-1 and IFN-y, alters protease/antiprotease balance, resulting in degradation of basement membrane protein by

endothelial cells [62]. The rise in serum level or in local concentration suggest the involvement of TNF-a in several inflammatory diseases such as rheumathoid arthritis [63], autoimmune diseases [64], renal allograft rejection [65] and graft-versushost disease [66]. However, homeostatic mechanisms modulate the role of TNF-a in inflammation. In fact, specific inhibitors for TNF-a activity [67 - 691 and synthesis under TNF-a stimulation of an anti-inflammatory cytokine, the transforming-growth-factor$ (TGF-P) [48], have been described. TGF-fi suppresses hydrogen-peroxide-releasing capacity of macrophages and the cytokine-mediated adherence of leukocytes to endothelium [69, 701. Tissue remodeling. TNF-a may also coordinate tissue remodeling. Indeed, TNF-a, as well as lymphotoxin, stimulates bone [71] and cartilage resorption [72]. Furthermore, TNFa inhibits the synthesis of proteoglycans [72]. The loss of proteoglycans, that occurs in several joint diseases, results in the impairment of cartilage function. TNF-a, however, may also participate in the mechanisms of normal tissue remodeling or of healing from inflammatory injury. In fact, by acting as a growth factor, TNF-a stimulates fibroblast and mesenchymal cell proliferation directly [73] and induces the production of other cytokines that stimulate cell proliferation and matrix production [4]. In addition, TNF-a may increase the mitogenic effect of epidermal growth factor by increasing expression of cell surface receptors for this cytokine [74]. Furthermore, TNF-a may promote neoangiogenesis [75]. Infection and immunity. TNF-a may participate in host defense against pathogens and in the modulation of immune response. The ability of TNF-a to activate phagocytosis [76] and leukocyte recruitment [77] plays a role in the defense against bacterial and parasitic infections. PMN show enhanced microbicidal activity [78], superoxide anion production and degranulation [79] when stimulated by TNF-a. In addition, TNF-a enhances the toxicity of eosinophils [80] and macrophages [81] to schistosomula in vitro. Macrophages are synergistically activated to achieve this effect by IFN-?/ and by IL-2 to kill Mycobacterium avium [82]. Moreover, it was recently reported that TNF-a possesses antiviral activity [83] not exclusively mediated by induction of IFN-fl [84, 851. Indeed, TNF-a may antagonize, directly or in cooperation with IFNs, viral infections by inducing resistance in uninfected cells and by selectively killing viral-infected cells, thus preventing viral spread [84]. On T lymphocytes, the expression of TNF-a receptors positively correlates with cell activation [86]. In addition, TNF-a enhances proliferation of IL-2-dependent T-cells, expression of MHC antigens and high-affinity IL-2 receptors [86]. Finally, TNF-a may modulate the immune response by triggering the production of a number of other regulatory cytokines such as IL-1, IL-6, IFNs, TGF-a, TGFp, granulocyte-monocyte colony-stimulating factor, and platelet-derived growth factor (PDGF). Finally, TNF-a enhances the T-cell-mediated response in synergism to antigenic challenge through a direct effect on T cells [87].Recent studies have also implic 2 ted TNF-a as an autocrine growth factor in chronic B-cell malignancies [88] and as relevant mediator in mixed lymphocyte reactions [89] and in the early phase of graft-versus-host disease [66]. Cytotoxicity. Depending on the type of target cell and on the presence of metabolic inhibitors, TNF-a may mediate apoptotic or necrotic cell lysis [90]. Apoptosis is characterized by compaction and margination of nuclear chromatin with formation of dense masses, by condensation of cytoplasm and by appearance of cell surface blebs. Necrotic cell lysis is characterized by irregular clumping of nuclear chromatin,

6 swelling of all cytoplasmic compartments, appearance of a dense matrix in the mitochondria, disruption of cell membrane, disintegration of organelles and disappearance of chromatin. Some cancer cells are particularly sensitive to the cytotoxic activity of TNF-a, whereas other cells are totally insensitive [3, 911. Furthermore, TNF-a appears to be a mediator of cytocidal activity of natural cytotoxic cells [92] and of activated macrophages [93].

