Interleukin 1, interleukin 6 and tumor necrosis factor in infection, inflammation and immunity

Immunology Letters, 19 (1988) 183-192 Elsevier IML01111

Interleukin 1, interleukin 6 and tumor necrosis factor in infection, inflammation and immunity Klaus Bendtzen Laboratory of Medical Immunology 7544, Rigshospitalet University Hospital, Copenhagen, Denmark (Received 30 June 1988; accepted 16 August 1988)

1. Introduction Many signs of infectious diseases evolve as a result of stimulation of cellular immunity, and abnormal regulation of cellular immune reactions may lead to autoimmunity and even to chronic diseases. These include systemic rheumatic diseases such as rheumatoid arthritis (RA) and organ-specific endocrine diseases, for example insulin-dependent diabetes mellitus (IDDM) and various thyroid diseases. Disorders of cell-mediated immunity may also contribute to the development of multiple sclerosis, coeliac disease, active chronic hepatitis, and some fibrotic skin-, liver- and lung diseases. T lymphocytes govern the induction and regulation of cell-mediated immune reactions, and proteins and glycoproteins produced by T lymphocytes (lymphokines) initiate and control the immune response [1, 2]. However, antigen activation of T cells requires physical interaction with antigenpresenting cells, and these cells also produce mediator molecules crucial for T cell activation and the subsequent triggering of antibody-producing B lymphocytes/plasma cells. These lymphocyte-activating mediators together with the lymphokines are collectively termed cytokines. Hence, cytokines are essential transmitters of cell-to-cell communication in both physio-

Key words: Review; Macrophage; Cytokine; Interleukins; Tumour necrosis factor; Gene structure; Autoimmune disease

Correspondence to: K. Bendtzen, MD, Lab. Med. Immunol. 7544, Rigshospitalet University Hospital, 20 Tagensvej, DK-2200 Copenhagen N, Denmark.

logical and pathophysiological immune processes. In many cases they also function as hormones providing information between the immune system and other tissues and organs. They are active at extremely low concentrations (10-z° to 10-15 M) via binding to specific high affinity receptors on a large number of target cells. Most cytokines probably act in the vicinity of the production site, but some of the mediators modulate functions of cells at distant sites via blood and lymph circulation. Fig. 1 shows the dynamics of production and function of cytokines during an immune reaction in a vascularized tissue. This review will focus on the increasing evidence for the involvement of macrophages (M~) and their polypeptide mediators in infectious and inflammatory processes. Usually, these processes serve to successfully eliminate invading microorganisms from the host. However, an inappropriate production or function of the M~) cytokines may contribute to the development of structural and/or functional damage to cells and tissues.

2. Macrophages Phylogenetically, M(~ are old cells - more than 500 million years old. They play a central role in the defence against microbial and neoplastic diseases, and they are important in a wide variety of tissue repair processes. Some M(~ functions are carried out continuously, such as removal of aged erythrocytes. Other functions are performed periodically, and these functions need prior activation of the M(~. The cells may be activated directly, for instance by contact with a microorganism or its products, such as endotoxins

0165-2478 / 88 / $ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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characterized human M~b hormones. They are all members of a growing family of immune cytokines which includes the IFNs and some hemopoietic growth factors. The M4~ cytokines are also produced during conditions o f 'stress', and they are important mediators of fever, the acute-phase reaction, and the generation of cachexia as a result of chronic infectious, inflammatory and neoplastic diseases. 2.2. Production of lL-L IL-6 and TNFet

Fig. 1. Cellularinteractionsand cytokineproduction during antigen presentation to T and B lymphocytes. The cytokines produced by both immune and non-immune ceils may enter the blood stream and as hormones give rise to effects in distant tissues. Modifiedfrom [2] with permissionfrom HarwoodAcademic Publishers, GmbH. M~, macrophage; Th, T helper lymphocyte; B, B lymphocyte;F, fibroblast; NK, natural killer cell; DR, MHC class II molecule;IL-2, interleukin2; IL-I IF, interleukin 1-inducing factor (= IL-67).

