ORGANIZACIÓN INTERNACIONAL DE ESTANDARIZACIÓN

Insulin-dependent diabetes mellitus (IDDM) is histologically characterised in its early stages by a mononuclear cell infiltration of the pancreatic islets denoted "insulitis" (Von Mering, 1889) accompanied by selective p cell destruction. The presence of such an inflammatory infiltrate suggested an autoimmune aetiology for IDDM. Furthermore cell- mediated autoimmunity may play an important role in the pathogenesis of the disease resulting in specific 6 cell degeneration and insulin insufficiency in both the human and animal models of IDDM. The initiating event and the role of specific immune cells in mediating 6 cell damage have yet to be delineated. Examination of new-onset type-1 diabetes patients has demonstrated many abnormalities in T cell subsets in both the periphery and the pancreas. Elevated killer ceU (K) activity (Pozzilli gf a/, 1979), imbalance in T cell subsets (Gupta, et a l, 1982; Jackson e ta l, 1982; Donen e ta l, 1984; Rodier e ta l,

1984 ) or the presence of activated T cells (la'*’) (Alviggi et al, 1984; De Berardinis et al,

1988A) have all been demonstrated in the peripheral blood of type 1 diabetic patients. The frequency of insulitis in IDDM patients with recent disease onset observed by different studies is inconsistent In a study by Gepts (Gepts, 1965) 16 of 23 pancreata from patients with recent onset IDDM showed insulitis, however Doniach and Morgan (Doniach and Morgan, 1973) did not observe this phenomenon in the 13 patients they examined. The extent of insulitis within the pancreata of individual IDDM patients is variable, not all islets are equally affected and insulitis is more pronounced among young patients. Such discrepancies in the frequency and extent of insuhtis observed in human IDDM are reflected in the spontaneous animal model of the disease and thus appear to be due to the disease pathology. Indeed studies by Gepts (Gepts, 1965) revealed in some pancreatic lobules islets devoid of p cells and insuhtis, in others many large intact islets with only a few lymphocytes peri-islet, and yet others with partial insulin content and lymphocytic infiltration. Given these findings of a very patchy histopathology it is therefore not surprising that results from different studies are not in accordance. Additionahy once P ceh destruction is complete ie. at chnical presentation of type 1 diabetes

about 70% of the islets contain no insuhn-secreting P cells and no infiltration. This is presumably because the antigenic stimulus, the B ceh, has been removed by autoimmune destruction. There is a report suggesting insuhtis particularly affects those islets where residual p cehs are sthl present (Fouhs etal, 1986) suggesting that some beta ceh antigen

remains.

Very few studies have been reported regarding pancreatic lymphocyte infiltrates in the prediabetic period of the human owing to the difficulty in performing pancreatic biopsies. Bottazzo found Ig bearing lymphocytes, NK cells and all subsets of T lymphocytes, although mainly Tc/s (CD8+) cells, in the pancreatic lesions of a newly diabetic child (Bottazzo et al, 1985). Analysis of the pancreata from long term IDDM patients with recurrent disease who had received pancreatic transplants from their discordant, identical twins demonstrated the presence of macrophages and T cells (mainly CD8+) in the insulitis lesions (Sibley etal, 1985).

Studies have also shown that T cells from diabetic patients can inhibit glucose and theophylline induced insulin release from murine islets and are cytotoxic to rat islet cells (Boitard et al, 1982; Charles et al, 1983; Boitard et al, 1984). Indeed many studies have indicated that T cells play a fundamental role in the development of IDDM, particularly in the spontaneous animal models, the NOD mouse and BB rat. NK cells have also been implicated in P cell destruction in the BB rat (MacKay et al, 1986). Insulitis commences at about 5-6 weeks of age in the NOD mouse and is observed in more than 90% of both male and female NOD mice at 200 days. Development of overt diabetes due to 6 cell destruction is observed generally in 70-80% of females and 10-20% of males at 210 days (Tochino, 1987).

Experimental evidence suggests that T lymphocytes play a major role in the development of diabetes in the spontaneous models of the disease as neonatal thymectomy prevents diabetes in both the NOD mouse (Ogawa et al, 1985) and the BB rat (Like et al,

1982). Treatment with anti-thymocyte serum or anti-Thy-1.2 antibody markedly suppresses the development of overt diabetes in NOD mice (Harada et al, 1986). Thus thymus dependent cell-mediated autoimmune mechanisms are responsible for the pathogenesis of insulitis in NOD mice, the primary change leading to the development of diabetes. In addition mononuclear cell infiltration of the lacrimal and submandibular glands is delayed by the same treatment regimes which diminish insulitis, suggesting that infiltration in various glandular tissues of NOD mice is also produced by T cell mediated autoimmunity (Hayward etal, 1988).

