Journal of Autoimmunity (1992) 5 (Supplement A), 11-26

The Forces Driving A u t o i m m u n e Disease

Ivan M. Roitt, Patricia R. Hutchings, K i m I. Dawe, Nazira S u m a r , Katherine B. B o d m a n and Anne Cooke* Dept. of Immunology, University College & Middlesex School of Medicine, London, and *Dept. of Pathology, University of Cambridge, UK

T h e r e a r e two c l a s s e s o f a u t o i m m u n e d i s e a s e , o r g a n - s p e c i f i c a n d n o n o r g a n specific o r s y s t e m i c . T h a t cells p r o d u c i n g a u t o a n t i b o d i e s a r e s e l e c t e d b y a n t i g e n is s t r o n g l y s u g g e s t e d b y t h e p r e s e n c e o f m u t a t i o n s a n d h i g h a f f i n i t y a n t i b o d y . T - c e l l s a r e p i v o t a l in a l l f o r m s o f a u t o i m m u n i t y as e v i d e n c e d b y t h e t h e r a p e u t i c b e n e f i t o f a n t i - T - c e l l m o n o c l o n a l s s u c h as anti-CD4, and the frequent development of high affinity IgG autoantib o d i e s . T h e p r o d u c t i o n o f a n e r g i c T - c e l l s b y t h e use o f n o n - d e p l e t i n g a n t i - C D 4 in t h e p r e s e n c e o f a n t i g e n is d i s c u s s e d w i t h p a r t i c u l a r r e f e r e n c e to its p o t e n t i a l f o r i m m u n o l o g i c a l i n t e r v e n t i o n i n a u t o i m m u n e d i s e a s e . I t is p o s s i b l e to i d e n t i f y T - c e l l e p i t o p e s in o r g a n - s p e c i f i c a u t o i m m u n i t y u s i n g p a t h o g e n i c T - c e l l c l o n e s o r h y b r i d o m a s to i d e n t i f y t h e p e p t i d e sequences which are reactive. Antigen-specific therapy may ultimately be b a s e d on s u c h p e p t i d e e p i t o p e s . T h e s p e c i f i c i t y o f t h e T - c e l l s in s y s t e m i c a u t o i m m u n i t y is s t i l l o b s c u r e , b u t t h e r e is s o m e e v i d e n c e t h a t r e a c t i v i t y w i t h c e r t a i n g e r m - l i n e i d i o t y p e s c a n l e a d to t h e d e v e l o p m e n t o f s y s t e m i c a u t o i m m u n i t y . T h e p o s s i b i l i t y o f s t i m u l a t i n g B l c e l l s specific f o r a u t o antigens such as DNA becomes feasible if a complex of antibody and DNA is t a k e n u p b y t h e s e s p e c i f i c B - c e l l s a n d p r o c e s s e d i d i o t y p e is p r e s e n t e d to T-helpers specific for those idiotype epitopes. E v i d e n c e is p r e s e n t e d t h a t t h e r e m a y b e p r e - e x i s t i n g d e f e c t s in t h e t a r g e t o r g a n in c e r t a i n o r g a n - s p e c i f i c d i s o r d e r s , a n d t h e e v i d e n c e f o r a g l y c o s y l a t i o n d e f e c t in t h e I g G i n p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s is e x p l o r e d . I t is n o t e d t h a t t h e s p o u s e s o f p r o b a n d s w i t h r h e u m a t o i d a r t h r i t i s also t e n d to h a v e t h i s g l y c o s y l a t i o n d e f e c t a n d t h i s r a i s e s t h e p o s s i b i l i t y o f a n effect d u e to a n e n v i r o n m e n t a l f a c t o r , s u c h a s a m i c r o b i a l i n f e c t i o n . Molecular mimicry of autoantigens by microbes can stimulate autor e a c t i v e cells b y t h e i r c r o s s - r e a c t i v i t y . I t is e m p h a s i z e d t h a t c r o s s - r e a c t i o n w h i c h gives r i s e to t h e p r i m i n g o f a u t o r e a c t i v e T - c e l l s c o u l d give r i s e to t h e establishment of a chronic autoimmune state. In animals with normal r e g u l a t o r y i m m u n e s y s t e m s , s u c h i n d u c e d a u t o i m m u n i t y is u l t i m a t e l y c o r r e c t e d a n d i t is o n l y in a n i m a l s w h e r e t h e r e a r e d e f e c t s in r e g u l a t i o n , Correspondence to: Professor I. M. Roitt, Dept. of Immunology, University College & Middlesex School of Medicine, Arthur Stanley House, 40-50 Tottenham Street, London W1P 9PG, UK. 11 0896-8411/92/0A0011 + 16 $03.00/0

© 1992 Academic Press Limited

12

Ivan M. Roitt e t al. that a u t o i m m u n i t y persists. Thus, there are m a n y factors giving rise to autoimmunity, and the diseases are rightly regarded as multifactorial in origin.

