Analysis of islet inflammation in human type 1 diabetes

Abstract

The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post-mortem from 29 patients (mean age 11.7 years) with recent-onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8(+) cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68(+)) were also present during both early and later insulitis, although in fewer numbers. CD20(+) cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138(+) plasma cells were infrequent at all stages of insulitis. CD4(+) cells were present in the islet infiltrate in all patients but were less abundant than CD8(+) or CD68(+) cells. Forkhead box protein P3(+) regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8(+) cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20(+) cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3(+) T cells do not appear to be required for beta cell death.

Fig. 1

Immunostaining of individual immune cells in serial sections of a single islet from a patient with recent-onset type 1 diabetes (T1D). The micrographs reveal the extent of endocrine cell immunostaining and the immune cell complement in serial sections of one representative islet. Sections were stained with anti-sera raised against glucagon (a), insulin (b), CD45 (c), CD3 (d), CD4 (e), CD8 (f), CD20 (g) and CD68 (h) respectively. All images were taken at ×200 magnification.

Fig. 2

Relationship between insulin immunopositivity and the complement of defined subsets of immune cells in the islets of patients with recent-onset type 1 diabetes (T1D). The number of immunopositive CD4⁺, CD8⁺, CD20⁺ and CD68⁺ cells were counted in a total of 279 islets across 29 patients with T1D. These were then correlated with insulin immunopositivity, as quantified by morphometry. The islets were subdivided according to their percentage insulin-positive area, which was taken to be representative of the residual beta cell mass. Each point is the mean ± standard error of the mean for the number of relevant immune cells detected in each islet section. The number of islets analysed in each insulin-positive category is given in parenthesis.

Fig. 3

Immunodetection of forkhead box P3 (FoxP3)⁺ or CD138⁺ cells in inflamed islets from patients with recent-onset type 1 diabetes (T1D). The micrographs reveal the presence of FoxP3⁺ T cells (a) within a single islet from a T1D patient. (b) FoxP3⁺ staining of a section of human tonsil as a positive control. CD138⁺ (c) and insulin (d) immunostaining respectively, on serial sections of a single representative islet from a second patient with T1D. (a) ×400 magnification; all other images taken at ×200 magnification.

Fig. 4

Immunodetection of natural killer (NK) cell markers in inflamed islets from patients with recent-onset type 1 diabetes (T1D). The micrographs demonstrate the immunostaining patterns for anti-CD56⁺ (a), CD57⁺ (b) and Pen5⁺ (c), respectively, in heavily inflamed islets from a single patient with recent-onset T1D. An example of a nerve that is stained positively for CD56 is highlighted by a red arrow in (a). While endocrine cells are stained by all these NK cell antibodies, lymphocytes which are clearly visible within each islet by virtue of their dense nuclei and small size do not stain (examples are indicated by black arrows in a, b and c). All images were taken at ×200 magnification.