Conflicting data have been reported concerning the percentage of lymphocyte subsets infiltrating the pancreas. A longitudinal study by Signore of the NOD endocrine pancreas showed that CD4"*", MHC class n"*" and surface IgM"*" cells are the predominant subsets detectable in insulitis (Signore etal, 1989). Additionally he noticed that 30 % of T

cells expressed surface H-2 receptors implying that they were activated. Once all the 6 cells had been destroyed, lymphocytes abandoned the islets. Hanafusa also investigated longitudinal changes in lymphocyte subsets in the NOD and pancreas found a predominant infiltration by activated T lymphocytes including ThA (helperAnducer) and Tc/s (cytotoxic/suppressor) observed in the early stages of insulitis (Hanafusa etal, 1987). NK cells were also detected in the lesion. In this study, Ig bearing cells were shown to increase in number with progression towards insulitis. Whereas T lymphocytes were localized close to islets, B cells appeared adjacent to blood vessels and around T cell clusters. The percentage of splenic T lymphocytes was also markedly increased in the initial stage of insulitis suggesting that T rather than B lymphocytes participate in the development of insulitis, accompanied by marked splenic T cell proliferation which corresponded to the beginning of T cell infiltration into islets. Like demonstrated activated T cells in the diabetic BB rat pancreas but B lymphocytes were observed infrequently (Like

et al, 1979, 1986). Walker also observed mainly Th, Tc/s and macrophages in the prediabetic BB rat (Walker gW , 1988A). Although studies in the human, NOD mouse and BB rat conflict within species and between species the basic principle remains that the insulitis consists of T cells (both of the Th/i and Tc/s phenotypes), monocytes and B cells to varying degrees. It remains to be clarified what the role of each particular cell type is, and if indeed they can be assigned a role or are merely present in the lesion due to nonspecific inflammatory recruitment Therefore it is essential to observe the disease manifestations during its early phases to eliminate the latter possibility.

The essential requirement for T cells in disease manifestations in the NOD mouse was observed by Wicker et al (Wicker et al, 1986), who demonstrated that adoptive transfer of splenocytes from overtly diabetic NOD mice induces diabetes within 12-22 days in 95% of irradiated (650-750 rads) mice older than 6 weeks of age. If either T cell subset is depleted from the donor cell inoculum then neither severe insulitis, nor diabetes, results (Miller etal, 1988). Such an adoptive transfer system provides an excellent model, inducing diabetes within a defined and reproducible time frame to allow manipulation of the immune system aimed at regulating the onset of diabetes. The neonatal adoptive transfer system described by Bendelac is also dependent on both L3T4+ and Lyt-2+ T cell populations (Bendelac et al, 1987).

In this chapter I have characterised the normal pancreatic infiltrate in the male and female NOD mouse longitudinally by immunohistochemistry of cryostat sections with monoclonal antibodies to T cell subsets, class I MHC antigens, class n MHC antigens, macrophages and B cells. My findings for the T ceU subset analysis are similar to those of

previous authors but it is essential to characterise the time course of infiltration for each NOD mouse colony as the disease incidence is so variable. Normally, pancreatic 6 cells of mice constitutively express low levels of class I, but do not express class II antigens (Faustman et al, 1980). However given that over-expression of class I MHC proteins on pancreatic islet cells is characteristic of autoimmune type I (insulin dependent) diabetes mellitus in humans who die soon after diagnosis (Bottazzo etal, 1984, 1985; Foulis etal,

1987A,B), it was of interest to examine if this phenomenon occurred in the NOD spontaneous animal model of IDDM. It is difficult to discover whether class I MHC antigen over-expression is due to the presence of inflammatory cells or whether both are secondary to another event such as virus infection. These are aU important questions that are difficult to resolve in humans because the pathology is invariably far-advanced at clinical presentation and pancreatic tissue is rarely available. However class I MHC antigen over-expression is also present in the animal models of Type I diabetes in the BB rat ( Ono

eta l ; 1988) and in the multi-low dose STZ mouse (Campbell et al ; 1988A, Cockfield e t al ; 1989). These observations and my own studies on the NOD mouse provide the opportunity to relate the expression of class I MHC antigens to other pathological changes associated with p cell destruction. Much controversy surrounds the expression of MHC

class n on pancreatic P cells in both the human and the NOD mouse. My studies in the transfer system have hopefully, delineated more precisely the class II MHC antigen expressing cells within pancreatic infiltrates.