(lipopolysaccharides (LPS)) and other cell wall components. This direct means o f M(~ activation is in higher animals improved considerably by a more indirect form of activation, via lymphokines [1, 2]. Thus, interferons (IFN) -et and -3' are important activators of M4~, but other M~-activating lymphokines exist. Phagocytosis itself and, perhaps more importantly, immune- or lymphokine-mediated activation trigger the M(~ to synthesize and release a number of biologically active molecules, including prostaglandins, proteolytic enzymes, complement components, etc. [1-3]. Four o f these factors, interleukin let (IL-let), interleukin 1/3 (IL-1/3), interleukin 6 (IL-6), and tumor necrosis factor-et (TNFet), have far-reaching biological and pathophysiological significance [2-6].

2.1. Macrophage-derived polypeptide mediators Tables 1 and 2 summarize the biology of the best184

IL-1, IL-6 and TNFet belong to a small group of polypeptides o f M r 1 7 - 24 kDa produced primarily by activated M~b. However, the capacity to elaborate IL-1 and IL-6, in particular, is not confined to Mq~s (Table 1). NK cells and B lymphocytes are already known to produce IL-1, and fibroblasts were the ceils originally shown to produce IL-6 (= IFN/32 [6, 7]. In fact, all nucleated cells may perhaps produce these peptides, if the proper stimulatory signal is provided. Nevertheless, cells o f the Mq~ lineage appear to be the quantitatively most important. Thus, IL-1/3 mRNA in activated human M~s may constitute up to 5070 o f total mRNA; approximately 1°70 may be TNFet mRNA [3, 4]. The liberated amounts o f the peptides depend upon the stimulus, because transcription of the genes - at least those for IL-1 and TNFet - is not necessarily followed by translation and secretion o f the hormones [4]. TNFet is produced almost exclusively by Mq~, but appropriately stimulated T lymphocytes may also elaborate this cytokine. TNFet is genetically and structurally closely related to a T lymphocytederived cytokine, TNF~, also termed lymphotoxin [8, 9]. Both species of TNF are of Mr 17 kDa, but TNF/~ in particular tends to aggregate to higher molecular weight forms. IL-6 is produced by many different types of cells both constitutively and as a result o f stimulation with various stimuli, including nonspecific lymphoid mitogens, various activators o f monocytes, including some cytokines (Table 1) [6]. The primary translation products of the IL-1, IL6 and TNFet genes contain single-chain polypeptides consisting of 271 (IL-let), 269 (IL-1/~), 212 (IL-6), and 233 (TNFet) amino acids with mw's about 30 kDa (Fig. 2) [3, 6, 8, 10-13]. Expression in E. coli o f the C-terminal 169 (IL-let), 153 (IL-lfl), 184 (IL-6), or 157 (TNFet) amino acids results in peptides of

TABLE 1 The best-characterized human macrophage cytokines. Acronym

Principalsources

Inducers

IL-lc~/~ (= LAF)

monocytes/macrophages NK cells B cells (and T cells?) dendritic cells Langerhans' cells (skin) keratinocytes? endothelial cells astrocytes synovial cells smooth muscle cells mesangial ceils melanoma cells monocytes/macrophages keratinocytes T cells? various cancer cells monocytes/macrophages T and B cells fibroblasts endothelialcells keratinocytes myxoma cells synovial cells carcinomas osteosarcomas multiple myelomacells

LPS staphylococcal exotoxins Klebsiella glycoproteins phorbol esters calcium ionophores immune complexes, C3a and C5a proteoglycans collagens II, IX urate crystals trauma, adherence, phagocytosis TNFa (IFNy decreases IL-1 production)

TNFa

IL-6 (= IFNB2) (= BSF-2) (= H P G F ) ( = BCDF)

LPS phorbol esters IFNy LPS polyclonal mitogens phorbol esters Staph. aureus Cowan I UV-irradiation cytokines (IL-1, TNF, IFNBI, PDGF) poly (I) (C) calcium ionophores cAMP and cAMP-generatingagents

Abbreviations: IL, interleukin; LAF, lymphocyte-activatingfactor; NK, natural killer cell; TNF, tumor necrosis factor; IFN, interferon; BSF, B cell-stimulatoryfactor; HPGF, hybridoma/plasmacytomagrowthfactor; BCDF, Bcell differentiation factor; LPS, lipopolysaccharide.