Two classes of autoimmune disease

It is convenient to group a u t o i m m u n e disease under two main headings, as first proposed by us m a n y years ago [ 1]. In the organ-specific diseases the i m m u n e process is directed to antigens within a specific organ and as a result of aggressive action, leads to organ-specific lesions. Examples are H a s h i m o t o ' s disease of the thyroid, pernicious anaemia affecting the stomach and myasthenia gravis involving autoimmunity to the acetylcholine receptors in the muscle endplate. In contrast, the nonorgan specific diseases, involving a systemic autoimmunity, lead to the production of antibodies to autoantigens with a widespread organ and tissue distribution. T h e r e is, therefore, a more widespread location of the lesions, often considered to be representative of i m m u n e complex disease although this m a y be an over-simplified view. T h e rheumatological disorders, such as rheumatoid arthritis, S L E , scleroderma, dermatomyositis, Wegener's granulomatosis, etc., are the main contributors to this group of disorders. Fundamentally different mechanisms may underlie the two groups of diseases as evident from the fact that there is frequent overlap of diseases within each group but far less between the groups. Currently, there are cogent advocates of the view that a pre-existing a u t o i m m u n e state is intrinsic to the i m m u n e system and that a regulated network of lymphocytes linked through autoantigen and idiotype anti-idiotype interactions forms a part of the normal physiology of the i m m u n e system [2]. F r o m this standpoint, failure to regulate this physiological a u t o i m m u n e network leads to the emergence of pathogenic a u t o i m m u n i t y as evidenced by the appearance of destructive autoreactive T-cells and high affinity I g G autoantibodies.

B-cells producing autoantibodies are selected by antigen

T h i s is not an entirely self-evident proposition since B-cells can be stimulated by polyclonal activators and by idiotypic interactions. Perhaps the most convincing evidence that B-cells are selected by antigen is the existence of somatic mutations [3] and high affinity in the autoantibody response. T-cell driven somatic mutation of B-lymphocytes can only lead consistently to a high affinity antibody response, if the appropriate B-cell mutants are selected by antigen itself in germinal centre structures. Additional support for the antigen-driven hypothesis comes from the occurrence of autoantibody responses to antigen clusters. T h e cluster m a y be represented by an intra-molecular linkage, as seen, for example, in the occurrence of several epitopes on the same autoantigenic molecule [4]. It is difficult to envisage a mechanism which could account for the co-existence of antibody responses to different epitopes on the same molecule, except through a linkage dependent on the structure of the antigen molecule itself. T h e same considerations apply to auto-

Forces driving a u t o i m m u n e disease

13

T a b l e 1. Neonatal thyroidectomy ( TdX) prevents autoantibody

formation in Obese Strain (OS) chickens

Experiment 1

Treatment TdX None TdX Sham TdX Hemi-TdX None

Incidence of thyroglobulin autoantibodies 0/5* 7 18/25 J

P=0.011

1/15 ] 11/24 6/9 5/12

P=0.021

*No. animals positive/totalno. in group. Data from L. C. P. de Carvalhoet al. 1982.J. Exp. Med. 155, 1255.

immunity directed against different molecular components of intracellular organelles such as nucleosomes or antigens linked within the same organ, such as thyroglobulin and thyroid peroxidase within the thyroid gland. In a sense these arguments are circumstantial and possibly the most direct evidence for the contention that autoimmunity is antigen driven, has been obtained by studies in the obese strain chicken which spontaneously develops thyroid autoimmunity with a chronic thyroiditis leading to complete atrophy of the gland and hypothyroidism. I f the thyroid gland as a source of antigen is removed at birth, the chickens grow up without developing thyroid autoantibodies (Table 1), i.e., in the absence of autoantigen, thyroid autoimmunity does not arise. Furthermore, once the disease and the thyroid autoimmunity have been established, removal of the thyroid then leads to a gross decline of thyroid autoantibodies, usually to undetectable levels, again implying that maintenance of autoimmunity is antigen driven rather than depending upon some non-antigen source.

T - c e l l s a r e p i v o t a l in o r g a n - s p e c i f i c a u t o i m m u n i t y

T h e evidence for this is overwhelming, at least in experimental models of disease. For example, neonatal thymectomy inhibits the induction of experimental allergic encephalomyelitis by myelin basic protein in Complete Freund's adjuvant. T h e spontaneous thyroiditis in the obese strain chicken is held in check by a combination of neonatal thymectomy plus Draconian injections of a turkey anti-chicken T-cell serum (Table 2). Another spontaneous model of autoimmune disease, the diabetes in N O D mice, is crucially dependent upon the activity of T-cells since treatment with monoclonal anti-CD4 or anti-CD8, or preferably both, can completely inhilSit the development of hypoglycaemia. Antigen-specific T-cell clones can themselves be shown to be capable of causing disease as has been demonstrated in experimental autoimmune encephalomyelitis [5] and thyroiditis, and in N O D mice. Clearly, it is far more difficult in the human to obtain direct evidence for pathogenic T-cell action and perhaps the nearest we can get at the m o m e n t is the demonstration

14

Ivan M. Roitt e t al. Table 2. Neonatal thymectomy ( T x ) plus anti-T-cell serum abrogates the spontaneous development of autoimmunity in O S chickens

Treatment None Tx Tx + normal serum Tx + anti-T serum

Thyroiditis

Thyroglobulin autoantibodies

12/15" 8/8 6/8 1/8

7/16 4/8 5/8 0/7

*No. animals positive/totalno. in group. Data from L. C. P. de Carvalhoet al. 1981.J. Immunol. 126, 750.

that thyroid-specific T-cells are actually present in the thyroid of patients with thyrotoxicosis. T-cell clones were isolated which were driven by class II associated antigens present on the thyroid cells of the patients, and many but not all, had specificity for peptides derived from the thyroid peroxidase molecule [6].

