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H. Ruddle
of Medicine, tumor
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Current
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Introduction The relationship between tumor necrosis factor (TNF-9; also known as cachectin) and lymphotoxin (TNF-p) has’ been unclear. Although they are members of a duplicated gene family that carry out many of the same activities in vitro and bind to the same cell surface receptors, their limited sequence homology (34%), occasional differences in target cell response, and real differences with regard to cell of origin, have suggested that the distinctions between the two molecules should be emphasized. Though TNF-a functions as a trimer [l], it was not previously clear whether TNF-j3 also formed oligomers, and whether the active sites of the molecules were similar. More fundamental questions were those concerning the reasons for the redundancy of the two molecules. Much of the uncertainty has been due to the difficulty in obtaining large quantities of biologically active TNF-j3 to allow functional comparisons. Also confusion concerning the relationship of the two cytokines has been reflected in the nomenclature. Authors have been unclear whether to emphasize their similarities and call them TNF-a and TNF-P, or their differences and call them tumor necrosis factor and lymphotoxin. (In this review, the TNFu and TNF j3 nomenclature will be used, even when other terms are employed in the primary literature.) In the past year, new data have conhrmed their homologous structures and receptor interactions but have continued to emphasize distinctions in their molecular regulation. No clear unifying picture has emerged concerning the mechanisms of their multiple activities, ranging from killing to induction of differentiation, and this area will not be addressed in detail in the present review. Clues to their biologic roles
1992, 4:327-332
will emerge with the ability to follow their inheritance patterns and disease associations with genetic markers and their manipulation in animal models.
Structure-function
relationships
of TNF-a and
TNF-p TNF-a is either maintained as a_celI surface molecule through its long’ (79 amino acid) ‘leader peptide [2] or released from macrophages and T cells. Until recently it was presumed that TNF-p with its conventional signal peptide was only produced in a secreted form. In the past year, however, a form of TNFj3 has been identified that associates with another protein with a molecular weight of 33 kD on the cell membrane [3,4-l. Recent advances in production and purification of human TNF p [ 5-,6-] have resulted in a clearer understanding of the biophysical similarities between the two cytokines. TNF-LX has been known for some time to form biologically active trimers [l] and TNF-j3 has now been shown to form similar trimers [ 6.1. The TNFP trimer molecule binds to both the 55 kD and 75 kD TNF receptors. The comparison of engineered mutants of TNF-CLand TNI-j3 has provided an insight into their receptor interactions. Several sites crucial for TNI-a biologic activity have been localized to loops at the base of the molecule [7*]. When TNF-P regions were subjected to similar analysis, it was shown that mutation of Asp50 (comparable to TNI-a hot spot loop that includes amino acids 29-36) or Tyr108 (comparable to TNI-a hot spot loop that includes amino acids 84-91)
Abbreviations CSF--cerebrospinal
fluid; HMC-high mobility group; L%Iipopolysaccharide; MS-multiple sclerosis; PHA-phytohaemagglutinin; RFLP-restriction fragment length polymorphism; TN&tumor necrosis factor. @ Current
Biology
ltd ISSN 0952-7915
327
328
lymphocyte
activation
and effector
functions
also results in loss of activity. These data suggest that the interaction of TNFcx and TNF-P with their receptors is similar. Thus far, these analyses have concentrated on the cytotoxic effects of the molecules and probably only detect interactions with the p55 receptor. It is not yet certain whether comparable sites will prove to be as important in interactions with the higher molecular weight receptor.
unclear; they may serve to inactivate or protect soluble TNF but this has not yet been resolved. Future studies employing both the natural shed receptors, and genetically engineered forms that have higher affinity and more favorable pharmacokinetics [ 16*], may be useful in treatment of sepsis and autoimmune disease.
Molecular
regulation
of TNF-a and TNF-P
Receptors Two types of TNF receptor have been identified and the cDNAs of the human forms have been cloned [8,9]. Unfortunately, the nomenclature for these molecules is still evolving, the low molecular weight form has variously been termed: ~55, Type B, Type II, Type 1; and the high molecular weight form has been termed: ~75, Type A, Type I and Type 2. In the present review, a nomenclature indicative of their molecular mass will be employed. In the past year, analysis of the TNF receptors has been expanded to the mouse, and data regarding their regulation, mapping, differences in species specificity and signal transduction have accumulated. Three groups have described the cDNA cloning of murine TNF receptors [IO*,II*,I2**]. As in the human, the two receptors are members of, and homologous to, a large family of proteins characterized by cysteine-rich repeats. Other members include nerve growth factor receptor, a B-lymphocyte activation molecule and a soluble form of the TNF receptor encoded by an open reading frame of Shope fibroma virus [ 131. Although both p55 and p75 bind TNF-c~ and TNF-P, the basis of species specificity previously described in TNF-induced thymocyte proliferation can now be understood. A murine CT6 cell line predominantly expresses p75 [10*,12**,13] and can be induced to proliferate after exposure to murine but not human TNF-cl [ 10*,12**], Therefore, p75 is implicated as a mediator of proliferation signals (at least in CT6); Barrett et al. [lo-] also imply that killing of WEHIby human TNF-cr or TNF-0 is mediated through ~55. Despite these observations, some similar functions may be mediated by the receptors, as both are capable of activating the NF-xB transcription factor after interaction with ligand [14-l. The two types of TNF receptor are regulated differently. Also the ratio of p55 to p75 on different cell types and their response to inductive signals vary. As discussed above, most cells express both types of receptor, though murine thymocytes predominantly express p75 [12-l, The receptors are encoded by genes on dilferent chromosomes; p55 is encoded by a gene on mouse chromosome 6 [ 11.1 and human chromosome 12 [ 151, while p75 is encoded by a gene on mouse chromosome 4 and human chromosome 1 [ 11.1. The signals regulating expression of the two receptors are different. For example, dibutyric CAMP treatment up-regulates p75 but not p55 [ 14.1. The analysis of molecular mechanisms of TNF receptor expression awaits the isolation of genomic clones. Considerable data exist concerning the presence of shed receptors in serum and urine but their biological role is
One of the more fascinating aspects of TNF-rx and TNFp concerns their regulation. Considerable emphasis in the past has been placed on their differences, namely the production by stimulated macrophages of TNF-c~, but not TNFD. Despite much evidence to the contrary, there have been frequent assertions that lymphocytes produce only TNF-P, whereas in fact, several studies have shown both TNI-a and TNI-p production by various T-cell and even B-cell populations. During the past year, English and his colleagues [17-l analyzed differences in the kinetics of TNF-c( and TNF-b mRNA accumulation by human peripheral blood T cells. They showed a high baseline transcription of TNF-a mRNA, an even higher transcription rate after activation and a short mRNA half life (30 min). TNF-p mRNA however, was transcribed at an undetectable baseline level with an increase after stimulation by concanavalin A and phorbol myristate acetate, although not to the levels of TN&X; its half-life was 5.5 h. These data highlight the differences in molecular regula tion of these genes, which occur even in the same cells under the same activation conditions. We have observed similar differences in TNF-cl and TNF-b transcription [ 181 and mRNA half lives (I Millet, NH Ruddle, abstract Al606, FASEB J 1992) in anti-CD3 activated murine T-cell clones. Controversy continues to surround the role of the four putative 5’ NF-xB sites in TNF-cl gene regulation. Drouet and colleagues [ 191 found that all four sites bind NF-xB and act as enhancers of TNF-CYgene expression, and that the presence of all four sites is crucial for lipopolysaccharide (LPS) activation in mouse macrophages. On the other hand, Goldfeld and colleagues [20-l demonstrated that TNF-c( mRNA induction in human T and B cell lines was not dependent on the presence of these NF-XB sites. These authors reached similar conclusions regarding the ability of LPS or virus to induce TNF-a in a fibroblast cell line. Although it is clear that the NF-xB sites can act as enhancers for heterologous promoters, their actual role in TNF-cl transcription awaits clarification. The role of 3’ sequences in TNF-cl regulation has also been studied. One study [21*] demonstrated that sequences in the 3’ untranslated region of the gene acted to repress TNF-cl promoter activity. Another study [22**] suggests that replacement of 3’ sequences of the human TNFc1 gene with globin sequences results in inappropriate regulation of TNF in transgenic mice; this gives rise to an arthritis not seen when the entire human TNF-cl gene was included. It is not clear whether these 3’ sequences alfect transcription, mRNA stability or translation. Thus far, no evidence for negative regulatory elements in TNFp 3’ sequences has been found, although previously ob-
Tumor necrosis factor (TNF-cl) and lymphotoxin (TNF-P) Ruddle
servations concerning negative regulatory elements in 5’ sequences have been published [23]. Analysis of TNF-P regulation followed previous observations concerning the importance of the 5’ NF-xB site in ‘I cells [23-251. One study [2&*] demonstrated that the NI-xB site alone was not sufficient for constitutive promoter activity in a pre-B cell line, but additional 5’ sequences were necessary. These sequences, which consist of a long stretch of poly(dAdT) bind a protein high mobility group (HMG)-I, and probably act to stabilize chromatin structure rather than as a transcriptional activator per se. The role of this sequence in ‘I’NFP production by activated T cells is currently under investigation. No etidence for a role for such a poly(dAdT) track in TNF-a regulation has emerged so far.