17-21 kDa with retained biological activities. It is assumed that IL- 1ct and -/3, and TNF~, are synthesized and transported to the cell membrane as larger precursor molecules. Because these precursors lack an amino acid signal sequence, they are probably not secreted by mechanisms considered characteristic o f secretory peptides. It is believed that the active peptide fragments o f IL-lot and -/3, andTNFot are generated at the cell surface by limited proteolysis. In contrast, the IL-6 precursor contains a hydrophobic signal sequence o f 28 amino acids (Fig. 2). The genes for TNFot and TNF/3 are both located in the class III region o f the major histocompatibility complex (MHC), i.e. the H L A region in man (Fig. 2) [8-14]. Recent studies in our laboratories have shown that alleles within the H L A - D R region

may control the production and/or the secretion o f T N F a [14a]. Blood mononuclear cells obtained from H L A - D R 2 positive individuals show a significantly diminished TNFot response in vitro after challenge with LPS. Interestingly, this diminished response was normalized by preincubation o f the cells with rlFN% When testing for the production o f IL-loL and IL-1/~ under similar conditions, we were unable to correlate the magnitudes of these responses to any specific H L A type. In this case, preactivation with rlFN3, significantly diminished the IL-1 responses to LPS. So, the production of T N F ~ but not that o f IL-lct and fl, seems to be governed by genes in the H L A - D R region, and opposite effects on Mq~ cytokine production are exerted by IFN3,.

185

TABLE 2 Functions of IL-1, IL-6 and TNFc~ which m a y be important in infection and immunity. Ibl

I~6

TNFa

+ + + +

++ ++ +

+ ++ ++

+

+

++

+ + + + + + + +

+

++ ++

+ ++ + +

Cytostatic/cytotoxic in vitro to: pancreatic islet/3-cells (activates at low concentration) thyrocytes (activates at low concentration)

+ +

-/+

-

Induction in vivo of." fever (via hypothalamus), slow-wave sleep, anorexia wasting (muscle protein and fat degradation) acute-phase-protein synthesis reduction in blood Zn 2+ , Fe 2+ and albumin, and elevation in blood Cu 2+ reduction in hepatocyte cytochrome p450 activity A C T H release leukocytosis (immature neutrophils from bone marrow stores) lymphopenia increased insulin release resistance against infections a n d irradiation hypotension capillary leakage

++ + + + + + + + + + + +

+

++ ++ +

Activation in vitro of." T, B lymphocytes and NK cells monocytes (production of thromboxane, PGE2, colony-stimulating factors, IL-1) neutrophils (adherence, degranulation, t h r o m b o x a n e and superoxide production) endothelial cells (production of IL-I, IL-6, PGI2, procoagulant-, leukocyte adhesion-, and class I M H C molecules) chondrocytes (production of collagenase, plasminogen, PGE2, and decrease of cartilage proteoglycan) osteoclasts (via osteoblasts?) fibroblasts (growth, production of PGE2 and collagenase) hepatocytes (acute-phase-protein production, and decreased production of albumin) nerve cells

-

++

++

+ + ++ ++

Abbreviations: M H C , major histocompatibility complex. A C T H , adrenocorticotropic hormone; PG, prostaglandin.

i

115 AA

I

15~ nA

I

IL-6

m..7 28

T,F,

1

164 AI~ ,

t AA

t

'I I"-76

Chr'.6 AA

I

157

All

I Ill OP DO DR

I

III B C •

2.3. Receptors for IL-1, IL-6 and TNFo~ and lym-

phocyte activation

sequences coding the Mature protein

Fig. 2. C h r o m o s o m a l localization and structure o f the genes encoding IL-1/3, IL-6 and TNFo~ The m R N A ' s of the mature horm o n e s (solid bars), and the putative promoter sequences are also shown [8, 10-13]. The M r of the mature proteins are: IL - I a and -t3, 17.5 kDa; IL6, 24 kDa (glycosylated); TNFo~ 17 kD.

186

The genomic structure of human IL-6, as well as the amino acid sequence of the mature protein, show slight but significant homology with granulocytecolony stimulating factor (G-CSF). Furthermore, the positions of four cysteine residues match those of GCSF, suggesting a similarity also in the tertiary structure of the two cytokines [6, 12].