T - c e l l e p i t o p e s in e x p e r i m e n t a l a u t o a l l e r g i c t h y r o i d i t i s With the knowledge that T-cell epitopes involve linear sequences of amino acids forming relatively small peptides, it is now feasible to seek to identify the T-cell epitopes responsible for autoimmune reactions with the view to developing possible immunotherapeutic intervention based upon such peptides which could be readily synthesized. Accordingly, with our interest in the thyroiditis produced by immunization with thyroglobulin in Complete Freund's adjuvant, we have attempted to identify the relevant T-cell epitopes and although the task is somewhat daunting in view of the fact that the molecule is made up of two chains each containing 2,760 amino acids, we were fortunate in having made the observation that thyroglobulin lacking iodine did not produce pathogenic autoimmunity [7]. Since thyroglobulin has just four sites of thyroxine hormonogenesis, we concentrated our efforts on the peptides which covered these four regions and together with Mario Geysen and his colleagues, synthesized a series of 12-mer peptides overlapping by 11 residues, which systematically covered these sites. Thyroglobulin-specific T-cell hybridomas derived from clones or lines which were themselves capable of inducing thyroiditis on transfer to a naive recipient, were used to identify the T-cell epitope. Figure 1 shows clearly that the epitope is localized to the peptide sequence covering the thyroxine at residue 2,553. T h e thyroxine side-chain is extremely bulky and must protrude quite prominently from the M H C class II groove, but nonetheless its presence is mandatory for antigenic activity since replacement of this residue by any of the 20 natural amino acids leads to loss of recognition of the peptide by the T-cell hybridoma [8]. T h e pathogenetic relevance of this thyroxine containing peptide is shown by the fact that cells taken from donors immunized with peptide in Complete Freund's adjuvant (CFA), and then cultured in vitro with the peptide, become highly activated and can

Forces driving autoimmune disease T4 Amino ocid residue

T4 = 5

T4 = 2555

Human Tg peptite

15

I L - 2 retease (CTLL cpm x I0 -3)

(-N Terminus) N I F E T4 QVD AQPL I FE T4QVDAQPLR F ET4QVDAQPLRP E T4QVDAQPLRPC T4QVDAQPLRPC E

40 I

80 I

120 I

40 I

80 I

120 I

YSLEHSTDDT4AS SLEHSTD DT4ASF LEHSTDDT4ASFS EHSTDDT4ASFSR HSTD DT4ASFSRA STD DT4ASFSRAL TDDT4ASFSRALE D DT4ASFSRALEN D T4AS FS RALENA T4ASFSRALE NAT ASFSRALENATR

T4 = 2567

T 4 = 2746

RALENATRDT4FI ALENATRDT4FI LENATRDT4FI ENATRDT4FI NATRDT4FI ATRDT4FI TRDT4FI RDT4FI DT4FI

C CP CPI CPI CPI CPI CPI

I I D I DM I DMA

LLSLQEPGSKTT4 LSLQEPGSKTT4S SLQEPGSKT T 4 S K ( - C Terminus) CH9

ADA2

Figure 1. Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T-cells.

transfer significant thyroiditis when injected into histocompatible recipients (unpublished observations). Therapy with non-depleting anti-CD4 Having identified an important T-cell epitope of thyroglobulin, it is clearly of interest to investigate possible means for exploiting the peptide in therapeutic strategies and we are particularly interested in the possibility of targeting the peptide to induce unresponsiveness in the antigen-specific T-cell population. We are encouraged by the observations of Waldmann and his colleagues [9] showing that treatment with a non-depleting monoclonal anti-CD4 in the presence of antigen, induces a state of anergy in the T-cell population, which can be sustained by repeated exposure to antigen which, in the case of autoimmune diseases, might be adequately provided by natural presentation of autoantigenic moieties. The precise mechanisms underlying the production of anergy are perhaps still controversial, but there is no doubt that given the right timing of the anti-CD4 and antigen injections, the treatment is effective and holds great promise. We have found in our system, that treatment with thyroid antigen in CFA together with a non-depleting anti-CD4 does in fact induce unresponsiveness with respect to the development of thyroiditis in response to subsequent challenge with a thyroiditogenic injection. Even more exciting, are the observations by A. Cooke and her colleagues (unpublished) suggesting that T-cells from N O D mice, which are already primed against pancreatic antigens, can be held in check by treatment with nondepleting anti-CD4 monoclonals.

16

Ivan M. Roitt e t al. Human 16/6-I- onIi-DNA

Anti- DNA, anti-Sm

Schoenfeld and Mozes 1 9 9 0

~

>

mouse 16/6 glomerulonephritis

>

Anti- RNP, onfi-DNA

LC CDR3 peptide from anti-(anti-RNP)

Pucelti e/aL 1990

Figure 2. Is S L E an 'idiotype disease'?