Role in disease Abnormalities in TNFa or TNF-l3 expression have been implicated in several diseases. In many instances, abnormally high levels of these cytokines are seen, while in others lower than normal production is seen. An, understanding of the basis of these associations and correlation with particular diseases may be useful in elucidating pathogenesis, and expression of TNF may be used as a marker of particular diseases. This information could form the basis for both therapy and diagnosis. In the past year, data have accumulated that implicate both TNF-a and TNI-l3 in multiple sclerosis (MS). Two studies [27*,28*] reported elevated levels of TNF-a in cerebrospinal fluid (CSF) of a high proportion of MS patients especially those in active disease. TNF-a levels in CSF were found to be a more reliable indicator of clinical status than serum levels. TNI-p was not analyzed. In a histologic analysis TNFa and TNFp were both detected in MS lesions, but in different cells [29”]. In an in vitro extension of these observations, TNI-l3 was found to be a considerably more potent killer of oligodendrocytes than TNFcr 1301. On the other hand, it was suggested that TNF-cl and TNFP play only a minor role in peptide-induced experimental allergic encephalomyelitis [ 31 I, indicating that considerably more data must be accumulated before the role of these cytokines in autoimmune neurologic inllammatoty diseases is fully understood. The activity of TNF-p as an osteoclast activating factor and its elevation in serum, freshly isolated cells and long-term lines of patients with adult T-cell leukemia implicates it in the hypercalcemia that frequently accompanies this malignancy [32-l. ‘INI-cx was not analyzed in these studies, but it has previously been reported to be elevated slightly, if at all, in long-term T-cell lines from such patients.
Genetic polymorphisms The study of the association of TNI-a and TNF-fi with disease at the genetic level requires the existence of genetic markers. In the past year, considerable advances have been made in the characterization of previously described polymorphisms, and in the identification of new variants of human TNFp. .Hybridization of Nco I digested DNA with TNI-a or TNF p probes resulted in a polymorphic fragment of DNA, either 5.5 kilobases (kb) or 10.5 kb in length [33]. This polymorphism was due to a substitution of a G for an A in intron 1 of TNI-fl, giving rise to the new Nco I site [34*], Studies analyzing the association of the Nco I polymorphism with disease have produced variable results. No association of either form was found with a&losing spondylitis [35]. The 10.5 kb allele was associated with an increased frequency of insulin dependent diabetes mellitus especially when inherited with B15+, DR-4+ haplotypes [ 36**]. On the other hand, Abraham and colleagues [34*], proposed that the 8.1 kb ancestral haplotype (5.5 kb allele) is associated with autoimmune disease, especially rheumatoid arthritis. Studies have been carried out to determine whether changes in the levels of TNF-a or TNI-p are associated with either allele. Messer and colleagues [ 37**] found that phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells from individual homozygous for the 5.5 kb allele expressed higher levels of TNI-P biologic activity than individuals homozygous for the 10.5 kb allele:No differences were seen with regard to TN-a. On the other hand, Pociot et al. [36*-l reported that monocytes of individuals heterozygous for the 5.5-10.5 kb allele produce less TNF-a than individuals homozygous for the 10.5 kb allele. TNF-l!l was not analyzed: These observations, though intriguing, require considerable additional analysis before l?rm conclusions can be reached concerning the association of the Nco I polymorphism with propensity to autoimmune disease and levels- of TNF-a and TNI-l3 production. The allele with the 5.5 kb Nco I fragment also includes a substitution of an A for a C in the coding sequence of mature TNF-l3, resulting in an asparagine instead of threonine at position 26 [37**], However, it is not yet known whether this substitution affects biologic activity. A polymorphism in the 3’ region of the TNF-p gene has also been detected after digestion with Eco Rl [38]. Although this restriction fragment length polymorphism (RFIP) is seen at a low frequency and has not been associated with disease, the fact that it does not as sociate with either Nco I allele [37**] allows its future use as a genetic marker. An exciting development in the genetic analysis of TNF polymorphisms has come from the discovety of microsatellites associated with the gene complex. These are DNA sequences consisting of varying lengths of TC/GA or AC/GT repeats, they are considerably more polymorphic than the RFIPs, and provide exciting future tools
329
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Lvmuhocvte
activation
and effector
functions
for study. Four such microsatellites have been described [3!?*]. One microsatellite, called TNFc, consists of two alleles (one containing nine TC repeats the other containing ten repeats), and is located in intron 1 of the TNF-p gene, downstream from the site of the Nco I polymorphism. Two others, TNFa and TNFb, are located 3.5 kb upstream (telomeric) of the TNI-p gene and have seven and 13 alleles [40-l respectively. The association of these polymorphisms with disease and/or levels of TNFCLor TNF-0 is yet to be established but will certainly be intriguing.
Structure that Binds to both Tumor Necrosis Factor Receptors. J Biol Cbem 1991, 266:386%3&?69. The authors present evidence for the trimeric structure of TNI-p and its association with both p55 and p75 receptors, indicating structural similarities between the two TNF forms. J, PRANGET, FIERS W: Localization VAN OSTADE2, TAVERNIER of the Active Site of Human Tumor Necrosis Factor (hTNF) by Mutational Analysis. EMBO J 1991, 4827-836. This paper highlights the importance of specilic sites at the base of the TNF-a molecule for receptor binding and emphasizes the importance of physical crosslinking of the receptors for biologic activity. 7. .
8.
LOETSCHER H, PAN YCE, IAHM HW, GEN’IZ R, BR~CKHAUSM, W: Molecular Cloning and Expression TABUCHIH, LESS~AUER
of the Human 55 kDa Tumor Necrosis Factor Receptor. Cell 1990, 61:351-359. 9.
Conclusion Data obtained in the past year have resulted in the al&mation of the biophysical similarities of TNF-ol and TNF-p and their receptor interactions. Insights into the molecular basis of the regulation of the cytokines continue to accumulate and indicate profound differences. A role for TNF-ct and TNF-p in disease pathogenesis has been suggested, particularly in the case of MS. The identification of a microsatellite polymorphism will be a useful genetic marker system in the study of disease associations.
References and recommended reading Papers of particular interest, published within the annual period of rev&v, have been highlighted as: . of special interest .. of outstanding interest 1.
JONES EY, STUARTDI, WAXER NPC: Structure Necrosis Factor. Nature 1989, 338:225-228.
2.
KR~EGLER M, PEREZC, DEFAYK, ALBERT I, Lu SD: A Novel Form of TNF/Cachectin is a Cell Surface Cytotoxic Transmembrane Protein: Ram&cations for the Complex Physiology of TNF. Cell 1988, 53:45-53.
3.
BROWNING JL, ANDROIEWICZ MJ, WARE CF: Lymphotoxin
4. ..
of Tumor
and an Associated 33 kDa Glycoprotein are Expressed on the Surface of an Activated T Cell Hybridoma J Immuno11991, 147:123&1237.
BARRETTK, TAYLOR-FISHWICK DA, COPE AP, KI%ONF,RGHIS AM, GRAY PW, FELDMANN M, FOXWELLBMJ: Cloning, Expression and Crosslinking Analysis of the Murine p55 Tumor Necrosis Factor Receptor. Eur J Immunol 1991, 21:164%1656. The authors demonstrate that human TNF-a does not bind to murine p75 TNF receptor and that a functional difTerence exists between the receptors. TNF interacts with p55 to induce killing of a WEHI 164 cell line, and p75 is associated with TNF induced proliferation of a T-cell line. 10. .
