2 Immunity and autoimmunity in diabetes mellitus

2

Immunity and Autoimmunity in Diabetes Mellitus

M O R T E N C H R I S T Y T O R S T E N D E C K E R T J O R N N E R U P

The syndrome of diabetes mellitus comprises at least five distinct nosological entities as shown in Table 1. These conditions differ from one another in clinical features, genetics (see Chapter 1) (Creutzfeldt, K/Sbberling and Neel, 1976) and, therefore, most likely also in aetiology and pathogenesis.

Some characteristics of insulin-dependent (IDDM) and non-insulin- dependent diabetes (NIDDM) are summarized in Table 2. Autoimmunity is a typical feature of insulin-dependent diabetes irrespective of age at onset but, according to present knowledge, not of any other type of diabetes, so that this chapter will deal mainly with IDDM. Questions of the immunogenicity of insulin preparations and of host defence against infection will be discussed separately.

AUTOIMMUNE DIABETES MELLITUS

Human Pathology

Since the beginning of this century pathologists have searched for clues to the aetiology and pathogenesis of diabetes mellitus in the microscopic structure of the pancreas. No specific feature, pathognomonic of diabetes mellitus as such, or of any type of diabetes, has yet been found. Nevertheless, these studies have provided much knowledge on which an understanding of the development of diabetes may eventually be based. Gepts (1965, 1976) and Gepts and Pipeleers (1977) have reviewed the histology and histopathology of the human pancreas in diabetes.

Beta-cell destruction A reduction in the number of islets of Langerhans and in the size of the average islet has been demonstrated by light microscopy in the pancreas of patients with insulin-dependent diabetes. By the use of specific staining techniques (e.g. Gomori's aldehyde-fuchsin staining of insulin-containing beta cells and Grimelius silver staining of glucagon-producing alpha cells) it has been shown that these changes are secondary to a disappearance of beta

Clinics in Endocrinology and Metabolism--Vol. 6, No. 2, July 1977. 305

306 MORTEN CHRISTY, TORSTEN DECKERT AND JORN NERUP

Table 1. The five nosologically distinct types of diabetes mellitus

1. Insulin-dependent (juvenile-onset) diabetes mellitus = IDDM 2. Non-insulin-dependent (maturity-onset) diabetes mellitus = NIDDM 3. Maturity-onset type of diabetes mellitus in the young (MODY) a rare dominantly inherited

mild type of diabetes mellitus = MODY (Tattersall, 1976) 4. Diabetes mellitus or carbohydrate intolerance associated with certain genetic syndromes

(Rimoin, 1976) 5. Secondary diabetes

cells, p r o b a b l y due to beta-cel l des truct ion , while a l p h a ce l l s~and o t h e r n o n - i n s u l i n p r o d u c i n g cells s e e m to be p r e s e r v e d . E v e n in t he in i t ia l p h a s e of i n s u l i n - d e p e n d e n t d i abe t e s , t he n u m b e r of b e t a cells is r e d u c e d to 10 p e r c e n t or less a n d wi th t ime b e t a cells d i s a p p e a r c o m p l e t e l y in m o s t p a t i e n t s ,

Table 2. Some characteristics of insulin dependent (IDDM) and non-insulin dependent diabetes (NIDDM)

IDDM NIDDM

Clinical featu res Ketosis Usual Rare Weight Non-obese Often obese Age at onset Usually <30 Usually >40 Onset Rapid-gradual Insidious Duration at onset of late Usually several years May be present at diagnosis complications Remission Often occurs ? Absent

Epidemiology Incidence 13-14/10 s (<30 years) peak at 65 years

peak at 12-14 years Prevalence 0.5 per cent 2 per cent Sex Slight male predominance Female predominance Seasonal variation Present ? Absent

Pathology Islet mass <10 per cent Only moderate reduction Beta-cell mass <10 per cent Insulitis at onset Present in 50-70 per cent ? Absent

Immunology Antipancreatic cell- mediated immunity Antipancreatic humoral immunity Association with other endocrinopathies

Genetics Concordance in identical twins Morbidity risk of DM at specified ages in 1st degree relatives of probands with (Degnbol and Gren Hansen, 1977) Association with HLA

35-50 per cent at onset

60-85 per cent at onset

Frequent

<50 per cent

Age 50:5 per cent Age 60:15 per cent

Present

<S per cent

S per cent

Infrequent

Almost invariable

Age 30:0.4 per cent Age 60:5 per cent

Absent

IMMUNITY AND AUTOIMMUNITY IN DIABETES MELLITUS 307

although in a few a small - - but still significant - - mass of beta cells remains and functions.

These results are consistent with histochemical, extraction and secretion studies. The insulin content of the pancreas and islets from IDDM patients is less than 10 per cent of normal values and basal and post-stimulatory insulin responses in newly diagnosed patients with insulin-dependent diabetes are almost completely absent (Cerasi and Luft, 1977).

Measurement of C-peptide provides new possibilities for assessing insulin production in patients on insulin. Steiner et al (1977) in a recent review conclude that beta-cell secretory capacity decreases gradually during the first few years of IDDM in some patients, while others show no secretion at all at the time of diagnosis. During periods of remission secretion may resume, while during episodes of ketoacidosis secretion is not detectable, and after five years of IDDM C-peptide secretion has been absent in all patients so far examined.

lnsulitis Most authors have found insul i t i s , i.e. infiltration of mononuclear cells, probably lymphocytes, in and around the islets in 50 to 70 per cent of patients coming to autopsy within a few months after the onset of IDDM (Gepts and Pipeleers, 1977). No such changes have been found when IDDM has persisted for more than one year. In the individual patient not all islets are equally affected. In a recent series, between 10 and 75 per cent of the islets were affected (Egeberg et al, 1976). In most the infiltration was very discrete, but in some it was distorting the whole architecture of the islet. In this series, with diabetes of only a few weeks duration, the number of islets appeared normal compared to age-matched controls, but selective destruction of beta cells was clearly seen.

Electronmicroscopy confirms the finding of insulitis and beta-cell destruction. The majority of peri- and intrainsular infiltrating cells resemble small lymphocytes. Beta cells are found to be degranulated and in various stages of pyknosis (Gepts, 1977).