T - c e l l s a r e c r i t i c a l in s y s t e m i c a u t o i m m u n i t y

Non-depleting anti-CD4 treatment is also extremely effective when used in models of systemic autoimmunity such as the spontaneous SLE, which develops in the New Zealand Black x White F1 cross [10]. Beneficial effects of anti-CD4 therapy are also seen in patients with rheumatoid arthritis in who there is a preponderance of activated T-cells in the diseased synovium. This emphasizes the point that T-cells are pivotal not only in organ-specific autoimmunity but also in the non-organ specific group of diseases. This is further supported by the finding that IgG autoantibodies in these disorders show somatic mutations and high affinity, properties which are very much dependent on the co-operative action of helper T-cells. Furthermore, knowing the role of M H C molecules in presentation of antigen to T-cells, associations such as those of rheumatoid arthritis with certain polymorphic sequences common to the H L A - D R 1 and -DR4 molecules, should be taken to imply a basic contribution by T-cell responses in the pathogenesis of disease. What is painfully obvious is that in systemic autoimmunity we have very little idea of the identity of the antigens recognized by the T-cells. One possibility which has been seriously muted, is that the T-cells do not see conventional antigen at all, clearly the case with D N A responses, but instead are devoted to the recognition of idiotype and in this view lupus, for example, would be an 'idiotype disease'. Some support for this view has been obtained experimentally, as shown in Figure 2. Here we describe experiments in which a human monoclonal anti-DNA obtained from a patient with SLE, and bearing the germ-line idiotype labelled 16/6, is injected with CFA into BALB/c mice. T h e animals develop anti-DNA, immune complex glomerulonephritis, and produce their own 16/6 positive antibodies [11]. H u m a n 16/6 could only elicit mouse antibodies with the same idiotype through the intermediary stage of an anti-16/6, either an antibody or a T-cell. In fact, T-cells with specificity for 16/6 idiotype were isolated and claimed by these authors to induce disease when transferred to new recipients. In another set of experiments, Schwartz and his colleagues [12] sequenced a monoelonal antibody from M R L / l p r mice which recognized the idiotype on an anti-ribonuclear protein and found that immunization with a peptide

Forces driving a u t o i m m u n e disease Processed

X Y

,

~

CompLex

Upt akeand processi byB-eel 1onti -Y ng

~ ~ e l

17

X

T

T-hI f r rocs

(e.g.Histones/DNA or16/6+Ab/DNA)

/~,nti-Y

I

Figure 3. T-cell help for one component in an intermolecular complex aids antibody formation to another component.

Drive) ~

~. Recognition . ~

Figure 4. Interaction between idiotype and autoantigen networks may account for specific autoantibody clusters in systemic autoimmunity.

derived from the third complementarity-determining region of the light chain, elicited the production of anti-RNP. Since the immunogen was a peptide, it is highly likely that the first cells to respond were T-cells. We thus have evidence that T-cells recognizing appropriate idiotypes may provoke the formation of antibodies typical of lupus. Nonetheless, we earlier produced evidence showing that the B-cells were probably being selected by conventional autoantigen. T h e two ideas are not mutually exclusive since T-cell help for one component in an intermolecular complex can provide help for antibody formation to the second component as envisaged in the scheme in Figure 3. For example, D N A complexed with antibody bearing the 16/6 idiotype (present as a 'natural antibody'?) would be taken up by B-cells specific for anti-DNA through the surface receptor, and processed internally to yield idiotype related peptide which could be expressed on the surface in association with M H C class I I. This could be recognized by T-helpers for 16/6 which, if they escape from normal regulation, would help the B-cell to become an antibody-forming cell secreting the immunoglobulin for which it was originally programmed, namely anti-DNA. Given a natural interlocking network of idiotypes and autoantigens (Figure 4) as postulated much earlier in this article, such a mechanism would contribute to the formation of specific autoantibody clusters in systemic autoimmunity including the examples shown in Figure 2. Is t h e r e a p r e - e x i s t i n g d e f e c t in t h e t a r g e t o r g a n ?

We should now turn our attention to evidence indicative of a pre-existing defect in the organs which become the target of autoimmune disease. Here, the evidence from

18

I v a n M. R o i t t e t al.

Immunosuppressed

I00 ,'7

o x

Outbred K

RLC Normal

RLR

RLR OS

CS

Figure 5. Abnormal thyroid 131iuptake in obese strain (O S) chickens and the related Cornell strain (CS) compared with normal strains. The endogenous T S H production was suppressed by administration of thyroxine. Thus, one was measuring TSH-independent 13~Iuptake. Sundick et al. (1979) [13].