11. .
GCQDWIN RG, ANDERSONB, JERZY R, DAVIS T, BRANNAN CL,
12.
L~wrs M, TAR’IAGLIA L4, LEEAA, BENNETTGL, RICEGC, WONG GHC, CHEN EY, G~EDDEL DV: Cloning and Expression of
COPE~AND NG, JENKINSNA, SMITHCA: Molecular Cloning and Expression of the Type 1 and Type 2 Murine Receptors for Tumor Necrosis Factor. Mol Cell Biol 1991, 11:302Cb3026. The authors isolate cDNA clones for both murine TNF receptors. In contrast to the ligands whose genes are tightly linked, the receptors are encoded by genes on separate chromosomes. ..
cDNAs for Two Distinct Murine Tumor Necrosis Factor Receptors Demonstrate One Receptor is Species Specific. Proc Nat1 Acud Sci USA 1991, 88:283@2834. The authors isolated cDNAs for both murine TNF receptors. By Northern blot analysis, they demonstrated that mRNA for p75 but not p55 is expressed in a murine T-cell line, CT6. By transfection into a cell line that normally express only low amounts of TNF receptor, they demonstrated that murine p75 receptors bind murine but not human TN&X. These observations are important because they provide a molecular explanation for a previously observed species specificity in only one kind (T-cell proliferation) of biologic assay. 13.
ANDROLEWICZ MJ, BROWNING JL, WARECF: Lymphotoxin is Ex-
pressed as a Heterodimeric Complex with a Distinct 33 KD Glycoprotein on the Surface of an Activated Human T Cell Hybridoma. J Biol Cbem 1991, 267:2542-2547. The authors present definitive data that TNI-p associates early in biosynthesis with a 33 kDa protein and the complex is expressed at the cell surface. This is of special interest because it indicates that both TNF-u and TNi-b can be membrane-associated, but the biochemical basis of that association differs.
SCHAL.LTJ, LEWIS M, KOUER KJ, LEE A, RICE GC, WONG GHW, GATANAGAT, GRANGERGA, LENTZR, RAAB H, KOHR WJ, G~EDDEL DV: Molecular Cloning and Expression of a Receptor for Human Tumor Necrosis Factor. Cell 1990, 61:361-370.
SMITH CA, DAVIST, WIGNALL JM, DIN WS, FARRAH T, UPTONC, MCFADDEN G, GOODWINRG: T2 Open Reading Frame for the Shope Fibroma Virus Encodes a Soluble Form of the TNF Receptor. Biockm Biopbys Res Comm 1991, 176:335-342.
108 are Essential for Receptor Binding and Cytotoxic Activity of Tumor Necrosis Factor Beta (Lymphotoxin). Prot Eng 1991, 4~785-791. The authors present the first mutational analysis of TNF-p. The data indicate that sites homologous to TNF-a active sites are also crucial for TN&p receptor binding and biologic activity.
HOHMANN H-P, BROCKHAUS M, BAEUEFZEPA, &MY R, KOLBECK R, VANLC~NAPGM: Expression of the Types A and B Tumor Necrosis Factor (TNF) Receptors is Independently Regulated, and Both Receptors Mediate Activation of the Transcription Factor, NF-xB. J Biol Chem 1991, * 265:22403_22417. In a study of human TNF receptors, the authors demonstrate that T&e A (~7.5) TNF receptor, but not Type B (~55) expression is increa.s?d in HI60 cells by treatment of cells with dibutyric CAMP.TNF and antibody to p55 induce NFxB activity in HEp2 cells and antibody to p75 induces NFxB activity in HI&l cells. The key point of the paper is the demonstration of differences between p75 and p55 in receptor regulation while at least one of their biologic activities, NFxB induction, remains similar.
6.
SCHOENFELD HJ, POESCHLB, FREYJR, L~ETSCHER H, HUNZIKER
W, Lusnc 4 ZULAUFM: Efficient Purification of Recombinant Human Tumor Necrosis Factor 0 from Escherichia coli Yields a Biologically Active Protein with a Trimeric
15.
.
5. ..
GOH CR, LOHS-C, PORTER AG: Aspartic Acid 50 and Tyrosine
14.
.
DERREJ, KEMPER 0, CHER~F D, NOPHARY, BERGERR, WALLXH
D: The Gene for Type 1 Tumor Necrosis Factor Receptor (TNF-Rl) is Localized on Band 12~13. Hum Genet 1991, 87~231-233.
Tumor
16. .
L~ETSCHER
H, GENTE R, ZULAIIF M, Lusnc A, TARUCHI H, SCHLAEGERE-J,. BROCKHAUS M, GALLATE H, MANNEBERGM, LESSIAIIERN: Recombinant 55-kDa Tumor Necrosis Factor (TNF) Receptor. J Biol C%em 1991, 266:18324-18329. A chimeric bivalent fusion protein consisting of the extracellular portion of p55 TNF receptor and the Fc region of human Igy3 binds human TNF-a and TNFB with high affinity, prevents TNF-a and TNF-p binding to p55 and p75 TNF receptor and blocks cytotoxicity. The importance of this observation is the potential clinical advantage of this high affinity TNF receptor protein in inhibiting TNF-a or TNF~P in, for example, autoimmune disease. 17. ..
ENGLISH K, WEA\TR
WM, WILSON CB: Differential Regulation of Lymphotoxin and Tumor Necrosis Factor Genes in Human T Lymphocytes. J Biol Chem 1991, 266:710%7113. This paper demonstrates differences in TNF-a and TNFB regulation in a single population of cells, peripheral blood T cells, stimulated with concanavalin A and phorhol mytystate acetate. This study is imp portant because it partially elucidates the mechanism of the differences in the kinetics of TNF~a and TNF-B mRNA accumulation; namely the low transcription rate and high stability of TNF~B mRNA, and the high transcription rate and low stability of TNF-a mRNA.
18.
FERRERJNR. SARRT, &KENASE PW, RUDDU? NR: Molecular Regulation of Tumor Necrosis Factor-a and Lymphotoxin Production in T CeIIs: Inhibition by Prostaglandin E2. J Rio1 Cbem 1992, 267:9443-9449.
19.
DRO~JET C, SHAKHOV AN, JONGENEEI. CV: Enhancers and Transcription Factors Controlling Inducibility of the Tumor Necrosis Factor-u Promoter in Primary Macrophages. .I Immunol 1991, 147:1694-1700.
20. .
GOLDFED AF, STROMINCER JL, DOYIE C: Human Tumor Necrosis Factor CLGene Regulation in Phorbol Ester Stimulated ’I T and B CeII Lines. J Exp Med 1991, 174:73-81. This paper is of interest because it disagrees with other work that had emphasized the importance of the 5’ NFxB sites in the regulation of TNF-a in macrophages and suggests that the mechanism of TNF-a regulation in lymphocytes may involve different regulatory sequences.
21. .
KRUYS V, KEMMERK, SHAKHOVA, JONGENEEL V, BEUTLER B: Constitutive Activity of the Tumor Necrosis Factor Promoter is CanceIIed by the 3’ Untranslated Region in Nonmacrophage CeII Lines; a Trans-dominant Factor Overcomes This Suppressive Effect. Proc Natl Acad Sci USA 1992, 89:673477. Data are presented that underscore the complexity of TNF-a regulation, and suggest negative regulation of promoter activity by non-coding 3’ sequences. 22. ..
KEFFERJ, PROBER?‘L, CAZLARISH, GEORGOPOIILOUSS, K~~IARIS E, KIOUSSISP, K0l.l.14~ G: Transgenic Mice Expressing Tumor Necrosis Factor: a Predictive Genetic Model of Arthritis. z!34BO J 1991, 10:40254031. This paper is the first study to demonstrate that inappropriate expression of TNF-a in a transgenic mouse system results in an identifiable pathology. The data also appear to confirm the importance of 3’ sequences in regulation of TNF~u production. The use of a human TNF~a construct facilitates detection of the transgene although the effects of the transgene are limited to cells expressing the p55 receptor (see also l10*,12**1). 23.