Doubts about the significance of insulitis were raised by Doniach and Morgan (1973), who were unable to detect it in the pancreases of 13 juvenile diabetics who had died in coma between 1907 and 1930.

The exact role of insulitis in the pathogenesis of IDDM remains unknown; an attractive possibility is that an autoimmune reaction participates in a selective non-cicatrical beta-cell destruction. Insulitis can be induced in several ways in experimental animals, as summarized later in this chapter (Table 10), and the elucidation of its significance is most likely to come from experimental models of IDDM.

Beta-cell regeneration

In his series of young insulin-dependent diabetics, who died within six months of the onset of symptoms, Gepts (1965) reported signs of islet celt'pr01iferation from existing islets and probably also de novo from duct epithelial cells. He assumed that strong stimulant factors had been acting in the pre-clinical phase to induce formation of new beta cells. For obvious reasons studies of


regeneration of beta cells in humans are almost impossible, but from work on experimental animals and the scanty observations in humans Hellerstr~Sm, Andersson and Gunnarsson (1976) conclude that cells with a capacity to differentiate into insulin-secreting beta cells exist, and that mature beta cells can divide under certain circumstances.

New islets and new beta cells are probably formed throughout life, although with a considerable variation of speed. Thus, the total beta-cell mass reflects a balance between renewal and destruction. The factors responsible for the growth of the endocrine pancreas are unknown but, based on experimental work in animals, these authors believe that hyperglycaemia, a high caloric intake, probably some hormones and sulphonylureas increase the rate of replication.

The balance between destruction and regeneration of beta cells is clearly of crucial importance for the understanding of the pathogenesis and time course of IDDM. That restoration of insulin secretion takes place in human diabetes was demonstrated by Steiner et al (1977) in their studies of C-peptide secretion during remission and by Park, Soeldner and Gleason (1974) who examined insulin secretory capacity in young ketosis prone diabetics during clinical remission. In animals there is a strong genetic influence on the capacity for regeneration of beta cells (Hellerstr~Sm, Andersson and Gunnarsson, 1976) and this may also be the case in man (Boquist et al, 1974).

Thus, clinical insulin-dependent diabetes may become manifest when beta cell regeneration cannot compensate for destruction, either because the regeneration capacity is already reduced by inheritance and is exhausted after a single injury to the endocrine pancreas, or because regeneration cannot catch up with a continued, possibly accelerating, destruction of beta cells. It is tempting to hypothesize that the diagnosis of IDDM in patients not showing signs of remission is actually made at the end of a 'remission period', - - the crucial difference being that beta-cell destruction in the initial attack(s) was not extensive enough to cause overt diabetes.

Therefore, the important questions in relation to pathogenesis of IDDM might be (a) what is the beta-cell destructive mechanism(s)? (b) which exogenous or endogenous factors can 'trigger' beta-cell destruction? and (c) what controls regeneration of beta cells?

T h e r o l e o f a v i r u s ?

For years it has been claimed that virus might induce beta-ceU destruction. It must be admitted that direct evidence is weak (Gepts, 1976; Andersen et al, 1976). However, Greider et al (1977) have reported the interesting observation that perinuclear inclusions of virus-like particles could be detected by electronmicroscopy in pancreatic duct cells of both juvenile and adult onset diabetics, in a few patients with islet hyperplasia, and in some autopsy specimens from patients suffering from non-pancreatic diseases.

The significance of this finding is not clear, but these particles may be an interesting parallel to the proliferation of C-type virus in beta cells of mice in a new experimental model of IDDM (Table 10) (Like and Rossini, 1976).


Association of IDDM with autoimmune endocrinopathies Clinical observations provide evidence for the association between IDDM and autoimmune endocrinopathies more often than would be expected by chance alone. Diseases in question are idiopathic Addison's disease (IAD), Graves disease, primary myxoedema, Hashimoto's thyroiditis, hyper- gonadotropic hypogonadism, idiopathic hypoparathyroidism and perhaps pernicious anaemia.

The prevalence of IDDM in IAD and thyroid autoimmune disorders is 30 to 50 times that of the individual diseases in the background population (Nerup et al, 1977).

The reverse situation, prevalence of other autoimmune endocrinopathies in IDDM, is less well documented, but there is reason to believe that it is four to five times higher than in the background population. Furthermore there is evidence to suggest that subclinical diabetes (i.e. carbohydrate intolerance) can be found with an even higher prevalence in the autoimmune endocrinopathies (K/Sbberling and Kattermann, 1974; MacCuish and Irvine, 1975).

Remarkable similarities exist in the microscopic appearance of the lymphoid infiltration and endocrine target cell destruction between the autoimmune endocrinopathies and IDDM, thus providing further indirect evidence that autoimmunity plays some part in the pathogenesis of IDDM and also that some common factor operates in all the endocrinopathies listed above.

Such similarities are found in human disease as well as in experimental endocrinopathies. A few cases of simultaneous 'autoimmune infiltrations' in islets and other endocrine glands have been reported (Gepts, 1976). More striking but still indirect evidence comes from the findings of high prevalences of certain organ-specific auto-antibodies in diabetic sera (MacCuish and Irvine, 1975). Table 3 summarizes three of the most recent studies. Thyroid and gastric antibodies are found most commonly in young female IDDM patients. The prevalence of these antibodies in older diabetic patients is

Table 3. Prevalence of thyroid, gastric and adrenal antibodies in IDDM patients

Authors

Percentage antibody positive

Number of Thyroid Parietal patients Thyroglobulin cytoplasmic cell Adrenal

(controls) antibody antibody antibody antibody

Whittingham et al (1971) 400 NT 14 21 NT

(400) 9 10 Nerup and Binder

(1973) 133 10 20 16 2 (128) 8 3 5 0

MacCuish and Irvine (1973) 250 9 17 18 NT

(250) 7 8 8 Combined data 9 16 19 2

7 7 9 0

NT = not tested.


much closer to that of the control populations, but here again the occurrence of the thyroid and gastric antibodies is associated with the female sex and insulin- dependency.

Adrenal antibodies have been found in a small proportion of IDDM patients (Nerup and Binder, 1973).