Molecular mechanisms of action Little is known about the molecular mechanisms responsible for the multiple biological activities of TNF-a. However, the first step, as is the case for most polypeptide hormones, is the binding to specific cell-surface receptors. The TNF-a receptor complexes activate a variety of biological activities that produce different effects depending on the cell target.

Type I

1_ NH 2

A: cyrteine-rich domain B: transmembrane domain C: proline-rich

192 229

265

I415

COOH

TNF receptors

Fig. I . Structure of TNF-a receptor according to Kohno et al. [lo61

Stable trimers formed by three identical TNF-a polypeptides bind to a single class of high-affinity sites on cell surfaces extracellular domain of type I TNF-a receptor has a marked with a Kd in the picomolar range [12, 94, 951. TNF-a binding similarity with that of the soluble receptor present in human to cell-surface receptors correlates with cell stimulation [96]. urine [101, 1021. Type I1 TNF-a receptor [105,106] is a 46-kDa protein (439 Some authors have identified both high-affinity (Kd = 0.1 pM) and low-affinity (& = 0.1 nM) binding sites on the residues) characterized by an extracellular domain (235 amino same cell [95]. Recent studies indicate that TNF-a receptor is acids) with 22 Cys residues, a transmembrane segment (30 a protein with a molecular mass of approximately 300 kDa amino acids) making a single helical span, and an intracellular [94], possibly composed of dissimilar subunits [93]. The bind- domain (174 amino acids) without sequences typical of protein ing subunit of the receptor, as estimated by cross-linking stud- kinases or phosphorylation sites corresponding to substrates ies, has a molecular size ranging over 54- 175 kDa [93, 96, for these enzymes. The structure of type I1 receptor is similar 971. Another subunit that does not bind TNF, named Fas to that of type I, but the amino acid sequence of the two antigen, is associated with the receptor [98]. This molecule extracellular domains is different. This domain has some simihas been suggested to be involved in the signal transduction larity with the extracellular domain of nerve growth factor pathway [98]. A recent study [97] suggests the existence of two receptor (31 % amino acids identity), with CDw40 protein major receptor types for TNF-a: a myeloid cell-type receptor (38.5% amino acids identity), and with T2 protein, a transcrip(& = 0.07 nM) and an epithelial cell type receptor (Kd = tionally active open frame from Shope fibroma virus (40% 0.3 nM). These two receptor types differ in size, glycosylation amino acid identity). and in their peptide maps [99, 1001. Both type I and I1 TNF-a receptors bind TNF-a and The soluble form of TNF-a receptor detected in urines of lymphotoxin [103, 1051 with approximately equal affinity. healthy subjects [101,102] and in sera of cancer patients [lo31 Scatchard analysis reveals two binding sites with high affinity is probably a ‘shed’ form of cell receptor. This soluble receptor (& = 0.66 nM) and low affinity (& = 19.6 nM) in TSA 201 may compete with the cell-surface receptor in the binding of cells transfected with the cDNA of type I receptor [103]. A TNF-a, thus acting as physiological inhibitor. Two types of similar high-affinity (& = 0.18 nM) and low-affinity (& = the TNF receptor cDNA have been cloned, indicating the 10.1 nM) binding of TNF-a was observed in COS cells transpresence of two species of TNF-a binding proteins [102fected with the cDNA of type I1 receptor. The number of the 1051, named type I [104-1061 and type I1 [105, 1061 receptor receptors expressed on the cell surface is increased by IFN-y (Fig. 1). Type I receptor is a 55-kDa protein encoded by a [12, 1071, while it is decreased after binding of TNF-a [lo81 or single gene with at least three exons. The encoded protein treatment with IL-1 [109], endotoxin [110] and phorbol ester shows a typical structure of cell-surface receptor characterized [109, 1111. by a signal peptide at the beginning of the molecule, a potential The rapid turnover of TNF-a receptor depends on its transmembrane domain of 21 amino acid residues that separ- internalization and intracellular degradation. The prompt ates an extracellular domain of 182 residues including 24 Cys degradation was correlated with the presence of a large numand three potential sites for N-glycosylation, and an intracellu- ber of residues of Pro, Glx, Thr and Ser in the intracellular lar domains of 223 residues. The spacing of Cys residues in domain of type 1 receptor [102, 104, 1121. The experiments the extracellular domain is periodic and forms four subdo- indicating the down-regulation of TNF-a receptors by mains, each tightly cross-linked by disulfide bonds. It is poss- phorbol esters suggest a role for protein kinase C. However, ible that these four subdomains contain the ligand binding the cytoplasmatic domain of TNF-a type I and I1 receptors sites. The extracellular domain of TNF-a receptor has a net does not have a consensus sequence typical of proteins phoscharge opposite to that of the specific ligand, suggesting an phorylated on Ser and Thr [102- 1051. Since microtubuleelectrostatic interaction. The cytoplasmic domain does not depolymerizing agents cause loss of TNF receptor [110], it appear to be homologous to the catalytic domain of the Tyr- was suggested that the protein-kinase-C-dependent downor Ser/Thr-specific protein kinases or to nucleotide binding regulation of TNF-a receptor expression be mediated by a proteins. The amino acid sequence (residues 20- 180) of the phosphorylation of cytoskeleton structures [I 131. In contrast,