FASHENASJ, TANG W-L, SARR T, RUDDLIZNH: The Murine Lymphotoxin Gene Promoter. Characterization and Negative Regulation. J Immunol 1990, 145:177-183.
24.
PAUL NL, LENAFXX) MJ, NOVAK KD, SARRT, TANG W-L, RUDDLE NH: Lymphotoxin Activation by Human T-cell Leukemia Virus Type I-infected Cell Lines: Role for NK-xB. J Viral 1990, 64:5412.
25.
26. ..
MESSERGE, WEISS H, BAFXIERLE PA: Tumor Necrosis Factor b (TNF-p), Induces Binding of the NF-xB Transcription Factor to a High-affinity xB Element in the TNFj3 Promoter. Cytokine 1990, 2:389. FA~HENA SJ, REEVES R, RUDDLE NH: A Poly (dA-dT) Upstream Activating Sequence Binds High Mobility Group I Protein
necrosis
factor
(TNF-09 and lymphotoxin
(TNF-0)
Ruddle
and Contributes to Lymphotoxin (Tumor Necrosis Factorp) Gene Regulation. Mol Cell Biol 1992, 12:894-903. The importance of this paper is the demonstration of a new regulatory element in the TNF-P gene that appears to function in at least one type of celI (pre B cells) which produces TNFp mRNA constitutively. This element is bound by a protein, HMG-1, that acts by stabilizing chromatin structure rather than by enhancing transcription directly. 27. .
TSUKADA
N, MNAGI K, MATSUDA M, YANAGISAWA N, YONE K: Tumor Necrosis Factor and Interleukin-1 in GF and Sera of Patients with Multiple Sclerosis. J New-01 Sci 1991, 102:23&234. The authors use a sensitive enzyme linked immunosorbent assay assay to detect TNFa in CSF of MS patients; detection of TNFu correlates with exacerbation. Lower levels were found in sera. No interleukinla or interleukin-lp were detected. This paper is important because it suggests a correlation of TNF levels with a clinical state and it em phzsizes the importance of analyzing a source of material (CSF) other than serum. 28. .
SHAIUEI:
MK, HENTGESR: Association Between Tumor Necrosis Factor-u and Disease Progression in Multiple Sclerosis. Ntru’En@ J Med 1991, 325i467-472. This paper is important because it reports higher levels of TNF-a in CSF of patients with chronic progressive MS, compared with levels in patients with stable disease. It also presents evidence for a role of TNFa in either MS disease pathogenesis, or at least its usefulness as a marker of the disease state. SEIMAJ K, RAINE CS, CANNELLA B, BROSNAN CF: Identification of Lymphotoxin and Tumor Necrosis Factor in Multiple Sclerosis Lesions. J Clin Invest 1991, 87:94+954. This paper is important because it demonstrates the production of TNF a and TNF~j3 by different cells in MS lesions. TNF-p was found in CD3+ lymphocytes and Leu M5+ microgial cells at the edge of the lesion, whereti TNFa was found in zxrocytes and macrophages. 29. ..
30.
SELM.$JK, RAINE CF, FAROCQ M, NORTON WT, BROSNAN CF: Cytokine Cytotoxicity Against OIIgodendrocytes. Apoptosis Induced by Lymphotoxin. .I Immunol 1991, 147:1522-1529.
31.
MEIUULLJE, KONO DH, CLAYTONJ, ANW DG, HINTON DR, HOFMAN FM: Intlammatory Leukocytes and Cytokines in the Peptide-induced Disease of Experimental AIIergic Encephalomyelitis in §JL and BlO.PL Mice. Proc Natl Acad Sci USA 1992, 89~576578. Y,
OTSUKA
M,
KWAZURU
ISH~RASHIK, ‘IsBrnu~~
.
Y,
33.
WERDGC, CHAKINDD: Genetic Variability at the Human Tumor Necrosis Factor Loci. J Immunol 1990, 145:127%1285.
IWAHASHI M,
K,
CH~MON
32.
UTSUNOMNA
A,
HANDA
S, SAKURAMIT, ARIMA T: Tumor Necrosis Factor-p in the Serum of Adult T CeU Leukemia with HypercaIcemia. Blood 1991, f7:2451-2455. This paper indicates that levels of TNl-b are elevated in the serum of patien& with a hone lesion, implicating cytokine with known osteoclast activating activity in the pathogenesis of the syndrome.
ABRAIW LJ, Du DC, ~~EIX K, DAWKINSRL, WHITEHEADAS: Haplotypic Polymorphisms of the TNF-!3 Gene. Immune genetics 1991, 33:50-53. The authors characterize polymorphisms of the TNF~P gene by sequence analysis and correlate them with ancestral haplotypes. They demonstrate Linkage between a sequence difference in intron 1 of TNFfi and a new Nco I site. Another polymorphism results in an amino acid substitution at position 26 in TNF~P. This study is important because it defines the molecular basis of the polymorphisms, and associates these with known haplotypes. 34. .
35.
VERJANSGMGM, VAN DER LINDENSM, VAN EYS GJJM, DE WAAL LP, KIJLSTRAA: Restriction Fragment Length Polymorphism of the Tumor Necrosis Factor Region in Patients with Ankylosing Spondytitis. Arthritis Rbeum 1991, 34:486-489.
36.
POCIO’I’ F,
. .
H,
BAEK
L,
MOLVIG NERUP
J,
WAGENSEN
L,
WORSAAI.
H,
DAL~OYE
L: Tumor Necrosis Factor Beta Gene Polymorphism in Relation to Monokine Secretion and Insulin Dependent Diabetes Mellitus. Scund J Immunol 1991, 33:37-49.
331
332
LvmDhocvte activation and effector functions
This study is important because it suggests an association of a particular TNF-p allele (10.5 kb RFLF’defined by Nco I that maps to intron 1 of TNF-P) with a disease. The increased production of TNF-a by monocytes in such individuals is intriguing with regard to a possible mechanism of disease pathogenesis. MESSERG, SPAXLER U, JUNG MC, HONOU) G, BLAMER K, P.WE GR, RIE~MKJLLER G, WEISS EH: Polymorphic Structure of the Tumor Necrosis Factor (TNF) Locus: An Nco I Polymorphism in the Fist Intron of the Human TNF-p Gene Correlates with a Variant Amino Acid in Position 26 and a Reduced Level of TNF-!3 Production. J E3cp Med 1991, 173:20?219. The authors localized the Nco I polymorphism to intron 1 of TNF-p, and suggest that individuals homozygous for the a 5.3 kb Nco I fmgment produce higher levels of TNF-P than individuals homozygous for the 10.5 kb allele after stimulation of peripheral blood cells with PHA. TNF-alpha levels were not significantly different. This paper is important because it associates differences in TNF levels with different alleles, and the genetic linkage of the 5.5 kb and 10.5 kb Nco I site with an asp paragine and a threonine in position 26 of the mature TNF-P protein, respectively. 37. . .
38.
PAR~ANEN J, KOSKIMIES S: Low Degree of DNA Polymorphism in the HLAlinked Lymphotoxin (Tumor Necrosis Factor-o) Gene. Stand J Immunol 1988, 28:313-316.
SA, ~JDALOVA L4, KUPRGH DV, TURETXAYA RL: DNA Sequence Polymorphism at the Human Tumor Necrosis Factor (TNF) Locus. J Immunol 1991, 147:1053-1059. The authors detect sequence polymorphisms that mark the TNF complex. These observations are important because the polymorphism is so extensive (one form has 13 alleles) it will be more useful than the previously described bi-allelic variants.
39.
NEMXPAWV
. .
40.
JONGENEEL
.
SA, CAMBON-THOMSEN
CV, BRL&N~L, UDAXWO rq SEWN A, NEDOSPASOV A: Extensive Genetic Polymorphism in the Human Tumor Necrosis Factor Region and Relation to Extended HLA Haplotypes. Proc Natl Acad Sci ZJSA 1991, g&9717-9721. The authors demonstrate the relationship between extensive micro satellite polymorphisms and HL4 haplotypes, indicating their potential usefulness as genetic markers of HIA associated diseases.