Cell-mediated Autoimmunity

Antipanereatie cell-mediated autaimmunlty Although workers had looked for autoimmunity directed against the endocrine pancreas in patients with IDDM, Nerup et al (1971) were the first to produce direct evidence for the occurrence of cell-mediated autoimmunity. By means of the leucocyte-migration test (LMT) (Bendixen and S0borg, 1969), they were able to demonstrate in vitro the occurrence of cell-mediated immunity directed against antigens of the endocrine pancreas in IDDM.

In this test system migration of leucocytes after incubation with the relevant antigen is compared to migration of leucocytes incubated with control preparations. Migration inhibition is taken as evidence of cell- mediated immunity. Since granulocytes, lymphocytes, different lymphokines and other factors influence the test it is difficult to standardize and the results are difficult to interpret in relation to disease pathogenesis.

The in vitro reaction observed by means of the LMT in this study was proved to be a cell-mediated immune reaction by the fact that intracutaneous testing with the same preparation led to typical delayed type hypersensitivity reactions. Migration inhibition of non-diabetic leucocytes could be demonstrated in only one of the controls.

The leucocyte migration inhibition studies were subsequently confirmed and extended by other groups (Table 4).

The precise nature of the antigen(s) in the endocrine pancreas which induces migration inhibition is unknown. It is organ-specific, species-non- specific and different from insulin, since different insulins have not been shown to induce migration inhibition in vitro or delayed type cutaneous reactions in vivo in non-insulin-treated IDDM patients.

Richens et al (1973) and Richens, Ancil and Hartog (1976) have described the occurrence of an organ-specific migration inhibition of diabetic leucocytes by rat and human liver mitochondria which runs parallel to the changes in the reactivity against pancreatic antigens. Mitochondrial hypersensitivity has been reported in other autoimmune conditions (Wartenberg et al, 1973), but the existence of such a reactivity in diabetics was not confirmed by Nerup et al (1971) and MacCuish et al (1974b). In these studies some controversy exists as to whether migration inhibition tends to predominate in young patients of recent onset and to fade away with duration of disease (Nerup et al, 1974a) or whether no such tendencies can be observed (Irvine et al, 1976).

Huang and MacLaren (1976), in another test system, found evidence of cell-mediated antipancreatic autoaggression in IDDM patients, but they did not provide proof of organ-specificity. Using cultured human insulinoma cells they observed a four- to fivefold increase of cytoadherence and a threefold increase in cytotoxicity of lymphocytes from IDDM patients

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irrespective of the presence of serum from IDDM patients. Complement had no influence on cytotoxicity.

Endocrine pancreatic preparations have not been shown to be able to induce blast transformation in the lymphocyte transformation test. However, Irvine et al (1976) reported the interesting and controversial finding that bovine and porcine insulin induced lymphocytic transformation in quite a few IDDM patients, including some who had not yet received exogenous insulin. In contrast to the humoral immune response to insulin the B-chain and not the A-chain seemed responsible for blast transformation. This observation clearly needs confirmation.

At present no available data allow conclusions as to what extent reversible lymphocyte dysfunctions due to metabolic disturbances influence the results of the above mentioned tests.

Cell-mediated autoimmunity and late diabetic complications Recently Aviner et al (1976) reported the original observation that migration inhibition to uveoretinal antigen in the leucocyte migration test (LMT) was exhibited by about one-third of diabetics with untreated simple or proliferative retinopathy, and was correlated to severity and duration of this complication. No other studies of cell-mediated immunity directed against target organs of late diabetic complications have been published, although Nerup and Bendixen (1972, unpublished observations) observed migration inhibition to kidney antigens in a few patients with diabetic nephropathy. Much more work is needed to establish a role of cell-mediated autoimmunity in the pathogenesis of late diabetic complications, but this possibility is of course very fascinating.

Antipancreatic Humoral Autoimmunity

Evidence of cell-mediated autoimmunity in IDDM patients has been accumulating since 1971 but, despite an intensive search, the existence of autoantibodies directed against the endocrine pancreas was not reported until 1974, when Bottazzo, Florin-Christensen and Doniach (1974) detected islet-cell antibodies (ICA) in patients suffering from autoimmune poly- endocrinopathies. These findings were soon confirmed by MacCuish et al (1974a).

ICA was detected by means of the standard sandwich immunofluorescence technique using fresh postmortem pancreas from blood group O kidney transplant donors as the antigen and anti-IgG fluorescein isothiocyanate (FITC) labelled conjugates. There might be two reasons why these antibodies were not detected before. Firstly, the earlier workers had used u.v. micro- scopes with dark field condensors where the light was reflected from below by a mirror, thereby giving a much lower visibility power than present day instruments equipped with the Ploem epi-illumination. A second cause might have been the rather weak fluorescence produced by these antibodies when compared to the brightness of the cytoplasmic staining seen with thyrogastric and adrenal antibodies. By this technique standard islet-cell antibodies are dim even in rather high titres (Bottazzo, Doniach and

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Pouplard, 1976). Other technical problems were clarified by Lendrum and Walker (1975) in studying immunofluorescence of the exocrine pancreas. Blood group substances are secreted by exocrine acini making group A and B organs give fluorescence with many sera.

At present quite a few autoantibodies to components of the endocrine pancreas have been described (Table 5). However, the relevance of most of these to the aetiology and pathogenesis of diabetes still needs to be elucidated. With the exception of the ICA, only small series have been studied and the reports need confirmation.

Islet-cell Antibodies (ICA)

More than one thousand diabetics, mainly insulin-dependent, have now been examined for the presence of islet-cell antibodies (Table 6). These antibodies are of the IgG class and many show complement fixation (Lendrum et al, 1976c). ICA is organ-specific and reacts with all types of endocrine islet cells (Bottazzo, Doniach and Pouplard, 1976; Lendrum et al, 1976c). The antibody is directed against a cytoplasmic antigen, probably located in organelle(s) concerned with hormone production or secretion. Bottazzo, Doniach and Pouplard (1976) have elegantly demonstrated, by double immunofluorescence staining using FITC-labelled antihuman IgG antibody and rhodamine-labelled anti-hormone antibodies, that ICA is not directed against islet hormones. The precise nature and localization of the antigen(s) remains to be elucidated. The antigen is widely species-non-specific since human ICA cross-reacts with islets from rat, guinea-pig and monkey.