7 activators of protein kinase A (i.e. dibutyryl-CAMP) enhance synthesis and expression of TNF-a receptors on the cell surface [114]. Furthermore, recent studies have suggested that the production of TNF-a is controlled by protein kinases C and A [114]: the activation of protein kinase A would increase expression of the receptor coupled with an inhibition of TNFa production [115]; the activation of protein kinase C would enhance the synthesis of the cytokine but simultaneously would decrease the number of receptors [116, 1171. Guanine-nucleotide binding regulatory proteins ( G proteins) Considerable effort has been devoted to clarify the postreceptor mechanism of TNF-a. However, the available data are difficult to interpret, since they have been obtained on a variety of cells that respond differently to TNF-a. TNF-a may induce a rapid or late response in different types of cells. In some cells (i.e. endothelial cells), TNF-a promotes both types of response, suggesting the involvement of different receptors and/or the activation of different pathways of signal transduction. The transduced signals generated in the cells after the formation of TNF-a - receptor complex are in part controlled by G proteins that act as a modulatory link between the agonist and the enzyme systems, such as adenyl cyclase and phospholipase. The latter generate intracellular second messengers (reviewed in [118]). A GTP-on switch and a GDP-off switch regulate the transduction of receptor-initiated signals. In HL-60 cells, TNF-a might act by increasing GTPase activity of the inhibitory G protein and most probably of another 40kDa pertussin-toxin-sensitive G protein [119]. In endothelial cells, the TNF-a-induced increase in permeability [49, 1201, but not the suppression of thrombomodulin activity [49], is inhibited by pertussin toxin. These data suggest that certain cellular responses, but not all, are regulated by G proteins. Furthermore, it was suggested that a pertussis-sensitive GTPbinding protein other than a G protein may mediate the action of TNF-a [119].

Culcium/phosphatidylinositols/phosphutidylcholine The best known pathway leading to the activation of protein kinase C is the hydrolysis of phosphatidylinositols by phospholipase C enzyme. By this pathway, the cell produces two messenger molecules : inositol 1,4,5-trisphosphate, which promotes the release of calcium ions from the intracellular stores, and 1,2-diacylglycerol, which activates protein kinase C (reviewed in [121]). On the other hand, calcium ions and inositol phosphates do not seem to be important messengers involved in the mechanism of action of TNF-a. In particular in inflammatory cells, TNF-a does not increase the intracellular concentration of calcium and does not activate the breakdown of phosphatidylinositols [122- 1251. Furthermore, calcium channel blockers (i.e. diltiazen) do not inhibit the accumulation of GM-CSF mRNA induced by TNF-a [124].Inhibitors of phospholipase C or of diacylglycerol lipase do not prevent TNF-a-mediated cytotoxicity of L929 cells [126]. However, some relationship between TNF-a and calcium ions can not be excluded. In fact, the activation of PMN by TNF-a is blocked by buffering the intracellular calcium with quin-2 [125], suggesting that this cytokine can induce local changes in the concentration of cytoplasmic calcium ions [125]. Recently, Richter and coworkers demonstrated that the ‘spontaneous’ oscillatory activity of cytosolic Ca2 is required f i r granule secretion from adherent PMN stimulated by TNF+