NH Ruddle, Department of Epidemiology and Public Health, Yale LJni~ versity School of Medicine, 60 College Street, PO Box 3333, New Haven, Connecticut, US.4
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Current
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Introduction The relationship between tumor necrosis factor (TNF-9; also known as cachectin) and lymphotoxin (TNF-p) has’ been unclear. Although they are members of a duplicated gene family that carry out many of the same activities in vitro and bind to the same cell surface receptors, their limited sequence homology (34%), occasional differences in target cell response, and real differences with regard to cell of origin, have suggested that the distinctions between the two molecules should be emphasized. Though TNF-a functions as a trimer [l], it was not previously clear whether TNF-j3 also formed oligomers, and whether the active sites of the molecules were similar. More fundamental questions were those concerning the reasons for the redundancy of the two molecules. Much of the uncertainty has been due to the difficulty in obtaining large quantities of biologically active TNF-j3 to allow functional comparisons. Also confusion concerning the relationship of the two cytokines has been reflected in the nomenclature. Authors have been unclear whether to emphasize their similarities and call them TNF-a and TNF-P, or their differences and call them tumor necrosis factor and lymphotoxin. (In this review, the TNFu and TNF j3 nomenclature will be used, even when other terms are employed in the primary literature.) In the past year, new data have conhrmed their homologous structures and receptor interactions but have continued to emphasize distinctions in their molecular regulation. No clear unifying picture has emerged concerning the mechanisms of their multiple activities, ranging from killing to induction of differentiation, and this area will not be addressed in detail in the present review. Clues to their biologic roles
1992, 4:327-332
will emerge with the ability to follow their inheritance patterns and disease associations with genetic markers and their manipulation in animal models.
Structure-function
relationships
of TNF-a and
TNF-p TNF-a is either maintained as a_celI surface molecule through its long’ (79 amino acid) ‘leader peptide [2] or released from macrophages and T cells. Until recently it was presumed that TNF-p with its conventional signal peptide was only produced in a secreted form. In the past year, however, a form of TNFj3 has been identified that associates with another protein with a molecular weight of 33 kD on the cell membrane [3,4-l. Recent advances in production and purification of human TNF p [ 5-,6-] have resulted in a clearer understanding of the biophysical similarities between the two cytokines. TNF-LX has been known for some time to form biologically active trimers [l] and TNF-j3 has now been shown to form similar trimers [ 6.1. The TNFP trimer molecule binds to both the 55 kD and 75 kD TNF receptors. The comparison of engineered mutants of TNF-CLand TNI-j3 has provided an insight into their receptor interactions. Several sites crucial for TNI-a biologic activity have been localized to loops at the base of the molecule [7*]. When TNF-P regions were subjected to similar analysis, it was shown that mutation of Asp50 (comparable to TNI-a hot spot loop that includes amino acids 29-36) or Tyr108 (comparable to TNI-a hot spot loop that includes amino acids 84-91)
Abbreviations CSF--cerebrospinal
fluid; HMC-high mobility group; L%Iipopolysaccharide; MS-multiple sclerosis; PHA-phytohaemagglutinin; RFLP-restriction fragment length polymorphism; TN&tumor necrosis factor. @ Current
Biology
ltd ISSN 0952-7915
327
328
lymphocyte
activation
and effector
functions
also results in loss of activity. These data suggest that the interaction of TNFcx and TNF-P with their receptors is similar. Thus far, these analyses have concentrated on the cytotoxic effects of the molecules and probably only detect interactions with the p55 receptor. It is not yet certain whether comparable sites will prove to be as important in interactions with the higher molecular weight receptor.
unclear; they may serve to inactivate or protect soluble TNF but this has not yet been resolved. Future studies employing both the natural shed receptors, and genetically engineered forms that have higher affinity and more favorable pharmacokinetics [ 16*], may be useful in treatment of sepsis and autoimmune disease.
Molecular
regulation
of TNF-a and TNF-P
Receptors Two types of TNF receptor have been identified and the cDNAs of the human forms have been cloned [8,9]. Unfortunately, the nomenclature for these molecules is still evolving, the low molecular weight form has variously been termed: ~55, Type B, Type II, Type 1; and the high molecular weight form has been termed: ~75, Type A, Type I and Type 2. In the present review, a nomenclature indicative of their molecular mass will be employed. In the past year, analysis of the TNF receptors has been expanded to the mouse, and data regarding their regulation, mapping, differences in species specificity and signal transduction have accumulated. Three groups have described the cDNA cloning of murine TNF receptors [IO*,II*,I2**]. As in the human, the two receptors are members of, and homologous to, a large family of proteins characterized by cysteine-rich repeats. Other members include nerve growth factor receptor, a B-lymphocyte activation molecule and a soluble form of the TNF receptor encoded by an open reading frame of Shope fibroma virus [ 131. Although both p55 and p75 bind TNF-c~ and TNF-P, the basis of species specificity previously described in TNF-induced thymocyte proliferation can now be understood. A murine CT6 cell line predominantly expresses p75 [10*,12**,13] and can be induced to proliferate after exposure to murine but not human TNF-cl [ 10*,12**], Therefore, p75 is implicated as a mediator of proliferation signals (at least in CT6); Barrett et al. [lo-] also imply that killing of WEHIby human TNF-cr or TNF-0 is mediated through ~55. Despite these observations, some similar functions may be mediated by the receptors, as both are capable of activating the NF-xB transcription factor after interaction with ligand [14-l. The two types of TNF receptor are regulated differently. Also the ratio of p55 to p75 on different cell types and their response to inductive signals vary. As discussed above, most cells express both types of receptor, though murine thymocytes predominantly express p75 [12-l, The receptors are encoded by genes on dilferent chromosomes; p55 is encoded by a gene on mouse chromosome 6 [ 11.1 and human chromosome 12 [ 151, while p75 is encoded by a gene on mouse chromosome 4 and human chromosome 1 [ 11.1. The signals regulating expression of the two receptors are different. For example, dibutyric CAMP treatment up-regulates p75 but not p55 [ 14.1. The analysis of molecular mechanisms of TNF receptor expression awaits the isolation of genomic clones. Considerable data exist concerning the presence of shed receptors in serum and urine but their biological role is
One of the more fascinating aspects of TNF-rx and TNFp concerns their regulation. Considerable emphasis in the past has been placed on their differences, namely the production by stimulated macrophages of TNF-c~, but not TNFD. Despite much evidence to the contrary, there have been frequent assertions that lymphocytes produce only TNF-P, whereas in fact, several studies have shown both TNI-a and TNI-p production by various T-cell and even B-cell populations. During the past year, English and his colleagues [17-l analyzed differences in the kinetics of TNF-c( and TNF-b mRNA accumulation by human peripheral blood T cells. They showed a high baseline transcription of TNF-a mRNA, an even higher transcription rate after activation and a short mRNA half life (30 min). TNF-p mRNA however, was transcribed at an undetectable baseline level with an increase after stimulation by concanavalin A and phorbol myristate acetate, although not to the levels of TN&X; its half-life was 5.5 h. These data highlight the differences in molecular regula tion of these genes, which occur even in the same cells under the same activation conditions. We have observed similar differences in TNF-cl and TNF-b transcription [ 181 and mRNA half lives (I Millet, NH Ruddle, abstract Al606, FASEB J 1992) in anti-CD3 activated murine T-cell clones. Controversy continues to surround the role of the four putative 5’ NF-xB sites in TNF-cl gene regulation. Drouet and colleagues [ 191 found that all four sites bind NF-xB and act as enhancers of TNF-CYgene expression, and that the presence of all four sites is crucial for lipopolysaccharide (LPS) activation in mouse macrophages. On the other hand, Goldfeld and colleagues [20-l demonstrated that TNF-c( mRNA induction in human T and B cell lines was not dependent on the presence of these NF-XB sites. These authors reached similar conclusions regarding the ability of LPS or virus to induce TNF-a in a fibroblast cell line. Although it is clear that the NF-xB sites can act as enhancers for heterologous promoters, their actual role in TNF-cl transcription awaits clarification. The role of 3’ sequences in TNF-cl regulation has also been studied. One study [21*] demonstrated that sequences in the 3’ untranslated region of the gene acted to repress TNF-cl promoter activity. Another study [22**] suggests that replacement of 3’ sequences of the human TNFc1 gene with globin sequences results in inappropriate regulation of TNF in transgenic mice; this gives rise to an arthritis not seen when the entire human TNF-cl gene was included. It is not clear whether these 3’ sequences alfect transcription, mRNA stability or translation. Thus far, no evidence for negative regulatory elements in TNFp 3’ sequences has been found, although previously ob-
Tumor necrosis factor (TNF-cl) and lymphotoxin (TNF-P) Ruddle
servations concerning negative regulatory elements in 5’ sequences have been published [23]. Analysis of TNF-P regulation followed previous observations concerning the importance of the 5’ NF-xB site in ‘I cells [23-251. One study [2&*] demonstrated that the NI-xB site alone was not sufficient for constitutive promoter activity in a pre-B cell line, but additional 5’ sequences were necessary. These sequences, which consist of a long stretch of poly(dAdT) bind a protein high mobility group (HMG)-I, and probably act to stabilize chromatin structure rather than as a transcriptional activator per se. The role of this sequence in ‘I’NFP production by activated T cells is currently under investigation. No etidence for a role for such a poly(dAdT) track in TNF-a regulation has emerged so far.