The prevalence of ICA in the normal population is very low. Lendrum et al (1976b) found 1.7 per cent positive reactions in 177 non-diabetics and Irvine, Gray and McCallum (1976) in 0.5 per cent of 434 controls. By contrast Buschard et al (1976) found 60 per cent and Lendrum et al (1976c) 85 per cent ICA positivity in young IDDM patients at the time of diagnosis (Table 6). This percentage declines rapidly to 50 per cent or less during the first few weeks of the disease and then remains constant during the following year. After three years less than a fifth of the patients are still ICA positive. Thus the presence of islet-cell antibodies seems to be a transient phenomenon closely related to the time of onset of IDDM. Antibody titres in these patients were generally low (Bottazzo, Doniach and Pouplard, 1976).

On the other hand, islet-cell antibodies have been found several years before the development of diabetes in some patients with autoimmune polyendocrinopathy (Bottazzo, Florin-Christensen and Doniach, 1974; Irvine Gray and McCallum, 1976) or in first degree relatives of IDDM patients (Nelson and Pyke, 1976; Lendrum et al, 1976c) and in some insulin- dependent diabetics rather high titres of ICA persist unchanged for years. Very few patients who are ICA-negative at diagnosis become positive later (Lendrum et al, 1976c).

Interestingly, Christy et al (1976) and Morris et al (1976) found a relative increase of the HLA type B8 in those patients who remained ICA positive, although Lendrum et al (1976c) failed to confirm this observation.

Table 7 summarizes reports of the prevalence of ICA in non-diabetics at

IMMUNITY AND AUTOIMMUNITY IN DIABETES MELLITUS

Table 6. Prevalence of islet-cell antibodies (ICA) in diabetic patients

315

Percentage Authors Type of patients Number tested positive

Bottazzo, Florin-Christensen and Doniach (1974)

MacCuish et al (1974a)

Christy et al (1976)

Lendrum et al (1976c)

Buschard et al (1976)

Mfintefering, Biener and Hillebrand (1976)

Lendrum et al (1976a)

Diabetes in association with 30 33 autoimmune endocrinopathy

IDDM with adrenal antibody 20 25 positive Addison's disease

IDDM (mean duration 3.2 38 55 years)

IDDM 829 38 NIDDM 112 5.3

IDDM (duration 1 week) 20 85 IDDM (duration 4 weeks) not stated 50 IDDM (duration more not stated 10-20 than 1 year)

IDDM (at onset age <30) 50 58

IDDM (duration less than 40 70 3 months)

IDDM in identical twins 25 concordant pairs 50 16 29 discordant pairs 29 34

high risk of becoming diabetic, i.e. patients suffering from other autoimmune endocrinopathies, notably idiopathic Addison's disease (IAD), and first degree relatives of patients with IDDM.

At present little is known about the clinical significance of ICA but these groups of possible pre-diabetics seem well suited to help clarify the matter. So far only a few studies have been carried out.

Irvine, Gray and McCallum (1976) studied ICA positive patients with polyendocrinopathy and first degree relatives or patients with IDDM and found abnormal glucose tolerance (oral GTT in almost all cases) in about 40 per cent. This figure is not much higher than that expected in first degree relatives of diabetics (Kt~bberling and Kattermann, 1974). Not all subjects were ICA positive at the time of glucose tolerance testing, but all had been so on at least one previous test. One patient, known to have been diabetic during pregnancy and showing a constant high titre of ICA, had normal glucose tolerance. Del Prete et al (1976) found an abnormal glucose tolerance and a low insulin response in two ICA-positive women, one suffering from Hashimoto's thyroiditis and the other from Graves' disease. Christy et al (1977, in preparation) found no association between intravenous glucose tolerance and ICA in patients with Addison's disease.

Thus it is still questionable whether ICA is a marker of subclinical or potential diabetes and a pathogenic role of ICA in the development of IDDM


Table 7. Islet cell antibodies in non-diabetics at high risk

Percentage Authors Type of patient Number tested positive

Bottazzo, Florin-Christensen Autoimmune endocrinopathies 81 4 and Doniach (1974)

Bottazzo, Doniach and Autoimmune endocrinopathies 244 8 Pouplard (1976)

Christy et al (1976) Idiopathic Addison's disease 34 44

Buschard et al (1977, First degree relatives of 90 19 unpublished) IDDM patients

Irvine, Gray and McCallum Autoimmune endocrinopathies 954 4 (1976)

remains to be established. Since this antibody reacts with all types of endocrine islet cells and the pathological lesion typical of IDDM is selective beta-cell destruction, such a role may be rather unlikely.

Bottazzo, Doniach and Pouplard (1976) speculate that insulin-dependent diabetes may be divided into two distinct aetiological types; according to them, type 1 may be viral in origin. ICA in this type should be transient and of low titre with the antibody being secondary to virus-induced beta-cell destruction. Type 2 may be truly autoimmune and represent those cases which remain ICA positive with high titres.

MacLaren, Huang and Fogh (1975) demonstrated serum antibodies reacting with the cell surfaces of viable human insulinoma cells in 85 per cent of IDDM patients. These were IgG or IgM antibodies and produced a different fluorescence. They may represent an antigen--antibody system other than ICA. Huang and MacLaren (1976) later reported evidence of cytotoxicity of lymphocytes from °IDDM patients when added to these insulinoma cells. They may have detected an antibody-dependent K-cell killing of beta cells, but neither of these studies provide proof of specificity and the antibody binding may be completely non-specific (Lernmark et al, 1977). To establish that ICA or any of the other antibodies mentioned are of pathogenetic importance in IDDM, it is necessary to prove that the antibody associates selectively only with the beta cells of the islets. Direct lysis of beta cells or measurable damage to some basic beta-cell function should also be demonstrated in vivo in experimental animals or during in vitro assays using normal beta cells or islets. No correlation between humoral and cell- mediated antipancreatic autoimmunity has been found (Christy et al, 1976).