spontaneous models of autoimmunity is likely to be more persuasive. Looking first at the obese strain chicken, studies by Sundick and colleagues [13] revealed that the uptake of iodine into the thyroid glands of animals in which endogenous T S H had been suppressed by thyroxine treatment, was far higher than that seen in a variety of normal strains. Furthermore, this was not due to any stimulating effect of the autoimmunity, since immunosuppressed animals showed even higher uptake of radio-iodine (Figure 5). Interestingly, the Cornell strain, from which the obese strain was derived by breeding, showed yet higher uptakes of 131I even though the animals do not develop spontaneous thyroiditis. This is indicative of abnormal thyroid behaviour which in itself is insufficient to induce autoimmune disease. That the Cornell strain thyroid possesses an abnormality which may predispose to the development of autoimmune thyroiditis, is seen by experiments in which lymphoid cells from older obese strain chickens with thyroid disease, were transferred to other histocompatible recipients. Young obese strain chickens develop thyroiditis on receiving these lymphoid cells, but normal strains do not (Figure 6). However, thyroiditis did develop in the Cornell strain of chickens suggesting that a combination of the autoaggressive lymphoid cells from the diseased OS chicken with the abnormally susceptible thyroid of the CS strain, did provide the circumstances in which thyroiditis could develop [14]. Considerable excitement was generated by the findings of Bottazzo, Pujol-Borell and colleagues [15] that the thyroids of patients with autoimmune thyrotoxicosis expressed abundant M H C class II on their surface. As mentioned above, these surface class II molecules can present endogenous antigen to appropriate thyroidspecific T-cells, but a role for such antigen presentation in the initiation or perpetuation of autoimmunity is still debated. Although this issue is as yet unresolved, there remain certain pieces of evidence which indicate that a propensity to class II

Forces driving a u t o i m m u n e disease

Tronsfer lymphoid

/

Normol

:>

No thyroidifis

YoungOS

)-

Thyroiditis

)-

Thyroiditis

19

/

cells /

Old OS

CS

Histocompatible recipients

Figure 6. Primed O S cells transfer thyroiditis to strains with abnormal thyroids. Wick et al. (1987).

T a b l e 3. Obese strain ( O S ) thyroid cells in culture have a lowered threshold for class I I induction by I F N - y % Cells class II +ve Strain Normal CB OS

No.

3d/-IFN

2d/+IFN

3d/+IFN

5 5

neg neg

4 24

10 30

Data from G. Wick et al. (1987). Immunology Letters 16, 249-258.

expression, m a y be a feature of organs involved in certain spontaneous a u t o i m m u n e diseases. F o r example, Wick et al. [ 14] showed that cultured thyroid cells from obese strain chickens have a lowered threshold for the induction of surface class I I M H C by interferon-y ( I F N - 7 ) than did cells from normal strains (Table 3). A similar p h e n o m e n o n was observed by Cooke et al. [ 16] regarding the induction of M H C class I I by I F N - y on the 13 cells of the islets of Langerhans of diabetic-prone BB rats c o m p a r e d with their diabetes-resistant cousins (Table 4). W h a t is striking about these results, is that they parallel the events occurring in the h u m a n disease, since only the insulin-producing cells but not the glucagon and somatostatin producing cells in the diabetic pancreas, express class II. Although normal h u m a n islets do not express class I I when stimulated by I F N - y , a combination with turnout necrosis factor ( T N F ) does upregulate the class I I genes, but here the effect is seen on all three types of cell making up the islets of Langerhans. It is conceivable that in spontaneous disease, there m i g h t be an agent such as an endogenous virus which turns on the cells' own T N F genes and this combined with an external source of I F N - 7 , possibly derived f r o m infiltrating T-cells, could provide the trigger for expression of class I I genes thereby making the cells susceptible to a u t o i m m u n e disease processes.

20

Ivan M. Roitt e t al.

T a b l e 4. Class H induction in the insulin-producing cells in rats predisposed to spontaneous development of autoimmunity is more readily induced by I F N - 7 compared with normals Expression of class II on Islet cells Diabetic human* Normal humant Prediabetic BB rat Diabetic resistant rat Normal humant

Stimulant

Insulin cells

Somatostatin cells

Glucagon cells

None I FN-y IFN-y IFN-? IFN-y + T N F

+ + -+ + -+ +

----+ +

----+ +

*in vivo. tData from R. Pujol-Borrell et al. (1987). Nature 326: 304-306.

IgG g l y c o s y l a t i o n d e f e c t in r h e u m a t o i d a r t h r i t i s Although there may be defects in the organization of target organs in various autoimmune disorders, in only one instance has it been possible to discern an abnormality in the structure of the autoantigen itself and that is in the case of immunoglobulin G in rheumatoid arthritis, where autoimmunity to this molecule is a dominant feature. It is now clear that the I g G molecules in both juvenile and adult forms of rheumatoid arthritis are significantly hypogalactosylated in that the percentage of biantennary sugars which lack galactose (G(0)%) is significantly higher than in I g G from normal individuals (Figure 7). These differences may endow the molecule with a greater propensity for self-association of I g G rheumatoid factor, thereby enhancing immune complex formation in the joint, or it may increase the propensity of I g G to be handled in a way which predisposes to the encouragement of autosensitization. Be that as it may, the occurrence of a high G(0) percentage early in disease can be a valuable prognostic indicator [17]. We know that it is a consequence of reduced galactosyltransferase [ 18], the enzyme which adds galactose to terminal N-acetyl glucosamine in the biantennary sugar molecule, and conceivably, other molecules of importance including those released by T-cells, may show abnormal behaviour as a result of a lower galactose content. This is an obvious area for further study. It is well known that the severity of rheumatoid arthritis is reduced late in pregnancy with an exacerbation post-partum. What was not expected, was that the degree of galactosylation of the patient's I g G should change together with the disease state pre- and post-partum. I n a series of cases, G(0) To levels fell markedly near to term in parallel with amelioration of disease but soon after that, the disease became more severe and was associated with the rise in the G(0)% [19]. At the least, this suggests that the galactose content of the I g G is tracking the underlying pathological events with some fidelity. Another unexpected finding surfaced during a study on family members of probands with rheumatoid arthritis. It appeared that, unlike other family members without disease, the spouses actually had elevated percentage G(0) values (Figure 8).