The incidence of diabetes at 30 weeks of age in the NOD/CRC colony is 80% for females and <10% for males. Given that the full manifestation of diabetes is not evident until 30 weeks of age the adoptive transfer model provided me with an excellent experimental model for reproducing diabetes. Therefore I characterised the pancreatic infiltrates present in the irradiated recipient pancreas at weekly intervals after the transfer of diabetic spleen cells. Having completed this study the time interval between transfer and the first evidence of insulitis was ascertained thereby providing a time point or "window" for commencement of immunogenic regimes to prevent the onset of disease. Such investigations will be discussed in the next chapter.

5.2 Results

5.2. Ki") Spontaneous Progressive Pancreatic Infiltration In Female NOD Mice. Female NOD/CRC mouse pancreata were analysed at 1,3 and 5 months of age by immunohistochemistry of pancreatic cryostat sections by the methods previously described. It can be seen from Table 5.1 A that at 1 month of age 26% of islets from the pancreata of female NOD mice demonstrated the presence of peri-islet infiltration and 4% the presence of intra-islet infiltration when stained with MRC-0X6 which detects NOD I- A. However by 3 months of age 56% of islets from NOD female mice demonstrated moderate peri-islet infiltration and 28% moderate intra-islet infiltration with complete destruction of 23% of islets. This 6 cell destruction continued until at 5 months 44% of islets in female NOD mice had been destroyed with only 9% of islets being completely unaffected. MHC class II expression was not observed on 6 cells or the endocrine cells of female NOD pancreas at any of the time points analysed. A similar pattern of infiltration was observed following the fate of CD3+ T cells. At 5 months of age 60% of islets demonstrated moderate peri-islet infiltration and 28% moderate intra-islet infiltration (Table 5.2A). In addition to class 11+ ceUs and T cells, cells bearing the F4/80 marker (mature macrophages) also demonstrated a progressive infiltration of pancreatic islets in female NOD mice with 95% and 14% demonstrating severe peri- and intra-islet infiltration respectively at 5 months of age (Table 5.3A). Accompanying this mononuclear cell infiltration, expression of class I MHC antigens in the pancreas underwent a marked change. Normally class I MHC antigen is expressed on resident macrophages but during insulitis it is not only expressed on infiltrating cells but also on pancreatic endocrine and the surrounding exocrine, tissue. Such hyperexpression of class I MHC antigen was observed in 81-82% of islets in 3-5 month old female NOD mice (Table 5.4A).

5.2.1 fifi Spontaneous Progressive Pancreatic Infiltration In Male NOD Mice

Male NOD/CRC mouse pancreata were analysed at 1, 3, and 5 months of age by immunohistochemistry of pancreatic cryostat sections by the methods previously described. It can be seen from tables (5. IB, 5.2B, 5.3B, 5.4B) that the pattern of lymphocytic infiltration in the male NOD pancreas follows a more prolonged time course and is less severe when compared to age matched female NOD mice (Figure 5.1C,D). At 1 month of age only 10% of male NOD islets had peri-islet infiltration and 1% intra-islet infiltration when stained with MRC 0X 6 (Table 5.IB), compared with age-matched females which had 25% and 4% moderate peri- and intra-islet infiltration respectively. This trend could be demonstrated at later time points in the male NOD mouse as at 3 and 5 months 47% and 58% of islets respectively demonstrated no peri-islet infiltration compared

to female NOD mice in which only 32% and 9% respectively had no peri-islet infiltration. In addition, absence of intra-islet infiltration in the male ranged from 87-98% from 3-5 months whereas in the female ranged from 71-46%. Thus the male NOD mouse demonstrated a progressive increase in peri-islet infiltration with minimal-moderate intra­ islet infiltration. This is consistent with the observed low incidence of diabetes in the male of <10% compared to 70% for females which demonstrate predominantly severe intra-islet infiltration at 5 months of age. Such a pattern of predominantly peri-islet infiltration observed in the male NOD mouse could also be demonstrated at 5 months of age since only 3% of islets demonstrated moderate presence of T cells at intra-islet locations (detected with a mo. Ab to CD3 present on the surface of T cells) (Table 5.2B) and macrophages respectively (Table 5.3B). It has to be noted that there is a constitutive population of macrophages present at peri/intra-islet locations in both male and female NOD mice independent of progression to diabetes, which can accumulate to moderate numbers (70% in male NOD mice at 5 months of age) this will be described later in this chapter. However hyperexpression of class I MHC antigen could be observed in 57% of male NOD mice at 5 months of age whereas 81% of female NOD mouse islets demonstrated hyperexpression of class I MHC antigens (Table 5.4B). Thus all the elements of the immune response; T cells, macrophages and class 1 / II MHC antigen positive cells, are present at peri-islet locations in the male NOD mouse but in most animals do not progress to intra-islet sites.