a [127]. In fact, blockade of these spontaneous oscillations by anti-CD11b/CDlS-integrin antibodies inhibits granule secretion induced by TNF-a. Alternative pathways mediated by phospholipases other than phospholipase C can generate second messengers, including diacylglycerol. In the last few years, it has been observed that an increase in the turnover of phosphatidylcholine is an early event in the action of diverse agonists (reviewed in [128]). It may be speculated that TNFa induces diacylglycerol formation by the hydrolysis of phosphatidylcholine mediated by a specific phospholipase C [129, 1301 or by two sequential steps catalyzed by a phospholipase D, which promotes the accumulation of phosphatidic acid, and by phosphatidate phosphohydrolase [131]. In line with this hypothesis, the priming of PMN by TNF-a involves the activation of a phospholipase D [132].

Phosphorylation steps The most significant and earliest event occurring in different target cells challenged with TNF-a is a rapid increase of phosphorylation reactions, having as substrate both cytosolic or membrane proteins. TNF-a stimulates the phosphorylation of a 26-kDa protein in U937 (a myeloid cell line) and in CRLl500 (a breast cancer cell line) cells [133], a 27-kDa protein in human fibroblasts [133], several 28-kDa proteins in endothelial cells [134] and in ME-180 (a human cervical carcinoma cells) cell lines [135], and 75-kDa protein in HL-60 cells (a myeloid cell line). In HeLa cells, it has been shown that an antibody that cross-links type I receptors, as does TNF-a, induces the phosphorylation of a 27-kDa protein. The effect of TNF-a is rapid and transient: phosphorylation starts within few seconds and returns to the basal level after about 60 min [136, 1371. The role of protein phosphorylation is still unclear as it may or may not occur in different cell types independently of the biological actions of TNF-a [134- 1361. The analysis of phosphorylated amino acids of cytosolic and membrane proteins (i.e. epidermal-growth factor receptor) [135-1371 indicates that Ser, Thr and Tyr can be phosphorylated after TNF-a stimulation, and suggests that this cytokine activates Thr/Ser kinases and Tyr kinases and/or inactivates specific phosphatases. In human fibroblasts, TNFa causes a rapid intracellular accumulation of cAMP and an increase of protein kinase A activity [138]. H-8, an inhibitor of protein kinase A, partially inhibits the TNF-a-induced IL6 mRNA induction [138]. PMN spread on a fibrinogen-coated surface respond to TNF-a with a fall in cAMP [139]. Furthermore, agents that elevate cAMP inhibit TNF-a-induced cell spreading and actin polymerization [139]. The role of protein kinase C also remains to be clarified. Several reports indicate that TNF-a-induced activation of protein kinase C is cell-specific, rather than a ubiquitous event [138, 140 - 1431. TNF-a translocates protein kinase C from the cytosol to the membrane fraction in myeloid and lymphoid cell lines I141 - 1431, but not in other cells sensitive to TNF-a such as fibroblasts [138, 141, 1421. The role of protein kinase C is intriguing for other aspects. TNF-a primers PMN for enhanced protein-kinase-C-dependent responses [76, 1441 by increasing the synthesis of an 82-kDa protein, named as MARKCS (myristoylated, Ala-rich C kinase substratum), which is phosphorylated by protein kinase C [145]. Several inhibitors of TNF-a activities inhibit phosphorylation of proteins. On the other hand, phorbol esters that activate protein kinase C can induce resistance to killing by macrophages [146]. Protein kinases mav limit sustained cvtodasmic calcium fluxes that activate the c&ium-dependent endonuclease [ 1471.

The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammato...
1MB Sizes 0 Downloads 0 Views