Forces driving a u t o i m m u n e disease

21

$ 0

•

2

• • • •

0~ • O0 • • • O0 O 0 0 0

o o 0 0

-2

O

-4

I 20

0

I 40

I 60

I 80

Age (yeers)

Figure 7. The percentage of sugar chains in IgG which lack galactose (%G(0)) is higher in both juvenile and adult forms of rheumatoid arthritis compared with normal. Results are presented in terms of standard deviation units above and below the mean for an age-related normal population. Data from: Roitt, I. M. and Sumar, N. 1990. IgG and rheumatoid factor at a glance. Clin. Exp. Rheum. 8 (Suppl. 5): 89-91. "s C3 e)

E

2.0

E o

I.o

ToO o

o ~

0.0

~ -I.0 o -2.0 c~

Control populotion

RA probonds

Spouses

Non-RA reletives

RA relotives

Figure 8. Galactose deficiency is seen in the spouses of probands with rheumatoid arthritis. The definition of %G0 and the units in which they are presented is given in the caption to Figure 7. Data from: Sumar et al. 1991. Abnormalities in the glycosylation of IgG in spouses of patients with rheumatoid arthritis. A family study, ft. Autoimmunity 4: 907-914.

A further cohort of spouses of patients attending the clinic for RA confirmed this finding of decreased galactose content in IgG of the spouses. This indicates the influence of environmental elements, most likely, infectious agents.

Molecular mimicry of autoantigens There are many ways in which micro-organisms could influence autoimmunity, but t h e m e c h a n i s m w h i c h h a s a t t r a c t e d m o s t i n t e r e s t , is t h a t o f m o l e c u l a r m i m i c r y

22

Ivan M. Roitt e t al.

between micro-organisms and autoantigens including, of course, idiotypes. T h e r e are innumerable examples of cross-reactions at the B-cell level or of sequence homology at the T-cell level, between microbial molecules and autoantigens [20]. Ebringer has consistently championed cross-reactivity between H L A - B 2 7 and certain strains of klebsiella in connection with ankylosing spondylitis, and more recently cross-reactivity between Proteus mirabilis and DR4 in relation to rheumatoid arthritis [21]. Homology between the highly conserved heat shock proteins in mycobacteria and humans has also been highlighted as a potential contributory factor in the pathogenesis of the latter disease [2]. T h e scene was set many years ago independently by Weigle and Allison, in which they envisaged a situation in which autoreactive B-cells which did not normally respond to autoantigens because of tolerance at the T-cell level, could be activated by cross-reacting microbial antigens which offered T-cell helper epitopes to which the individual was not tolerant (compare Figure 10 which illustrates the same system but with heterologous erythrocytes in place of a cross-reacting bacterial antigen). T h e problem with this system, is that when the foreign microbe is eliminated by immune reactivity, there is no longer T-cell help and the autoreactive B-cell cannot be stimulated by endogenous autoantigen. Thus, a chronic autoimmune state cannot be established. On the other hand, cross-reactivity at the T-cell level may lead to chronicity because of the nature of interactions with primed vs naive T-cells. We can illustrate the point by quoting the studies of Clayton, Sercarz and colleagues [22]. T h e y used the experimental allergic encephalomyelitis system immunizing with myelin basic protein (MBP) in CFA. First they tolerized to the most immunogenic epitope, peptide I in the diagram (Figure 9), and then showed that cross-reacting M B P could induce brain lesions, whereas the mouse protein could not. T h u s , mouse peptide II, which is the second encephalitogenic epitope, given as the whole protein, could not induce the brain lesions through production of autoreactive T-cells, whereas heterologous guinea-pig M B P presented epitope II effectively and induced autoreactive T-cells capable of interacting with mouse M B P processed in the brain and which acts as the target for these cells and leads to the production of brain lesions. In other words, the heterologous M B P epitope is presented more effectively to the naive T-cells than the homologous protein, but once it has stimulated the cells they are then able to react with processed homologous M B P because they have a higher avidity for the antigen processing cell. This is not a consequence of any change in the receptor because T-cell receptors do not mutate, but rather a result of increased expression of accessory molecules such as CD2 and L F A - 1 , which give much stronger binding to the antigen presenting cells. T h u s , when primed, the T-cells can react with peptides from endogenous protein that normally would remain cryptic [22], i.e., not be presented in sufficient concentration to bind effectively and trigger the naive T-cell. Once triggered, the endogenous molecules can continue to stimulate the T-cells and so give rise to a chronic autoimmune state. R e g u l a t o r y d e f e c t s c o n t r i b u t e to t h e d e v e l o p m e n t o f a u t o i m m u n e d i s e a s e