HLA and IDDM

The human leucocyte antigens (HLA) represent the major human histocompatibility system. Four different loci (A, B, C, and D) controlling at least 64 HLA specificities are located within a small part of chromosome number 6


covering only 1.6 centimorgans. Thus each individual inherits one maternal and one paternal HLA-haplotype, usually without recombinations taking place. The genes are co-dominant so that the phenotype of each individual may express two specificities of the A locus, two specificities of the B locus and so on. The specificities in question are glycoprotein antigens on the cell surfaces of all nucleated cells, 'floating' freely in the cell membranes. Antigens of the A, B and C series are detected by serological methods (the microlymphocytotoxicity test) while antigens of the D series can only be detected by one-way mixed lymphocyte culture.

Studies of associations between the HLA system and disease represent an almost exploding field at present. Most of the data available up to the end of 1976 have been published recently (Dausset and Svejgaard, 1977).

Little is known of the mechanisms by which susceptibility to HLA- associated diseases is conferred. Several theories have been put forward and fall into three main categories: (1) Direct effects of the HLA antigens (e.g. interference with l igand--receptor interactions (Svejgaard and Ryder, 1976) or interactions with viral proteins in infected cells resulting in recognition by T cells and target cell damage (Doherty and Zinkernagel, 1975); (2) Effects of genes in the HLA region different from the HLA genes but probably functionally related to them, for example, adverse effects of immune-response genes (McDevitt and Bodmer, 1974); (3) Effects of genes in linkage disequilibrium with HLA by pure coincidence (e.g. theoretical specific 'diabetes-genes'), HLA then representing merely 'inert' genetic markers.

For a brief introduction to the HLA system the reader is referred to Svejgaard et al (1975) and for an extensive review of the function of the HLA system in human disease and a registry of disease associations to Dausset and Svejgaard (1977).

Since 1974 a definite association between IDDM and certain HLA-types of the B and D series has been found and confirmed (for a review see Nerup et al, 1977). This association clearly distinguishes IDDM from NIDDM. The strength of the association between HLA and any particular disease (but not the statistical significance) is usually expressed in terms of a so-called relative risk, which indicates how many times more frequently the disease occurs in a group of individuals carrying the HLA-antigen in question than in a group lacking it.

Table 8 shows the relative risks for the HLA antigens most frequently encountered in IDDM in Caucasians from Northern Europe. These data are derived from population studies, i.e. comparisons of HLA antigen frequencies in patients and normal controls. Such studies cannot prove a n aetiological role of HLA in IDDM, but do permit two conclusions: (1) since the relative risk for both D-series antigens, Dw3 and Dw4, exceeds the risk for the antigens of the B-series, the gene(s) associated with development of IDDM is located closer to the D locus than to the B locus; (2) the relative risk for BS/Bw15 and Dw3/4Dw4 heterozygous individuals is approximately twice the risk for individuals either heterozygous for the B or D antigen in combination with any other antigen (x in the table) or homozygous for any of these four antigens, therefore, there must be at least two genes predisposing to development of IDDM located in the HLA region.


Table 8. Relative risks o f l D D M for various HLA-B and D phenotypes

HLA phenotype Relative risk

Bwl5/x 2.5 Bw15 2.1 B8/x 2.5 B8 3.1 B8/Bwl5 9.8 Dw3 3.7 Dw4 4.9 Dw3/Dw4 9.4

Data on B-phenotypes compiled by Svejgaard and Ryder (1977). Data on D-phenotypes compiled by Nerup et al (1977). x indicates presence of another B-antigen.

Studies of the H L A - - I D D M association in families with diabetics in two or more generations have demonstrated that the phenotype I D D M segregates with a certain genotype, i.e. the HLA-haplotype of the index case. Sibship studies also emphasize the strong association between HLA and I D D M (Tabl 9 9) (Nerup et al, 1977).

Reports of association between certain clinical features of I D D M and HLA (i.e. seasonal variation of incidence, age at onset and severity of disease) are sparse and inconsistent, while a few investigators have also found an association between late diabetic complications and HLA. For example, ret inopathy was more common in HLA-B8 and Bw15 positive individuals.

Other studies have suggested that genetic factors may influence insulin antibody formation since the highest titres have been found in HLA-Bwl5 positive I D D M patients and the possession of HLA-B7 seems to protect against insulin ant ibody formation. This observation should be taken into consideration in assessing the antigenicity of the new highly purified insulin preparat ions (Nerup et al, 1977).

Informat ion about an association between HLA and the many viruses incriminated in the search for aetiological factors in I D D M (Gepts, 1976; Andersen, 1976) is still inconclusive.

The 'diabetogenic ' HLA types B8 and Dw3 are shared with several of the other au to immune endocrinopathies thus indicating a common genetic susceptibility.

Table 9. HLA-haplotypes in families with two or more insulin-dependent siblings

2 Haplotypes shared (HLA identity) 1 Haplotype shared No haplotype shared

Observed 61.5 per cent 30 per cent 8.5 per cent Expected 25 per cent 50 per cent 25 per cent

52 Diabetic probands with 57 diabetic siblings. Data from Nerup et al (1977). Statistics: X2 = 40.86, P <0.001.


ANIMAL MODELS OF IDDM

Nakhooda et al (1977) recently reported the development of a spontaneously diabetic Wistar rat characterized by normal weight, insulin deficiency, glucagon excess, ketosis and dramatic lymphocytic infiltration during active beta-cell destruction. This model of course provides very fascinating possibilities. Hitherto, studies of IDDM in laboratory animals have been undertaken in various experimental models. Table 10 briefly summarizes the major characteristics of the most interesting of these models.

Since these animal models have many features in common with IDDM in man, including genetically determined susceptibility, involvement of viruses, lymphocytic insulitis, cell-mediated and possibly also humoral autoimmunity, they have important implications for the study of the pathogenesis of IDDM.

Thus in conclusion of this section we can state, (1) that genes conferring susceptibility to development of IDDM are located in the HLA region, that these genes are necessary but not sufficient for diabetes to develop, (2) that there must be exogenous factors triggering beta-cell destruction, (3) the nature of the environmental factor(s) required is unknown, but it is anticipated to be of viral origin. Further, on the basis of pathological, clinical and immunological studies, it seems fair to claim, (4) that beta-cell destruction is likely to be a sequel of cell-mediated and/or humoral autoimmunity directed against the endocrine pancreas and (5) that genetic factors are likely to influence the clinical course of IDDM.