5.2.2 Diabetic Spleen Cell Transfer-Time Course of Inflammatorv Events

We utilised the adoptive transfer model of Wicker et al (1986) to facilitate a histological analysis of the temporal relationships between pancreatic lymphocytic and macrophage influx and the expression of MHC antigens and adhesion molecules in the prediabetic period before disease onset in recipient mice. Seventeen non-diabetic sublethally irradiated, male NOD mice aged 10-14 weeks were injected i.v. with 2x10^ spleen cells from diabetic adult donors and killed randomly at weeks 1, 2, 3 and 5 for histological analysis. Four age-and sex-matched control irradiated NOD mice were injected i.v. with 2x10^ syngeneic, non-diabetic spleen cells from adult mice and analysed 5

Histological changes occurring in the pancreas from the time of diabetic spleen cell transfer until the onset of diabetes.

5.2.2Ci) T cell sub-DODulations

All mice which received the non-diabetic spleen transfer remained normoglycemic with only 36% and between 9% and 18% of islets displaying peri and intra-islet infiltration respectively (Table 5.5). This minimal insulitis is that which would be expected of male NOD mice of this age.

Pancreata fi*om mice which had received diabetic spleen cells displayed neither lymphocytic infiltration nor evidence of beta cell destruction until week 2, however class II MHC antigen expression was observed on blood vessel associated cells adjoining intact islets at week 1. Class II expression on blood vessel-associated cells was not seen in the recipients of non-diabetic spleen cells at week 5 suggesting that this observation was a result of the homing of the diabetogenic effector cells. At 2 weeks after transfer 67-75% of islets analysed had peri-islet infiltration of both L3T4+ and Ly-2+ T lymphocytes (Figure 5.1 A, B). Lymphocytic infiltration of islets was rapid and 75% of recipients were diabetic 3 weeks after transfer. At this point only 19% of islets were not infiltrated with 60-65% demonstrating severe intra-islet infiltration. T cells in the inflammatory infiltrate were

(data not shown)

positive for IL-2 receptor expression. Five weeks after transfer all remaining recipients of diabetic spleen cells were diabetic and 58% of islets examined contained remnants devoid of beta cells with little remaining mononuclear cell infiltrate. Those islets retaining beta cells showed extensive intra-islet infiltration and destruction of islet morphology (Table 5.5). Residual islets were not observed in pancreata from non-diabetic spleen cell recipients and aU islets analysed had full insuhn content. Irradiation of recipients was found to be essential for disease transfer as non-irradiated recipients did not develop diabetes or manifest this pattern of intra-islet infiltration and beta cell destruction (Table 5.6). Few B cells were detected, those observed were at peri-islet locations

5.2.2 fii) T cell receptor VB usage bv Islet infiltrating cells

To characterise in further detail, the T cell subpopulations present in the pancreatic inflammatory infiltrates in the NOD mouse, histological analysis of the pancreas was performed using the monoclonal antibodies KJ16, 44.22.1 and KTl 1 which recognise T cell receptors using V68.1,2, V66 and V611 chains respectively. Examination of non­ diabetic 4 month old male NOD mice revealed that T cells representing all of these V6

chains were present in the pancreatic infiltrate. Such intra-islet infiltration observed was minimal with most infiltration being predominantly peri-islet.

cell receptor VB usage. Table 5.7 summarises the data from two different transfer experiments. Initially V68.1,2 bearing T cells were present in greater number in peri-islet locations than V66 and VBll bearing T cells which were almost undetectable at week 1. By week 2, V68.1,2, V66 and V Bll were all present peri-islet. This predominance of VB8.1,2 was also demonstrated at intra-islet locations at later time points. Since the heterogeneity and extent of VB usage also reflected the proportion of all three VB bearing T cells present in the peripheral blood of NOD mice, this implies that there was no unusual bias toward usage of these three VB chains (Table 5.7 ) Such a heterogeneous pattern of infiltration was also observed in islets of spontaneously diabetic female NOD mice.

5.2.2.riiil The Presence of Macrophages In The Inflammatorv Infiltrate.

The best characterised of the macrophage antibodies utilised is M l/70 which defines the glycoprotein antigen, macrophage 1 molecule (Mac-1) found on the surface of macrophages, monocytes, granulocytes and NK cells. The MAb F4/80 binds to F4/80 antigen present on mouse macrophages but not detectable on polymorphonuclear leukocytes or lymphocytes.

SER-4 is a MAb which recognises the sheep erythrocyte receptor present on