Role in disease Abnormalities in TNFa or TNF-l3 expression have been implicated in several diseases. In many instances, abnormally high levels of these cytokines are seen, while in others lower than normal production is seen. An, understanding of the basis of these associations and correlation with particular diseases may be useful in elucidating pathogenesis, and expression of TNF may be used as a marker of particular diseases. This information could form the basis for both therapy and diagnosis. In the past year, data have accumulated that implicate both TNF-a and TNI-l3 in multiple sclerosis (MS). Two studies [27*,28*] reported elevated levels of TNF-a in cerebrospinal fluid (CSF) of a high proportion of MS patients especially those in active disease. TNF-a levels in CSF were found to be a more reliable indicator of clinical status than serum levels. TNI-p was not analyzed. In a histologic analysis TNFa and TNFp were both detected in MS lesions, but in different cells [29”]. In an in vitro extension of these observations, TNI-l3 was found to be a considerably more potent killer of oligodendrocytes than TNFcr 1301. On the other hand, it was suggested that TNF-cl and TNFP play only a minor role in peptide-induced experimental allergic encephalomyelitis [ 31 I, indicating that considerably more data must be accumulated before the role of these cytokines in autoimmune neurologic inllammatoty diseases is fully understood. The activity of TNF-p as an osteoclast activating factor and its elevation in serum, freshly isolated cells and long-term lines of patients with adult T-cell leukemia implicates it in the hypercalcemia that frequently accompanies this malignancy [32-l. ‘INI-cx was not analyzed in these studies, but it has previously been reported to be elevated slightly, if at all, in long-term T-cell lines from such patients.
Genetic polymorphisms The study of the association of TNI-a and TNF-fi with disease at the genetic level requires the existence of genetic markers. In the past year, considerable advances have been made in the characterization of previously described polymorphisms, and in the identification of new variants of human TNFp. .Hybridization of Nco I digested DNA with TNI-a or TNF p probes resulted in a polymorphic fragment of DNA, either 5.5 kilobases (kb) or 10.5 kb in length [33]. This polymorphism was due to a substitution of a G for an A in intron 1 of TNI-fl, giving rise to the new Nco I site [34*], Studies analyzing the association of the Nco I polymorphism with disease have produced variable results. No association of either form was found with a&losing spondylitis [35]. The 10.5 kb allele was associated with an increased frequency of insulin dependent diabetes mellitus especially when inherited with B15+, DR-4+ haplotypes [ 36**]. On the other hand, Abraham and colleagues [34*], proposed that the 8.1 kb ancestral haplotype (5.5 kb allele) is associated with autoimmune disease, especially rheumatoid arthritis. Studies have been carried out to determine whether changes in the levels of TNF-a or TNI-p are associated with either allele. Messer and colleagues [ 37**] found that phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells from individual homozygous for the 5.5 kb allele expressed higher levels of TNI-P biologic activity than individuals homozygous for the 10.5 kb allele:No differences were seen with regard to TN-a. On the other hand, Pociot et al. [36*-l reported that monocytes of individuals heterozygous for the 5.5-10.5 kb allele produce less TNF-a than individuals homozygous for the 10.5 kb allele. TNF-l!l was not analyzed: These observations, though intriguing, require considerable additional analysis before l?rm conclusions can be reached concerning the association of the Nco I polymorphism with propensity to autoimmune disease and levels- of TNF-a and TNI-l3 production. The allele with the 5.5 kb Nco I fragment also includes a substitution of an A for a C in the coding sequence of mature TNF-l3, resulting in an asparagine instead of threonine at position 26 [37**], However, it is not yet known whether this substitution affects biologic activity. A polymorphism in the 3’ region of the TNF-p gene has also been detected after digestion with Eco Rl [38]. Although this restriction fragment length polymorphism (RFIP) is seen at a low frequency and has not been associated with disease, the fact that it does not as sociate with either Nco I allele [37**] allows its future use as a genetic marker. An exciting development in the genetic analysis of TNF polymorphisms has come from the discovety of microsatellites associated with the gene complex. These are DNA sequences consisting of varying lengths of TC/GA or AC/GT repeats, they are considerably more polymorphic than the RFIPs, and provide exciting future tools
329
330
Lvmuhocvte
activation
and effector
functions
for study. Four such microsatellites have been described [3!?*]. One microsatellite, called TNFc, consists of two alleles (one containing nine TC repeats the other containing ten repeats), and is located in intron 1 of the TNF-p gene, downstream from the site of the Nco I polymorphism. Two others, TNFa and TNFb, are located 3.5 kb upstream (telomeric) of the TNI-p gene and have seven and 13 alleles [40-l respectively. The association of these polymorphisms with disease and/or levels of TNFCLor TNF-0 is yet to be established but will certainly be intriguing.
Structure that Binds to both Tumor Necrosis Factor Receptors. J Biol Cbem 1991, 266:386%3&?69. The authors present evidence for the trimeric structure of TNI-p and its association with both p55 and p75 receptors, indicating structural similarities between the two TNF forms. J, PRANGET, FIERS W: Localization VAN OSTADE2, TAVERNIER of the Active Site of Human Tumor Necrosis Factor (hTNF) by Mutational Analysis. EMBO J 1991, 4827-836. This paper highlights the importance of specilic sites at the base of the TNF-a molecule for receptor binding and emphasizes the importance of physical crosslinking of the receptors for biologic activity. 7. .
8.
LOETSCHER H, PAN YCE, IAHM HW, GEN’IZ R, BR~CKHAUSM, W: Molecular Cloning and Expression TABUCHIH, LESS~AUER
of the Human 55 kDa Tumor Necrosis Factor Receptor. Cell 1990, 61:351-359. 9.
Conclusion Data obtained in the past year have resulted in the al&mation of the biophysical similarities of TNF-ol and TNF-p and their receptor interactions. Insights into the molecular basis of the regulation of the cytokines continue to accumulate and indicate profound differences. A role for TNF-ct and TNF-p in disease pathogenesis has been suggested, particularly in the case of MS. The identification of a microsatellite polymorphism will be a useful genetic marker system in the study of disease associations.
References and recommended reading Papers of particular interest, published within the annual period of rev&v, have been highlighted as: . of special interest .. of outstanding interest 1.
JONES EY, STUARTDI, WAXER NPC: Structure Necrosis Factor. Nature 1989, 338:225-228.
2.
KR~EGLER M, PEREZC, DEFAYK, ALBERT I, Lu SD: A Novel Form of TNF/Cachectin is a Cell Surface Cytotoxic Transmembrane Protein: Ram&cations for the Complex Physiology of TNF. Cell 1988, 53:45-53.
3.
BROWNING JL, ANDROIEWICZ MJ, WARE CF: Lymphotoxin
4. ..
of Tumor
and an Associated 33 kDa Glycoprotein are Expressed on the Surface of an Activated T Cell Hybridoma J Immuno11991, 147:123&1237.
BARRETTK, TAYLOR-FISHWICK DA, COPE AP, KI%ONF,RGHIS AM, GRAY PW, FELDMANN M, FOXWELLBMJ: Cloning, Expression and Crosslinking Analysis of the Murine p55 Tumor Necrosis Factor Receptor. Eur J Immunol 1991, 21:164%1656. The authors demonstrate that human TNF-a does not bind to murine p75 TNF receptor and that a functional difTerence exists between the receptors. TNF interacts with p55 to induce killing of a WEHI 164 cell line, and p75 is associated with TNF induced proliferation of a T-cell line. 10. .