It seems that the adventitious induction of autoimmunity by cross-reactivity through the mechanisms outlined above, may be regulated in normal animals. Let us look for

Forces driving a u t o i m m u n e disease Peptide

23

Peptide

'

Mouse MBP

Guinea-pigMBP Tolerizewith peptJdeI

Immunizewith MBP

V

Brain lesionsEAE

Guinea-pig

-I-+ Mouse

Guinea-pigMBP

I! Naive

Primed

S, LowK ~

oh K

Mouse MBP

0 Key: • MHC class 11" NV Processedpeptide.

Figure 9. Induction of T-cell autoimmunity with cross-reacting peptide. T h e commentary on the experiment is given in the text, b u t the bottom line is that naive T-cells cannot be stimulated by certain cryptic peptides of endogenous origin which either have a low affinity for the naive T-cell or are not presented at sufficiently high concentration to trigger them. T h e m o u s e protein is unable to present epitope II to naive T cells through inadequate processing. However, once primed, the T-cells can now react with even the inadequately processed M B P in the m o u s e brain through its higher avidity for the primed cells. Once the T cell is primed, what was previously an inadequate presentation of endogenous antigen, now becomes adequate for triggering since the primed cell has a higher avidity for the antigen presenting cell through its increased expression of accessory molecules such as C D 2 and LFA-1 which increase its adhesion to complementary molecules on the antigen presenting cell. If the cross-reacting antigen is capable of binding to B-cells, these Bcells m a y then focus the endogenous antigen on their surface in such high concentrations, that the final concentration of processed cryptic epitope m a y also be high enough to stimulate even naive T-cells. T h u s , there are m a n y routes by which cross-reactive antigens can stimulate autoreactive cells.

24

Ivan M. Roitt e t al. No help

Help

f

I

-.~

"\

/

/

\ Tolera

Figure 10. Cross-reacting rat red cells break tolerance to mouse erythrocytes in mice. T h e rat red cells cross-react with autoreactive B-cells and rat determinants recruit T-cell help denied to the autologous red blood cells.

Death

+++

8

/ O

CBA

I

2

3

4

5

Weeks Figure 1l. Persistence of autoimmunity induced by cross-reacting red cells is strain-related. T h e autoimmunity induced by cross-reacting rat red cells (compare Figure 10) is eliminated in normal mice but not in other strains such as N Z B and SJL w h e r e the regulatory control mechanisms, probably at T-cell level, are defective. Cooke, A. and Hutchings, P. (1984) Immunology51" 489-492.

example at the m o d e l in which rat red cells are used to break tolerance as a crossreacting antigen with m o u s e erythrocytes in the mouse. T h e system is shown in Figure 10, and to remind the reader, this is an example of cross-reactivity at the B-cell level, with T - c e l l help provided by n e w rat determinants to which the m o u s e is not tolerant. T h e resulting antibodies give rise to positive C o o m b ' s tests in which the presence of antibody on the red cells is revealed by a conventional antiglobulin reagent. If one continues to inject rat red cells at weekly intervals, the C o o m b ' s test b e c o m e s positive and reaches a m a x i m u m around 2 weeks, but thereafter declines in a

Forces driving autoimmune disease

25

normal mouse strain such as CBA (Figure 11). However, in other strains, where the tolerance mechanisms are less well developed, such as N Z B (which eventually spontaneously develops an autoimmune haemolytic anaemia) and even more so in the SJL, the autoimmune state does not resolve and in the case of the SJL, becomes so severe that the animals die shortly after the 4th injection probably as the result of anaphylaxis. T h u s , it appears that the normal mice had managed to regulate the induced autoimmune response and further studies reveal that the mice at 4 weeks contain T-cells capable of transferring active suppression to a naive recipient which will then fail to make autoantibodies when challenged with rat red blood cells. At the T-cell level in experimental models such as allergic encephalomyelitis induced by M B P and the adjuvant arthritis produced by F r e u n d ' s adjuvant alone, the diseases are also self-limiting apparently owing to the operation of T-suppressor systems. It is clear, therefore, that many different factors must operate together before a chronic autoimmune state becomes established, i.e., autoimmune diseases are multifactorial in origin. References