IMMUNOGENICITY OF INSULIN PREPARATIONS

Immunization procedure

For practical purposes immunization against insulin develops only after the injection of foreign insulin preparations (Deckert, 1971), although a very few cases of insulin immunization have been described without preceding insulin injection (FNling and Normann, 1972; Ohneda et al, 1974).

In addition to alterations in the immune system of the recipient induced by the subcutaneous injection of insulin, tissue changes are undoubtedly of importance for the immunogenicity of insulin preparations in man. The injection itself will give rise to small haemorrhages, connective tissue changes, accumulation of round cells and disintegration of the normal architecture of fat cells (Poulsen, 1967). Furthermore, after injecting regular insulin in neutral or acid solution the local insulin concentration will rise 106-fold from about 25 microunits/ml to about 25 units/ml. Furthermore, injection of regular insulin in acid solution leads to neutralization of the solution by tissue fluids so that the isoelectric point of porcine or bovine insulin is passed. Consequently, insulin will. precipitate, which might be of importance for its immunogenicity. If long-acting aqueous insulin preparations are injected, the suspension of amorphous or crystalline particles in the subcutaneous tissue will activate cells from the reticuloendothelial systems resulting in phagocytosis. In this connection the size of the particles might be important. This varies from near zero for the smallest particles in amorphous insulin suspensions, through a volume of about 10/~m 3 in NPH or PZI insulin

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crystals, up to about 2000/am 3 in zinc insulin crystals. The fate of NPH-insulin crystals, zinc-insulins, or globulin insulin after injection in the subcutaneous tissue is unknown, but it seems likely that the bonds between the retarding substances and the insulin molecule are split at the injection site before insulin resorption takes place. Last, but not least, the different polypeptides included in insulin preparations (insulin, insulin modifications and impurities) may activate T and B lymphocytes.

Measurement of immunization Alterations of the immune systems, i.e. T and/or B lymphocytes in humans after starting insulin therapy, are diagnosed by looking for signs of delayed hypersensitivity (skin test, lymphocyte transformation in vitro, leucocyte migration inhibition test in vitro (Federlin, 1975), insulitis (infiltration of round cells in and around the islets of Langerhans) (Kl~ppel, 1976)), and antibodies against insulin or other polypeptides included in insulin preparations (Schlichtkrull, 1976). Whereas quantitation and specification of delayed type hypersensitivity is rather difficult, humoral insulin antibodies can easily be measured quantitatively by radioimmunological methods, where the binding of insulin to immunoglobulins (IgG, IgE, IgA, IgM) is measured.

The ideal method for determination of humoral insulin antibodies does not exist. In order to characterize circulating insulin antibodies several methods are necessary. Furthermore, some methods are greatly influenced by the concentration of insulin in plasma, whereas other methods are free from this disadvantage (Andersen, 1977). In all the most commonly used methods, insulin antibodies are characterized by the amount of labelled or unlabelled insulin bound to immunoglobulins.

Unfortunately only a few comparisons between different methods have been done (Andersen, 1977), so that most publications can only be evaluated against their own control material.

Factors of Significance for the Immunogenicity of Insulin Preparations

The immunogenicity of insulin preparations depends on, (1) the presentation of immunogens and, (2) on factors in the diabetic patient.

Presentation of immunogens Most insulin preparations are manufactured from bovine or porcine pancreas. The immunogenicity of bovine insulin preparations in man is more pro- nounced than the immunogenicity of pure porcine insulin preparations (Andersen, 1973). This is also valid for porcine and bovine insulin preparations of comparably high purity (Chance, Root and Galloway, 1976). The cause of this difference is not entirely clear. There are two possible explanations. (1) The different primary structure of porcine and bovine insulin might be the main cause. Bovine insulin differs from human insulin by three amino acids, whereas porcine insulin only differs by one single amino acid - - the terminal amino acid in the B chain, an amino acid which can be removed without loss of biological activity of the remaining insulin molecule. (2) Bovine insulin has a higher tendency to polymerization than porcine


insulin. If one tries to neutralize this by making beef insulin preparations in a special way, reduced antibody formation results (B. Hansen, 1977, unpublished observations). More important than species specificity for the immunogenicity of insulin preparations is their purity (Brunfeldt and Deckert, 1964; Schlichtkrull, 1976; Chance, Root and Galloway, 1976). It has been found that even several times recrystalliz.ed insulin may contain traces of non-insulin components and several per cent of modified insulins (Schlichtkrull, 1976). On gel filtration of crystalline insulin, which is not specially purified, three factions are demonstrable, the 'a', 'b' and 'c' fractions (Chance, Root and Galloway, 1976). The 'a' fraction probably consists of non-insulin proteins from the pancreas, the 'b' fraction of proinsulin, intermediary insulin, and dimer insulin, and the 'c' fraction of arginine insulin, ethyl ester insulin, and insulin deamidated to a varying degree (Schlichtkrull et al, 1973). From an immunological point of view, the most important fractions of ordinary crystalline insulin are the 'a' and 'b' fractions, containing proinsulin (Schlichtkrull et al, 1975). Therefore, in order to produce non-immunogenic porcine insulins with prolonged action, it is important to purify recrystallized porcine insulin further, so that at least the 'a' fraction, containing insulin-foreign components, and the 'b' fraction are removed (so-called 'single-peak' or 'monotop' insulin). The reason why the 'a' fraction - - that containing insulin-foreign components - - may give rise to insulin antibody formation in people treated with recrystallized human insulin in neutral solution is presumably that this fraction contains proteins which have coupled to the insulin as a hapten or contains molecules having antigenic groups in common with insulin (Deckert and Grundahl, 1970). The explanation of the immunogenecity of the 'b' fraction is to be found in the difference of the C-peptide structure (and thereby proinsulin) of various animal species, a difference which is greater than the difference in insulin structure (Steiner et al, 1977).