11. .
GCQDWIN RG, ANDERSONB, JERZY R, DAVIS T, BRANNAN CL,
12.
L~wrs M, TAR’IAGLIA L4, LEEAA, BENNETTGL, RICEGC, WONG GHC, CHEN EY, G~EDDEL DV: Cloning and Expression of
COPE~AND NG, JENKINSNA, SMITHCA: Molecular Cloning and Expression of the Type 1 and Type 2 Murine Receptors for Tumor Necrosis Factor. Mol Cell Biol 1991, 11:302Cb3026. The authors isolate cDNA clones for both murine TNF receptors. In contrast to the ligands whose genes are tightly linked, the receptors are encoded by genes on separate chromosomes. ..
cDNAs for Two Distinct Murine Tumor Necrosis Factor Receptors Demonstrate One Receptor is Species Specific. Proc Nat1 Acud Sci USA 1991, 88:283@2834. The authors isolated cDNAs for both murine TNF receptors. By Northern blot analysis, they demonstrated that mRNA for p75 but not p55 is expressed in a murine T-cell line, CT6. By transfection into a cell line that normally express only low amounts of TNF receptor, they demonstrated that murine p75 receptors bind murine but not human TN&X. These observations are important because they provide a molecular explanation for a previously observed species specificity in only one kind (T-cell proliferation) of biologic assay. 13.
ANDROLEWICZ MJ, BROWNING JL, WARECF: Lymphotoxin is Ex-
pressed as a Heterodimeric Complex with a Distinct 33 KD Glycoprotein on the Surface of an Activated Human T Cell Hybridoma. J Biol Cbem 1991, 267:2542-2547. The authors present definitive data that TNI-p associates early in biosynthesis with a 33 kDa protein and the complex is expressed at the cell surface. This is of special interest because it indicates that both TNF-u and TNi-b can be membrane-associated, but the biochemical basis of that association differs.
SCHAL.LTJ, LEWIS M, KOUER KJ, LEE A, RICE GC, WONG GHW, GATANAGAT, GRANGERGA, LENTZR, RAAB H, KOHR WJ, G~EDDEL DV: Molecular Cloning and Expression of a Receptor for Human Tumor Necrosis Factor. Cell 1990, 61:361-370.
SMITH CA, DAVIST, WIGNALL JM, DIN WS, FARRAH T, UPTONC, MCFADDEN G, GOODWINRG: T2 Open Reading Frame for the Shope Fibroma Virus Encodes a Soluble Form of the TNF Receptor. Biockm Biopbys Res Comm 1991, 176:335-342.
108 are Essential for Receptor Binding and Cytotoxic Activity of Tumor Necrosis Factor Beta (Lymphotoxin). Prot Eng 1991, 4~785-791. The authors present the first mutational analysis of TNF-p. The data indicate that sites homologous to TNF-a active sites are also crucial for TN&p receptor binding and biologic activity.
HOHMANN H-P, BROCKHAUS M, BAEUEFZEPA, &MY R, KOLBECK R, VANLC~NAPGM: Expression of the Types A and B Tumor Necrosis Factor (TNF) Receptors is Independently Regulated, and Both Receptors Mediate Activation of the Transcription Factor, NF-xB. J Biol Chem 1991, * 265:22403_22417. In a study of human TNF receptors, the authors demonstrate that T&e A (~7.5) TNF receptor, but not Type B (~55) expression is increa.s?d in HI60 cells by treatment of cells with dibutyric CAMP.TNF and antibody to p55 induce NFxB activity in HEp2 cells and antibody to p75 induces NFxB activity in HI&l cells. The key point of the paper is the demonstration of differences between p75 and p55 in receptor regulation while at least one of their biologic activities, NFxB induction, remains similar.
6.
SCHOENFELD HJ, POESCHLB, FREYJR, L~ETSCHER H, HUNZIKER
W, Lusnc 4 ZULAUFM: Efficient Purification of Recombinant Human Tumor Necrosis Factor 0 from Escherichia coli Yields a Biologically Active Protein with a Trimeric
15.
.
5. ..
GOH CR, LOHS-C, PORTER AG: Aspartic Acid 50 and Tyrosine
14.
.
DERREJ, KEMPER 0, CHER~F D, NOPHARY, BERGERR, WALLXH
D: The Gene for Type 1 Tumor Necrosis Factor Receptor (TNF-Rl) is Localized on Band 12~13. Hum Genet 1991, 87~231-233.
Tumor
16. .
L~ETSCHER
H, GENTE R, ZULAIIF M, Lusnc A, TARUCHI H, SCHLAEGERE-J,. BROCKHAUS M, GALLATE H, MANNEBERGM, LESSIAIIERN: Recombinant 55-kDa Tumor Necrosis Factor (TNF) Receptor. J Biol C%em 1991, 266:18324-18329. A chimeric bivalent fusion protein consisting of the extracellular portion of p55 TNF receptor and the Fc region of human Igy3 binds human TNF-a and TNFB with high affinity, prevents TNF-a and TNF-p binding to p55 and p75 TNF receptor and blocks cytotoxicity. The importance of this observation is the potential clinical advantage of this high affinity TNF receptor protein in inhibiting TNF-a or TNF~P in, for example, autoimmune disease. 17. ..
ENGLISH K, WEA\TR
WM, WILSON CB: Differential Regulation of Lymphotoxin and Tumor Necrosis Factor Genes in Human T Lymphocytes. J Biol Chem 1991, 266:710%7113. This paper demonstrates differences in TNF-a and TNFB regulation in a single population of cells, peripheral blood T cells, stimulated with concanavalin A and phorhol mytystate acetate. This study is imp portant because it partially elucidates the mechanism of the differences in the kinetics of TNF~a and TNF-B mRNA accumulation; namely the low transcription rate and high stability of TNF~B mRNA, and the high transcription rate and low stability of TNF-a mRNA.
18.
FERRERJNR. SARRT, &KENASE PW, RUDDU? NR: Molecular Regulation of Tumor Necrosis Factor-a and Lymphotoxin Production in T CeIIs: Inhibition by Prostaglandin E2. J Rio1 Cbem 1992, 267:9443-9449.
19.
DRO~JET C, SHAKHOV AN, JONGENEEI. CV: Enhancers and Transcription Factors Controlling Inducibility of the Tumor Necrosis Factor-u Promoter in Primary Macrophages. .I Immunol 1991, 147:1694-1700.
20. .
GOLDFED AF, STROMINCER JL, DOYIE C: Human Tumor Necrosis Factor CLGene Regulation in Phorbol Ester Stimulated ’I T and B CeII Lines. J Exp Med 1991, 174:73-81. This paper is of interest because it disagrees with other work that had emphasized the importance of the 5’ NFxB sites in the regulation of TNF-a in macrophages and suggests that the mechanism of TNF-a regulation in lymphocytes may involve different regulatory sequences.
21. .
KRUYS V, KEMMERK, SHAKHOVA, JONGENEEL V, BEUTLER B: Constitutive Activity of the Tumor Necrosis Factor Promoter is CanceIIed by the 3’ Untranslated Region in Nonmacrophage CeII Lines; a Trans-dominant Factor Overcomes This Suppressive Effect. Proc Natl Acad Sci USA 1992, 89:673477. Data are presented that underscore the complexity of TNF-a regulation, and suggest negative regulation of promoter activity by non-coding 3’ sequences. 22. ..
KEFFERJ, PROBER?‘L, CAZLARISH, GEORGOPOIILOUSS, K~~IARIS E, KIOUSSISP, K0l.l.14~ G: Transgenic Mice Expressing Tumor Necrosis Factor: a Predictive Genetic Model of Arthritis. z!34BO J 1991, 10:40254031. This paper is the first study to demonstrate that inappropriate expression of TNF-a in a transgenic mouse system results in an identifiable pathology. The data also appear to confirm the importance of 3’ sequences in regulation of TNF~u production. The use of a human TNF~a construct facilitates detection of the transgene although the effects of the transgene are limited to cells expressing the p55 receptor (see also l10*,12**1). 23.