1. Hijmans, W., D. Doniach, I. M. Roitt, and E. J. Holborow. 1961. Serological overlap between lupus erythematosus, rheumatoid arthritis, and thyroid autoimmune disease. Br. Med. J. ii: 909-914 2. Cohen, I. R. and D. B. Young. 1991. Autoimmunity, microbial immunity and the immunological homunculus. Immunol. Today 12:105-110 3. Schlomchik, M., M. Mascelli, H. Shan, M. Z. Radic, C. Pisetsky, A. Marshak-Rothstein, and M. Weigert. 1990. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J. Exp. Med. 171:265-292 4. Vincent, A., P. J. Whiting, M. Schluep, F. Heidenreich, B. Lang, A. Roberts, N. Willcox, and J. Newsom-Davis. 1987. Antibody heterogeneity and specificity in myasthenia gravis. Ann. N. Y. Acad. Sci. 505:106-120 5. Ben-Nun, A. andI.R. Cohen. 1982. Experimentalautoimmuneencephalomyelitis(EAE) mediated by T cell lines: process of selection of lines and characterization of the cells. J. Immunol. 129:303-308 6. Dayan, C. M., M. Londei, A. E. Corcoran, B. Grubeck-Loebenstein, R. S. L. James, B. Rappaport, and M. Feldmann. 1991. Auto-antigen recognition by thyroid-infiltrating T-cells in Graves Disease. Proc. Natl. Acad. Sci. USA 88:7415-7419 7. Champion, B. R., D. C. Rayner, P. Byfield, K. Page, J. Chan, and I. M. Roitt. 1987. Critical role of iodination for T-cell recognition of thyroglobulin in experimental murine thyroid autoimmunity. J. Immunol. 139:3665-3670 8. Champion, B. R., K. R. Page, N. Parish, D. C. Rayner, K. Dawe, G. Biswas-Hughes, A. Cooke, M. Geysen, and I. M. Roitt. 1991. Identification of a thyroxine-containing self epitope of thyroglobulin which triggers thyroid autoreactive T cells. J. Exp. Med. 174: 363-370 9. Mathieson, P. W., S. P. Cobbold, G. Hale, M. R. Clark, D. B. G. Oliviera, C. M. Lockwood, and H. Waldmann. 1990. Monoclonal antibody treatment in systemic vasculitis. N. Engl. ,7. Med. 323:250-254 10. Carterton, N. L., C. L. Schimenti, and D. Wofsy. 1989. Treatment ofmurine lupus with F(ab')2 fragments of monoclonal antibody to L3T4. Suppression of autoimmunity does not depend on T helper cell depletion. J. Immunol. 142:1470-1475 11. Shoenfeld, M. and E. Mozes. 1990. Pathogenic idiotypes of autoantibodies in autoimmunity: lessons from new experimental models of SLE. F A S E B J . 4:2646-2651 12. Pucetti, A., T. Koizumi, P. Migliorini, J. Andre-Schwartz, K. J. Barrett, and R. S. Schwartz. 1990. An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice. J. Exp. Med. 171:1919-1930

26

I v a n M. R o i t t et al.

13. Sundick, R. S., N. Bagchi, M. D. Livezey, T. R. Brown, and R. E. Mack. 1979. Abnormal thyroid regulation in chickens with autoimmune thyroiditis. Endocrinology (Baltimore) 105:493-498 14. Wick, G., T. Kuhr, G. Kromer, and K. Hala. 1990. Genetic background, thyroid activity and autoimmune phenomena. In The Thyroid Gland, Environment and Autoimmunity. H. A. Drexhage, J. J. M. de Vijlder and W. M. Wiersinga, eds. Elsevier Science Publishers B. V., Amsterdam. pp. 23-32 15. Bottazzo, G. F., R. Pujol-Borrell, T. Hanafusa, and M. Feldmann. 1983. Hypothesis: Role of aberrant H L A - D R expression and antigen presentation in the induction of endocrine autoimmunity. Lancet ii- 1115-1119 16. Walker, R., A. Cooke, A. J. Bone, B. M. Dean, P. van der Meide, and J. D. Baird. 1986. Induction of class II M H C antigens in vitro on pancreatic B cells isolated from BB/E rats.

Diabetologia 29:749-751 17. Young, A., N. Sumar, K. Bodman, S. Goyal, H. Sinclair, I. Roitt, and D. Isenberg. 1991. Agalactosyl IgCr--an aid to differential diagnosis in early synovitis. Arthritis Rheum. (in press) 18. Axford, J. S., L. Mackenzie, P. M. Lydyard, F. C. Hay, D. Isenberg, and I. M. Roitt. 1987. Reduced B-cell galactosyltransferase activity in rheumatoid arthritis. Lancet ii: 1486-1488 19. Rook, G. A. W., J. Steele, R. Brealey, A. Whyte, D. Isenberg, N. Sumar, L. Nelson, K. B. Bodman, A. Young, I. M. Roitt, P. Williams, I. Scragg, C. J. Edge, P. Arkwright, D. Ashford, M. Wormald, P. Rudd, C. Redman, R. A. Dwek, and T. W. Rademacher. 1991. Changes in IgG glycoform levels may be relevant to remission of arthritis during pregnancy. J. Autoimmunity 4:779-794 20. Oldstone, M. B. 1989 Virus-induced autoimmunity: molecular mimicry as a route to autoimmune disease. J. Autoimmunity 2 (Suppl.): 187-194 21. Ebringer, A., S. Khalafpour, and C. Wilson. 1989. Rheumatoid arthritis and Proteus: a possible aetiological association. Rheumatol. Int. 9:223-228 22. Clayton, J. P., G. M. Gammon, D. G. Ando, D. H. Kono, L. Hood, and E. E. Sercarz. 1989. Peptide-specific prevention of experimental allergic encephalomyelitis. J. Exp. Med. 169:1681-1691