Receptors for IL-1, IL-6 and TNFo~ have been found on most of the cell types known to respond to the cytokines, and there is evidence for the presence of both high and low affinity IL-1 and IL-6 receptors [6, 15]. The receptor for human IL-1 on

murine EL4 thymoma cells has recently been shown to be a membrane-associated glycopeptide o f M r 82 kDa [16]. This receptor binds both the mature forms of I L - l a and IL-1/i Interestingly, it is also able to bind the precursor o f IL-lo~ but not that o f IL-1/3 [17]. This may explain why cell-associated 30 kDa I L - l a , probably identical with the IL-lot precursor, may be particularly efficacious in T cell activation [18]. It is also potentially interesting that both I L - l a and IL-1B are capable of down-regulating the expression o f the CD4 structure on human T cells [19]. The CD4 structure is directly engaged in antigen presentation, possibly through interaction with M H C class II molecules on antigen-presenting cells [20]. One may therefore speculate that the IL-1 receptor, binding to membrane-associated IL-1 a on the antigen-presenting cell, is physically or functionally related to the CD4 molecule (Fig. 3). It may prove clinically important that interference with the IL- 1-mediated 'second signal' for T cell activation may lead to antigen-specific tolerance. Thus, triggering the T cell receptor by a properly processed antigen, while preventing the ensuing ILl-induced activation o f these specifically reactive cells, will prevent the clone from once again responding to the same antigen in vitro [21] and in vivo [22]. The molecular characterization o f the human IL6 receptor has not yet been performed. However, the

HLA-DR

T cell receptor

-~

complete receptor may include two peptide chains [61. The TNFot receptor(s) is identical to, or closely related with, the receptor for the lymphocytederived TNF~ [4, 9, 23]. The receptor appears to be composed o f four cellular, non-covalently linked proteins (M r 138, 90, 75 and 54 kDa) [231. These may act independently or as multiple subunits, one of which (138 kDa) may be selectively responsible for the cytotoxic action of TNFu. 2.4. Other functions of IL-L 1L-6 and TNF~ IL-1, IL-6 and TNFct are pleiotropic in that they exert multiple functions on many different cell types (Table 2). Fever as a symptom has been known for thousands of years. It is mentioned in the Old Testament as a punishment inflicted by God, and Hippocrates (400 B.C.) knew that fever was a symptom o f disease. He even described fever patterns in various diseases long before the thermometer was invented by Wunderlich only a century ago! It has become clear that IL-1 is identical with the factor(s) originally described as endogenous or leukocytic pyrogen; i.e., hormone(s) of leukocytic origin which produced fever [2, 3]. Later, it has become evident that in addition to IL-I, TNFct and IL-6 are also potent endogenous pyrogens. However, it is not entirely clear whether all these cytokines act in a similar fashion in the brain during fever induction. The pathophysiology of fever is shown in Fig. 4. It is noteworthy that prostaglandin E 2 is a second mediator o f IL-1 and, possibly, TNFc~ and IL-6 in the thermoregulatory center of the brain. Thus, cyclooxygenase inhibitors such as aspirin ameliorate

IL-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

r

. . . . . . . . . . . . . . . . .

P a t h o p h y s i o l o g y of fever ................................................................ M ~ c r o o r g a n i s m s and m i c r o b i a l products (toxins) Products of i n f l a m m a t i o n and tissue i n j u r y Cytokines A n t i g e n - a n t i b o d y complexes

C

P r o d u c t i o n and

• Antigen Fig. 3. Model for the molecular interactions during antigen presentation to T lymphocytes.Membrane-boundIL-la (= ILlct precursor) and soluble IL-lfl both bind to the putative IL-l receptor(s), which may be physically and/or functionally associated with CD4.

I lineage I r e l e a s e to the blood of IL-I, IL-6 and I of p r o s t a g l a n d i n E in the h y p o t h a l a m u s I

A c t i v a t i o n of cells of the m a c r o p h a g e s

Induction

TNF

Muscle c o n t r a c t i o n Blood flow r e a r r a n g e m e n t and t a c h y c a r d i a

I

Fever ...............................................................

Fig. 4. 187

............................................................... Fever and loss of w e i g h t Muscle protein degradation Inductlon of sleep and loss of appetite D e m l n e r a l i z a t i o n of bone Blood leukocytosls

Decreased blood levels of iron and zinc Increased blood levels of copper Activation of hepatocytes (elevation of a c u t e - p h a s e proteins, and s e d i m e n t a t i o n rate) (decreased b l o o d level of albumin) Immune s t i m u l a t i o n ...............................................................