Whereas total removal of the proteins of the 'b' fraction and of arginine insulin and ethyl ester insulin does not appear to be an absolute necessity for preventing insulin antibody formation in man treated with porcine insulin in neutral solution (Andersen, 1973), very careful purification of porcine insulin in order to remove these components is needed to reduced antibody formation when porcine insulins with prolonged action are used (Andersen, 1975; Schlichtkrull et al, 1975; Poulsen and Deckert, 1977). Removal of these components is effected in various ways, resulting in a highly purified insulin ('mono-component', 'single-component', or 'rarely immunogenic (RI)' insulin). However, when a highly purified insulin is left to stand in solution, some of the insulin will again be transformed, so that disc electrophoresis after a few months will again show several fractions. However, it has been demonstrated by Schlichtkrull et al (1973), that injection of mono-component porcine insulin, even after storage for two years, does not lead to antibody formation in rabbits.

The content of proinsulin in highly purified insulin should be below 0.2 per mille (less than 0.2/ag proinsulin injected per day) as only porcine insulin preparation at such purity results in reduced antibody formation (Schlichtkrull, 1976). The names 'single-peak', 'single-component', 'MC', 'RI', 'monotop',


'chromatographically purified' etc. are not, by themselves, sufficient to characterize the purity of insulin preparations. The content of proinsulin and other impurities should be stated. Single-component insulins usually contain fewer impurities than single-peak insulins.

The careful purification of porcine insulin is of importance to the immunogenicity of the long-acting insulins, as has been demonstrated by a number of workers. The latest and largest series was published by Poulsen and Deckert (1977). However, highly purified insulin preparations containing bovine insulin of comparable purity are considerably immunogenic (Chance, Root and Galloway, 1976). Treatment of patients with 'dirty' insulin preparations can lead to the formation of specific proinsulin antibodies (Andersen, 1973), antibodies against some components (Schlichtkrull et al, 1975), and antibodies against vasoactive intestinal polypeptide (VIP), glucagon and pancreatic polypeptide (PP) (Bloom et al, 1976). By using highly purified insulin preparations the formation of antibodies against these components can be avoided (Bloom et al, 1976). Whereas the purity of insulin preparations is highly significant for their immunogenicity, the retarding substances play a minor and indirect role (Kulpe, 1958; Kern and Laugner, 1939; Grabar, 1961).

Factors in the recipient Different factors in the recipient play a role in the immunogenicity of insulin preparations. Diabetes as such does not seem to be'important (Deckert, 1964). Insulin extracted from pancreas of diabetic patients was shown to have the same biological, structural and immunological properties as human insulin extracted from non-diabetics (Brunfeldt, Deckert and Thomsen, 1969), and the ability of the immune system to respond to antigens seems not to be reduced in fairly well regulated diabetics (see below).

Treatment with cortisone or cyclophosphamide, however, reduced antibody formation (Korcakova, Titlbach and Nouza, 1972). The age of the patients also seems to be important, since the young have higher antibody titres than elderly patients (Andersen, 1972). Pregnancy, sex and infections seem not to play any role for the immunogenicity of insulin preparations (Andersen, 1972). The most important single factor is interrupted insulin treatment. Deckert and Grundahl (1970) demonstrated that, of 36 patients without detectable insulin antibodies, only those who had previously been treated with insulin developed antibodies after treatment with regular porcine insulin in neutral solution.

The duration of insulin treatment is of importance for the development of insulin antibodies. The maximal antibody titre after beginning treatment with porcine NPH-insulin was found four to six months later (Andersen, 1972).

Clinical Significance of the Immunogenicity of Insulin Preparations

Insulin antibodies play a role in the development of insulin allergy and insulin resistance, and seem to influence the daily insulin requirement. On


the other hand, their influence on the duration of remission, the stability of diabetes control, lipoatrophy and the development of late diabetic complications is more controversial.

Insulin allergy

Immediately after the introduction of insulin therapy, it was obvious that injection of insulin preparations might give rise to allergic reactions (Joslin, Gray and Root, 1922). However, these reactions have grown less common with the increasing purity of insulin preparations (Jorpes, 1949), and in patients treated exclusively with highly purified insulin preparations allergy has so far not been seen (Deckert, Andersen and Poulsen, 1974). It must be assumed, therefore, that insulin allergy is induced n o t by insulin itself but by polypeptide contaminants. However, once the patients are sensitized, even highly purified insulin may occasionally give rise to local allergic reactions (Galloway and Root, 1972; Federlin et al, 1974). A correlation between circulating insulin antibodies and allergic reactions is seen only with antibodies of the IgE type (Federlin et al, 1974) and not with IgG antibodies (Deckert, 1964). If the patient has troublesome insulin allergy the first step should be to investigate whether the symptoms can be overcome by changing to another insulin preparation. Attention should be directed to the species specificity and degree of purity of the insulin preparations. The pH, content of disinfectants and retarding substances are seldom of any importance (Oakley, 1976). In the Anglo-Saxon countries preparations containing insulin modified in different ways may be tried (Moloney, Aprile and Wilson, 1964; Kreines, 1965; Moloney and Jackson, 1973). If testing with highly purified insulins also induces a highly positive reaction, the patient must be desensitized, if he needs insulin therapy. Antihistamines are of no use, and cortisone is unnecessary. The desensitization process is carried out with a short-acting, highly purified insulin.

Insulin resistance

At one time insulin resistance was defined as the state where the daily dose of insulin had to exceed 200 iu to produce a clinical effect in non-ketoacidotic, insulin-treated persons (Martin et al, 1941). There is an increasing tendency to apply the term insulin resistance to cases in which the dose of insulin exceeds 100 iu/24 hours for several consecutive days. Not infrequently, insulin-resistant patients require several thousand units of insulin daily. In spite of the high dose of insulin, its effect is often minimal, and several patients succumb in a state of gradually developing ketoacidosis.

There are many causes of insulin resistance including intercurrent infections and the development of another endocrine disorder, particularly Cushing's syndrome or acromegaly. Gorden and colleagues at the National Institutes of Health (Flier et al, 1975; Kahn et al, 1976) have recently described a rare group of female patients with acanthosis nigricans and insulin resistance due to an alteration in the insulin--receptor interaction. They suggest that these patients can be divided into two groups: Type A, a syndrome in young women with signs of virilization in whom the receptor defect may be primary; and Type B, a~syndrome in older women with signs of


an immunological disease in whom there are circulating antibodies to the insulin receptor.

