Clin. exp. Immunol. (1992) 88, 410-413

Anti-endothelial cell antibodies in insulin-dependent diabetes mellitus A. G. WANGEL, S. KONTIAINEN*, T. SCHEININt, A. SCHLENZKA*, D. WANGEL & J. MAENPAAX Department of Bacteriology and Immunology, University of Helsinki, Departments of *Microbiology and JPaediatrics, Aurora Hospital, Helsinki, and tIV Department of Surgery University of Helsinki, Helsinki, Finland (Acceptedfor publication 18 February 1992)

SUMMARY The prevalence of IgG class antibodies to endothelial cells (AECA) was studied in 136 young patients with insulin-dependent diabetes mellitus by an enzyme immunoassay using human umbilical cord vein endothelial cells. The patients were divided into four groups according to the time between diagnosis and study and their results were compared with those in control children and blood donors. AECA became progressively more frequent with the duration of diabetes, being 4% in diabetics tested within 2 weeks of diagnosis and reaching 34% after an average disease duration of 11 -2 years. They were not more common in patients with neuropathy, retinopathy or nephropathy than in patients without these complications, but were associated with co-existing thyroid disease and IgA deficiency. The results suggest that in insulin-dependent diabetes mellitus AECA are associated with co-existing autoimmune disorders but not with diabetic microvascular disease. Keywords anti-endothelial cell antibodies insulin-dependent diabetes mellitus microangiopathy thyroid disease IgA deficiency

of diagnosis; group B, 20 children whose samples were taken 3-9 months after diagnosis; group C, 21 patients studied 1 year after diagnosis; and group D, 50 patients studied on an average of 11 2 years after diagnosis (range 5-0-18 1 years). Three groups of control subjects were included: (i) 54 children with endocrine disorders, excluding IDDM, who attended the endocrine clinic at the Aurora Hospital; (ii) 82 children whose blood samples had been obtained as part of routine preoperative screening for minor procedures at the Aurora Hospital or whose symptoms were regarded as psychosomatic in origin; and (iii) 99 volunteer blood donors from the Finnish Red Cross Blood Transfusion Service. The disorders included: juvenile autoimmune thyroiditis (n = 20) as defined by Kontiainen et al. [13], Graves' disease (n= 15), congenital hypothyroidism (n = 2), toxic goitre (n = 2) and growth disturbances (n = 10). Informed consent was obtained from the patients or their parents and the study was accepted by the Ethics Committee of the Aurora Hospital.

INTRODUCTION Evidence to suggest participation of autoimmune mechanisms in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) includes the presence of circulating antibodies to insulin [1] and to pancreatic islet [2] cells, of T cells infiltrating the islets [3] and of increased numbers of activated circulating T cells from which lines and/or clones cytotoxic to islet cells and proliferating in response to islets can be raised [4-6]. Since the long-term complications of IDDM are due at least in part to microvascular disease, and since anti-endothelial cell antibodies (AECA) are frequently found in diseases such as systemic lupus erythematosus (SLE), systemic sclerosis, rheumatoid vasculitis and microscopic poly-arteritis, in which vascular damage is prominent [7-12], it seemed appropriate to ascertain their prevalence in IDDM and their possible association with complications of diabetes. The results described in this report indicate that AECA do occur in IDDM and that they are more common in patients with long-standing diabetes and associated autoimmune disease, but not in patients with diabetic neuropathy, retinopathy or nephropathy.

Methods AECA were sought by a cellular immunoassay, similar to that described by Rosenbaum et al. [14]. Briefly, human umbilical vein endothelial cells were harvested by collagenase digestion

SUBJECTS AND METHODS Patients and controls Four groups of patients were studied: group A, 45 diabetic children whose blood samples had been collected within 2 weeks

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410

Anti-endothelial cell antibodies in diabetes

411

Table 1. Prevalence and mean levels of anti-endothelial cell antibodies (AECA) in diabetics and in control

subjects Age at time of study n

45 Group A 20 GroupB 21 Group C 50 Group D Control Children 82 Adult 99 Red Cross blood donors Children (endocrine, not IDDM) 54

Positive

(years, mean + s.d.)

%

n

10-1 +3-9 74+35 7 9+31 19 6+4 8

2 0 2 17

85+42

AECA

P

P

(Student's (Fisher's (U/ml, mean (s.d.)) exact test) t-test)

15 2 (6 9)

44 0 95 34

155(85) 18-3 (20 2) 32 6 (25-5)

0

0

125(65)

N/A

2

2

113 +49

6

11-1

0 03 009 0-03 10-6

012 099 0 004

10-6

94 (59) 15 1 (17-8)

022

0-003

P values were calculated from comparisons with control children. IDDM, insulin-dependent diabetes mellitus.

gentamycin. At this stage, the cells were detached by exposure to trypsin (0-2% in 0-05% EDTA, GIBco) and either subcultured or pooled and transferred to 96-well gelatin-coated Costar flatbottomed plates at a density of 1-2 x 104 cells/well. Cells were used between the second and fifth passages. When confluent, the cells were fixed for 5 min in 0-2% glutaraldehyde and washed three times for 3 min in PBS under sterile conditions. Plates prepared in this way and sealed with plate sealer tape (Flow Laboratories) could be stored for up to 2 months before use.

120 100

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6

Anti-endothelial cell antibodies in insulin-dependent diabetes mellitus.

The prevalence of IgG class antibodies to endothelial cells (AECA) was studied in 136 young patients with insulin-dependent diabetes mellitus by an en...
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