Fig. 5.

the pyrogenic action of IL-I, even though the hormone is still present in the blood. Other functions of IL-1 that do not require prostaglandins as second messengers are therefore intact. This explains why aspirin may lower the temperature o f a febrile patient without reducing the function o f his immune system, his tendency to sleep, or his acute-phase response. Fever is often part of the acute-phase reaction usually seen in conjunction with acute and chronic infectious and inflammatory diseases, and in cancer (Fig. 5) [2-6, 24]. IL-1 and IL-6, and to a lesser extent TNF~, induce hepatocytes to synthesize acutephase proteins, including serum amyloid A, Creactive protein, fibrinogen, haptoglobin, complement components and clotting factors. The elevated level o f fibrinogen, especially if accompanied by anemia, causes an increased erythrocyte sedimentation rate, a commonly used clinical parameter of 'inflammation'. Since IL-1 and TNFo~ are potent inducers of IL-6 in many cell types, including hepatocytes, it is possible that IL-6 is a second mediator o f the acute-phase response elicited by IL-I and TNFoL The above-mentioned clinical picture is often associated with disturbances in carbohydrate-, lipidand protein metabolism eventually resulting in wasting (cachexia). In rare situations, clotting abnormalities and shock may occur. It was previously thought that microbial products such as LPS were directly responsible for these symptoms if triggered by bacterial infection. This is now known to be incorrect, because LPS is a potent inducer of MO IL-1, IL-6 and T N F ~ and all pathophysiological processes associated with LPS188

induced shock can be reproduced by injection o f T N F a and to a lesser extent by IL- 1 [2-4]. Moreover, LPS does not induce shock in the endotoxinresistant C 3 H / H e J mouse. In these animals, a mutation decreases transcription of the LPS-induced TNFo~ gene and thus prevents the production of the hormone [4]. TNFa, therefore, appears to be centrally involved in LPS-induced shock. TNFc~ is probably also responsible for other phenomena associated with LPS-induced disease, such as metabolic acidosis, disseminated intravascular coagulation and cachexia (cachectin) [2, 4]. It is likely that some of these phenomena are mediated through TNFa-induced production of IL-1 or IL-6, or one or several other (unknown) hormones. TNFo~ has been identified as the mediator of the cytotoxic activity in serum of mice infected with mycobacteria and subsequently injected with LPS [25]. Certain tumor cells are highly susceptible to the toxic activity of TNF in vitro and in vivo, whereas normal cells usually survive treatment with the hormone. TNFa, or a TNFa-like cytokine(s), also appears to be involved in the defence against parasitic diseases such as malaria, most likely by its ability to activate neutrophils and eosinophils and/or by a direct effect on the hepatic stage of the disease [26]. IL-6 has been shown to be identical with B cellstimulating factor 2 [12] and a factor necessary for the growth of plasmacytomas and B cell hybridomas [7, 27]. The lack of IL-6 receptors on resting B cells [6] agrees with the fact that IL-6 alone fails to trigger B cell growth and differentiation. However, the receptors appear on activated B cells, and the cells are now able to respond to IL-6. IL-6 is also a cofactor in T cell activation, probably because it induces IL-2 receptors and functions as a second signal for IL-2 production [6]. It is therefore not surprising that IL-6 substitutes for IL-1 in the 'classical' mouse thymocyte costimulatory assay for IL-1 (Bendtzen K., unpublished data). Unregulated production of IL-6 may be pathogeneticaIly involved in the manifestation of several human diseases, including cardiac myxoma and various lymphoid malignancies such as multiple myelomas and various T and B cell lymphomas [6]. It is interesting that myeloma cells constitutively produce IL-6 and express IL-6 receptors, and that the proliferation of myeloma cells in vitro is inhibited by specific antibodies to human IL-6 [28]. Hence,

a dysregulated production and function of IL-6 may be a key event in the pathogenesis of at least some lymphoid proliferative diseases. IL-6, and possibly TNF~ may also be involved in other proliferative disorders. Using specific antibodies to human rTNFot and human rlL-6, we have recently demonstrated TNFot and IL-6 in skin biopsies [29, 29a]. The expression of both mediators is considerably augmented by UV-irradiation, and large amounts of IL-6 and TNFc~ may be found in psoriatic lesions. Interestingly, local treatment of the psoriatic lesions with a vitamin D 3 analogue results in clinical improvement, and biopsies performed after treatment showed unaltered expression TNF~ but an almost complete elimination of the expression of IL-6 in the upper epidermal layers [29a].