FASHENASJ, TANG W-L, SARR T, RUDDLIZNH: The Murine Lymphotoxin Gene Promoter. Characterization and Negative Regulation. J Immunol 1990, 145:177-183.
24.
PAUL NL, LENAFXX) MJ, NOVAK KD, SARRT, TANG W-L, RUDDLE NH: Lymphotoxin Activation by Human T-cell Leukemia Virus Type I-infected Cell Lines: Role for NK-xB. J Viral 1990, 64:5412.
25.
26. ..
MESSERGE, WEISS H, BAFXIERLE PA: Tumor Necrosis Factor b (TNF-p), Induces Binding of the NF-xB Transcription Factor to a High-affinity xB Element in the TNFj3 Promoter. Cytokine 1990, 2:389. FA~HENA SJ, REEVES R, RUDDLE NH: A Poly (dA-dT) Upstream Activating Sequence Binds High Mobility Group I Protein
necrosis
factor
(TNF-09 and lymphotoxin
(TNF-0)
Ruddle
and Contributes to Lymphotoxin (Tumor Necrosis Factorp) Gene Regulation. Mol Cell Biol 1992, 12:894-903. The importance of this paper is the demonstration of a new regulatory element in the TNF-P gene that appears to function in at least one type of celI (pre B cells) which produces TNFp mRNA constitutively. This element is bound by a protein, HMG-1, that acts by stabilizing chromatin structure rather than by enhancing transcription directly. 27. .
TSUKADA
N, MNAGI K, MATSUDA M, YANAGISAWA N, YONE K: Tumor Necrosis Factor and Interleukin-1 in GF and Sera of Patients with Multiple Sclerosis. J New-01 Sci 1991, 102:23&234. The authors use a sensitive enzyme linked immunosorbent assay assay to detect TNFa in CSF of MS patients; detection of TNFu correlates with exacerbation. Lower levels were found in sera. No interleukinla or interleukin-lp were detected. This paper is important because it suggests a correlation of TNF levels with a clinical state and it em phzsizes the importance of analyzing a source of material (CSF) other than serum. 28. .
SHAIUEI:
MK, HENTGESR: Association Between Tumor Necrosis Factor-u and Disease Progression in Multiple Sclerosis. Ntru’En@ J Med 1991, 325i467-472. This paper is important because it reports higher levels of TNF-a in CSF of patients with chronic progressive MS, compared with levels in patients with stable disease. It also presents evidence for a role of TNFa in either MS disease pathogenesis, or at least its usefulness as a marker of the disease state. SEIMAJ K, RAINE CS, CANNELLA B, BROSNAN CF: Identification of Lymphotoxin and Tumor Necrosis Factor in Multiple Sclerosis Lesions. J Clin Invest 1991, 87:94+954. This paper is important because it demonstrates the production of TNF a and TNF~j3 by different cells in MS lesions. TNF-p was found in CD3+ lymphocytes and Leu M5+ microgial cells at the edge of the lesion, whereti TNFa was found in zxrocytes and macrophages. 29. ..
30.
SELM.$JK, RAINE CF, FAROCQ M, NORTON WT, BROSNAN CF: Cytokine Cytotoxicity Against OIIgodendrocytes. Apoptosis Induced by Lymphotoxin. .I Immunol 1991, 147:1522-1529.
31.
MEIUULLJE, KONO DH, CLAYTONJ, ANW DG, HINTON DR, HOFMAN FM: Intlammatory Leukocytes and Cytokines in the Peptide-induced Disease of Experimental AIIergic Encephalomyelitis in §JL and BlO.PL Mice. Proc Natl Acad Sci USA 1992, 89~576578. Y,
OTSUKA
M,
KWAZURU
ISH~RASHIK, ‘IsBrnu~~
.
Y,
33.
WERDGC, CHAKINDD: Genetic Variability at the Human Tumor Necrosis Factor Loci. J Immunol 1990, 145:127%1285.
IWAHASHI M,
K,
CH~MON
32.
UTSUNOMNA
A,
HANDA
S, SAKURAMIT, ARIMA T: Tumor Necrosis Factor-p in the Serum of Adult T CeU Leukemia with HypercaIcemia. Blood 1991, f7:2451-2455. This paper indicates that levels of TNl-b are elevated in the serum of patien& with a hone lesion, implicating cytokine with known osteoclast activating activity in the pathogenesis of the syndrome.
ABRAIW LJ, Du DC, ~~EIX K, DAWKINSRL, WHITEHEADAS: Haplotypic Polymorphisms of the TNF-!3 Gene. Immune genetics 1991, 33:50-53. The authors characterize polymorphisms of the TNF~P gene by sequence analysis and correlate them with ancestral haplotypes. They demonstrate Linkage between a sequence difference in intron 1 of TNFfi and a new Nco I site. Another polymorphism results in an amino acid substitution at position 26 in TNF~P. This study is important because it defines the molecular basis of the polymorphisms, and associates these with known haplotypes. 34. .
35.
VERJANSGMGM, VAN DER LINDENSM, VAN EYS GJJM, DE WAAL LP, KIJLSTRAA: Restriction Fragment Length Polymorphism of the Tumor Necrosis Factor Region in Patients with Ankylosing Spondytitis. Arthritis Rbeum 1991, 34:486-489.
36.
POCIO’I’ F,
. .
H,
BAEK
L,
MOLVIG NERUP
J,
WAGENSEN
L,
WORSAAI.
H,
DAL~OYE
L: Tumor Necrosis Factor Beta Gene Polymorphism in Relation to Monokine Secretion and Insulin Dependent Diabetes Mellitus. Scund J Immunol 1991, 33:37-49.
331
332
LvmDhocvte activation and effector functions
This study is important because it suggests an association of a particular TNF-p allele (10.5 kb RFLF’defined by Nco I that maps to intron 1 of TNF-P) with a disease. The increased production of TNF-a by monocytes in such individuals is intriguing with regard to a possible mechanism of disease pathogenesis. MESSERG, SPAXLER U, JUNG MC, HONOU) G, BLAMER K, P.WE GR, RIE~MKJLLER G, WEISS EH: Polymorphic Structure of the Tumor Necrosis Factor (TNF) Locus: An Nco I Polymorphism in the Fist Intron of the Human TNF-p Gene Correlates with a Variant Amino Acid in Position 26 and a Reduced Level of TNF-!3 Production. J E3cp Med 1991, 173:20?219. The authors localized the Nco I polymorphism to intron 1 of TNF-p, and suggest that individuals homozygous for the a 5.3 kb Nco I fmgment produce higher levels of TNF-P than individuals homozygous for the 10.5 kb allele after stimulation of peripheral blood cells with PHA. TNF-alpha levels were not significantly different. This paper is important because it associates differences in TNF levels with different alleles, and the genetic linkage of the 5.5 kb and 10.5 kb Nco I site with an asp paragine and a threonine in position 26 of the mature TNF-P protein, respectively. 37. . .
38.
PAR~ANEN J, KOSKIMIES S: Low Degree of DNA Polymorphism in the HLAlinked Lymphotoxin (Tumor Necrosis Factor-o) Gene. Stand J Immunol 1988, 28:313-316.
SA, ~JDALOVA L4, KUPRGH DV, TURETXAYA RL: DNA Sequence Polymorphism at the Human Tumor Necrosis Factor (TNF) Locus. J Immunol 1991, 147:1053-1059. The authors detect sequence polymorphisms that mark the TNF complex. These observations are important because the polymorphism is so extensive (one form has 13 alleles) it will be more useful than the previously described bi-allelic variants.
39.
NEMXPAWV
. .
40.
JONGENEEL
.
SA, CAMBON-THOMSEN
CV, BRL&N~L, UDAXWO rq SEWN A, NEDOSPASOV A: Extensive Genetic Polymorphism in the Human Tumor Necrosis Factor Region and Relation to Extended HLA Haplotypes. Proc Natl Acad Sci ZJSA 1991, g&9717-9721. The authors demonstrate the relationship between extensive micro satellite polymorphisms and HL4 haplotypes, indicating their potential usefulness as genetic markers of HIA associated diseases.
NH Ruddle, Department of Epidemiology and Public Health, Yale LJni~ versity School of Medicine, 60 College Street, PO Box 3333, New Haven, Connecticut, US.4