One of the most common causes of insulin resistance is an excessive binding of insulin to circulating IgG insulin antibodies. Insulin resistance often co-exists with insulin allergy (Federlin, Ditschuneit and Pfeiffer, 1971). To confirm the diagnosis in non-allergic patients, an insulin sensitivity test must be done, using 0.1 units of a short-acting insulin injected intravenously per kg body weight and recording the blood glucose every 10 minutes. Normally, a distinct fall of the blood glucose occurs within 30 minutes. Moreover, the serum must be examined for insulin antibodies. In immunologically conditioned insulin resistance the insulin binding capacity of the serum is very high, but there may be exceptions. If the insulin sensitivity test fails to induce a fall of blood glucose, and insulin antibodies are demonstrable, the patient has immunologically conditioned insulin resistance.

There is no doubt about the positive correlation between insulin antibody titre and the daily dose of insulin in patients whose daily dose is high (Federlin, Ditschuneit and Pfeiffer, 1971). However, 'normal' insulin antibody titres also seem to influence the daily dose (Andersen, 1972).

Period of remission

That insulin antibodies presumably influence the length of the remission period is apparent from a study by Ortved Andersen (1975) who followed insulin treated diabetics during the period of remission after insulin therapy had been initiated. After a follow-up period of one year only two-fifths of the patients who had formed insulin antibodies were still in remission, whereas two-thirds of the patients who did not form insulin antibodies were still in remission. The explanation of the influence exerted by the insulin antibodies upon the length of the remission period may well be that circulating insulin antibodies are able to bind and eliminate endogenous insulin (Grodsky et al, 1966), and thereby lead to a more rapid exhaustion of the patients' remaining B-cell mass. Ludvigsson (1976) also found a negative correlation between remission of diabetes and insulin antibody concentrations in juvenile diabetics.

Control of diabetes

Whether the control of diabetes is facilitated in the sense of fewer blood glucose fluctuations on highly purified porcine insulin preparations cannot be decided with certainty. Andersen (1972) demonstrated that the quality of the diabetes control in young diabetics is significantly correlated to the insulin-binding capacity in-their serum, but this did not apply to elderly patients. Dixon, Exon and Hughes (1972) came to the opposite conclusion, viz. that patients with an unstable metabolic balance had small amounts of insulin antibodies or insulin antibodies of high avidity, whereas patients in stable metabolic balance had larger quantities of insulin antibodies of low avidity in the serum. In a recent publication, however Ludvigsson (1976) found a strong correlation between low concentration of insulin antibodies and quality of metabolic control.


Late diabetic complications It is uncertain whether circulating, dissolved insulin--insulin antibody complexes play a role in the development of late diabetic manifestations. F~511ing (1975) has shown that high molecular insulinlinsulin antibody complexes develop during immunization against insulin and that some of these complexes are able to bind complement. He could not rule out the possibility that the complexes might be harmful to small blood vessels. Wehner, Huber and Kronenberg (1973) found changes in the basement membrane of glomerular capillaries from mice treated with immunogenic insulin preparations, but not in mice treated with insulin preparations of low immunogenicity. However, these findings were not confirmed by Westberg and Michael (1972) or H~igg (1974). On the other hand Andersen (1976) found high insulin antibodies significantly more frequently in juvenile diabetics who developed proliferative retinopathy and/or nephropathy early in the course of diabetes. He thinks that insulin antibodies cannot be the cause of nephropathy or retinopathy in diabetics but that certain insulin-- insulin antibody complexes might influence the function of blood vessels and in this way play a part in the development of late diabetic manifestations.

Avoidance of insulin antibody formation Since insulin antibodies are potentially dangerous, their formation should be avoided if the costs are not too high. This is best achieved by using soluble regular porcine insulin preparations in neutral solution and/or slow acting insulin preparations manufactured from highly purified porcine insulin with a proinsulin content of less than 0.2 per mille. If the patient has never had insulin before, the chance of developing insulin antibodies will be about 10 to 20 per cent after one year, and the titres are usually low. Patients whose treatment is liable to be interrupted (e.g. pregnant women with minor degrees of diabetes, insulin treatment in tablet-treated diabetics during stress, operation etc.), and also juvenile diabetics, should be treated with these preparations.

Insulin antibodies present after treatment with bovine or mixed bovine-- porcine insulin preparations can usually be reduced by changing to highly purified porcine insulin preparations (Oakley, 1976). It is recommended that, immediately after changing the preparation, the daily insulin dose should be lowered by 10 to 20 per cent in order to avoid severe hypoglycaemia (Daggett, Mustaffa and Nabarro, 1976).

Insulin antibodies present after treatment with porcine insulin, which is not highly purified, cannot usually be reduced by changing to highly purified porcine insulin, although proinsulin antibodies can be eliminated (Frilling et al, 1976).

IMMUNE FUNCTION AND HOST DEFENCE

Subpopulations of T and B lymphocytes have been found to be normal in diabetic patients (MacCuish et al, 1974b). Lymphocyte transformation to non-specific mitogens such as phytohaemagglutinin (PHA) is generally accepted as an in vitro indicator of T-lymphocyte function and there have


been reports claiming that PHA-induced lymphocyte transformation is reduced in diabetics (Delespesse et al, 1974). However, most investigators relate an impaired PHA response in diabetics to the degree of metabolic control (MacCuish and Irvine, 1975), since the decreased PHA response can be restored to normal by normalizing the metabolic state of the individual tested.

The function of the reticuloendothelial system in diabetics also seems to be well correlated to quality of regulation (Berken and Sherman, 1974).

Bagdade (1976), in a brief review, comments upon granulocyte function in diabetes. He concludes that chemotaxis, adherence to endothelium prior to diapedesis into extravascular compartments, phagocytosis and intracellular microbicidal function are all largely dependent on metabolic control.

Thus no defect in host defence specific to any type of diabetes has been reported, but the available data provide ample evidence to advocate good metabolic control.

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2 Immunity and autoimmunity in diabetes mellitus

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