seen from in vivo animal experiments using recombinant IL-1 (rIL-1). Thus, human rIL-13 induces MO and neutrophil accumulation 24 h after injection, and this is associated with depletion of proteoglycan from the articular cartilage and an increase in the glycosaminoglycan content of the joint fluid [31]. The same signs of cartilage damage by ILl are seen in joints of rabbits previously depleted of polymorphonuclear leukocytes and Mq~ by systemic administration of nitrogen mustard [31]. This suggests that IL-1 itself is capable of stimulating resident cells of the joint, such as the chondrocytes, to cause proteoglycan depletion. Again, it is possible that some of the effects ascribed to IL-l may be mediated via local, IL-1-induced production of other cytokines, particularly IL-6.

3. Rheumatic diseases

4. A u t o i m m u n e endocrine diseases

IL-1, IL-6 and TNFa have many biological effects which qualify them as potentially important in the pathology leading to rheumatic diseases (Table 2) [2]. The most relevant actions of IL-lc~ and -3 might be their stimulatory effects on T and B cells (nonspecific production of inflammatory lymphokines and immunoglobulins), M4~ (e.g. MHC class II antigen expression and production of eicosanoids), chondrocytes (production of collagen type II), fibroblasts (production of collagen types I and II and eicosanoids), and osteoclasts (bone resorption). TNFot and IL-6 share many of these effects with ILl, although IL-6 does not cause bone resorption (Pedersen J. G. and Bendtzen, K, unpublished data). Several potential cellular sources of the cytokines are present in the arthritic joint, including M~, B lymphocytes, fibroblasts and vascular endothelial cells (see Table 1). The demonstration of IL-1 in synovial fluids from rheumatoid and osteoarthritic joints further suggests that IL-1 in particular may play an important role in the pathogenesis of arthritis [2, 30]. An unbalanced reaction between immunocompetent cells and accessory cells such as M~b, NK cells and polymorphonuclear leukocytes might continually induce IL-1 and other inflammatory substances, for example eicosanoids derived from both the cyclooxygenase and the lipoxygenase pathways. This would contribute to the chronic nature of diseases such as RA. The arthritogenic activity of IL-I is most clearly

Recently, the predominant species of human IL- 1, IL-13, was shown to be a potent suppressor of insulin production in vitro, possibly as a result of a direct and selective cytotoxic effect on pancreatic islet 3cells [32]. IL-la also reduces insulin production, but it is 10 times less potent than IL-13; in addition, ILl o~ is produced in much smaller quantities [2]. An increased insulin production is consistently observed at low concentrations of IL-13 (10-200 pg/ml = 5 x I0-13-10TM M), and although TNFt~ itself fails to affect B-cell function, the suppressive effect of IL- 13 is augmented significantly by TNFa [2, 32]. It is potentially interesting that IL-6 augments the release of insulin from normal rat islets (Buschard, K., Aaen, K. and Bentzen, K., manuscript in prep.). Thus, IL-6 may be able to protect islet B-cells against IL-1/3-induced damage. Alternatively, IL-6 may stimulate B-cells in a manner leading to cell damage and leakage of insulin. Local, IL-l-induced production of IL-6 may thus underly the biphasic insulin response observed with various concentrations of IL-1 [32]. Similar effects to those described above have been obtained when testing IL-13 on human thyroid cells removed during surgery of paraadenomatous glands [33]. The secretion of thyroglobulin and cAMP is markedly suppressed even by low concentrations of rlL-13 (15 pg/ml = 1 0 -12 M). The effect is augmented by TNFc~ and, in addition, by IFN-3,. The thyrocytes are not killed by IL-13, but their ability 189

to form follicles and accumulate glycogen in response to thyroid stimulating hormone is suppressed [34]. Once more, a stimulatory effect on thyroglobulin production is consistently observed at very low concentrations of IL-1/3 (1.5-150 fg/ml -10-16-10 -14 M). The above findings and considerations indicate a central role of M~b and, perhaps, NK cells and their products IL-1, TNFet and IL-6 in a number of autoimmune diseases [2]. Even endothelial cells, fibroblasts and other cells in the connective tissue could be involved through their production of cytokines such as IL-1 and IL-6. Moreover, low concentrations of IL-I and IL-6 may accumulate in target tissues by diffusion from the blood during conditions of stress, and these cytokines may therefore fulfill important beneficial functions by potentiating the secretion of insulin, thyroid and pituitary hormones under these circumstances (Fig. 1). The magnitude o f the autoimmune reactions leading to disease would be expected to depend upon the M H C class II types of the individual, and this therefore might account for the M H C association of many autoimmune disorders. However, the fact that the TNFet genes, as well as the TNF/3 genes, are located in the M H C class III region (Fig. 2) suggests that if the T N F genes are polymorphic, the diseaseassociated H L A types may be linked to allele(s) for the T N F ' s coding for an inappropriate secretion of TNFa/~. Another possibility would be that regulatory genes (for example repressor genes) may be involved in the expression o f the TNFct gene or the processes leading to the release of TNFa. This is supported by the fact that the gene seems to be controlled by short-lived repressors [35]. Also, low TNFc~ production is associated with an H L A - D R rather than an HLA-B type [14a]. Therefore, the association is probably not due to a variant of the promoter for TNFct, because the TNFct locus is closer to the HLA-B region [14] and, consequently, a stronger linkage disequilibrium would be expected between HLA-B and allelic variants in or around the TNFot gene. The negative association between H L A - D R 2 and the production of TNFot may be implicated in some HLA-associated diseases such as IDDM and RA [2]. In the latter two, H L A - D R 2 is associated with resistance to disease, and this might be explained if TNFo~ is directly or indirectly involved in the destruction of 190

islet/3-cells in IDDM and of cartilage and bone in RA. However, it is still not clear whether TNFtx, or IL-1 and IL-6 induced by TNFot, play a role in the development of these diseases as well as in multiple sclerosis and narcolepsy, both positively associated with HLA-DR2. 5. Therapeutic considerations Systemic administration of IL-1, IL-6 or TNFo~ causes fever, malaise and hypotension. In addition, TNFct may elicit disseminated coagulation and even shock. Since it has not yet been possible to relate specific functions to distinct sequences of the mature peptides, systemic administration of the hormones is not generally acceptable. Even so, several phase I clinical trials of systemically administered T N F a in cancer patients have been started, together with local treatment of solitary tumors. Considering the many putative pathophysiological functions of IL-1, IL-6 and TNFa, intervention with the production or action of the hormones might be of great benefit. The production of the mediators is prevented by high doses of glucocorticoids, which directly suppress gene transcription and, if the genes have been transcribed, the mobilization o f mRNA, at least in the case of IL- 1 and TNFot [4]. This is probably the reason why glucocorticoids given to an animal before injection of LPS prevents the development of shock. If administered after the translation process has started, glucocorticoids are usually much less effective. The effect of IL-1 on T cells in vitro is prevented by cyclosporin [36], and by vitamin D 3 (1,25 [OH]2 D3) and a synthetic vitamin D 3 analogue [37]. Whether cyclosporin and vitamin D 3 also prevent some of the other activities of IL-1 is not completely clear, although the metabolic inhibition of pancreatic ~5-cellfunction is counteracted by both cyclosporin [38] and vitamin D 3 (Buschard, K. and Bendtzen, K., unpublished data). 6. Conclusions T and B lymphocytes, Mq~ and NK cells play important roles in our defence against microorganisms, and there is strong evidence for a role o f these cells in the pathogenesis of certain rheumatic diseases and autoimmune endocrine diseases. The pep-

tide hormones (cytokines) produced by these cells, particularly IL-l~3 and TNF~, are potent immune stimulators and modulators of bone and synovial tissue cells as well as pancreatic islet ~-cells and thyrocytes. The newly described pleiotropic M4~ hormone, IL-6, has many properties in common with IL-1 and TNFa, and it may also be directly involved in infectious and autoimmune disease processes. The genes for TNFot and -~ are topographically associated with MHC, in man the H L A - D R and H L A B genes, and the HLA-DR2 allele imposes a low TNFot response in vitro. Treatment to prevent the production and/or function of IL- 1, IL-6 and TNFo~ may prevent the development, or ameliorate the symptoms, of many autoimmune diseases.

Acknowledgements This work was supported by the Willumsen Foundation, the Danish Medical Reserach Council and the Danish Biotechnology program.

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