ANTI-COLLAGEN TYPE IV IgG SUBCLASSES AND THE DEVELOPMENT OF DIABETIC MICROVASCULAR COMPLICATIONS

Transcription

1 Division of Biology, Department of Biology and Pathological Physiology University School of Medicine 1, St. Kliment Ohridski Street 58 Pleven, Bulgaria Original Scientific Paper ANTI-COLLAGEN TYPE IV IgG SUBCLASSES AND THE DEVELOPMENT OF DIABETIC MICROVASCULAR COMPLICATIO George Nicoloff Key words: diabetes mellitus, anti-collagen type IV IgG subclasses, diabetic vascular complications SUMMARY Levels of anti-collagen type IV IgG (ACIV IgG) subclasses were determined by ELISA in sera of 65 children with type 1 (insulin-dependent) diabetes mellitus (mean age 12.1±2.8 years, diabetes duration 5.5±2.8 years). Seventeen diabetic patients were positive for ACIV IgG subclasses, having been diabetic for more than 5 years. Thirty-four percent (22/65) of the patients had diabetic vascular complications, i.e. 1 retinopathy and 12 microalbuminuria, all of whom had diabetes duration for more than 5 years. Sixty-eight percent (15/22) of the patients with vascular complications were positive for ACIV IgG subclasses, i.e. 5% (/22) for IgG1, 13% (3/22) for IgG2, and 36% (8/22) for IgG3. ACIV IgG1 were independently associated with microalbuminuria (r=.35, p=.2), duration of diabetes (r=.35, p=.2), HbA1c (r=.33, p=.5), anti-age antibodies (r=.88, p=.3) and ACIV IgG3 with microalbuminuria (r=.39, p=.1). In conclusion, our study showed a significant relationship between serum ACIV IgG1 and IgG3 and development of diabetic microangiopathy. INTRODUCTION Patients with juvenile onset type 1 (insulindependent) diabetes mellitus are at a high risk of diabetic microangiopathy and vascular disease. The angiopathic complications usually developed after several years of type 1 diabetes (1). We recently found an increased serum level of the main protein of basement membrane collagen type IV in diabetic children with microalbuminuria (2). These soluble collagen type IV-derived peptides are a pathologic stimulus for the production of collagen antibodies. In our recent study we found the IgG1 to another main connective tissue protein, elastin, to show strong correlation with the development of diabetic retinopathy (3). Because there is evidence that IgG is deposited in the walls of small blood vessels in diabetes (4), in the present study we continued our investigation with the detection of serum anti-collagen type IV IgG (ACIV IgG) subclasses. Each IgG subclass (IgG1 IgG4) has different biological and physiochemical properties. The IgG subclass may be preferentially produced in response to different antigens and pathologic conditions. This study was undertaken to assess the relationship between ACIV IgG subclasses and vascular complications in children with type 1 diabetes mellitus. SUBJECTS AND METHODS Subjects The baseline study population consisted of 65 (31 boys and 34 girls) patients with type 1 diabetes mellitus (mean age 12.1±2.8 years) and a control group Diabetologia Croatica -3,

2 DIABETIC MICROVASCULAR COMPLICATIO of age- and sex-matched healthy children with no family history of diabetes, atherosclerosis or emphysema. The mean duration of diabetes was 5.5±2.8 years. Thirty-four percent (22/65) of the patients had diabetic vascular complications, i.e. 1 retinopathy and 12 microalbuminuria. All subjects were taking 2-4 s.c. insulin doses per day. Microalbuminuria was defined as persistent urinary albumin excretion rate (AER) in the range of 2 and 2 µg/min in sterile urine. None of the patients had a diagnosis of renal disease unrelated to diabetes. Methods ACIV IgG subclasses were detected by an enzymelinked immunosorbent assay (ELISA). All reagents were added in 1 µl volume per well. Polystyrene plates were coated with 1 µg/ml dilution of human CIV (SIGMA, USA) and incubated at room temperature for 18 h, and then washed with phosphate-buffered saline (PBS) + Tween 2. Control and patient sera (diluted 1:1 in.5% PBS + Tween 2) were added to the wells, incubated for 1 h at 37 C, and subsequently washed four times with PBS. A 1:1 dilution of human sera was utilized after preliminary experiments with serial dilutions of patient sera and immunoglobulins had been used to determine optimal conditions. A secondary antibody (mouse anti-human IgG1, IgG2, IgG3, IgG4; Sigma, USA) was then added at a dilution of 1:1 in.5% PBS + Tween 2, incubated for 1 h at 37 C and subsequently washed. A tertiary antibody (HRP-conjugated goat anti-mouse IgG; Sigma, USA) was added at a dilution of 1:1 in.5% PBS + Tween 2 and incubated for 1 h at 37 C, followed by PBS washing. Finally, o-phenylenediamine was added and incubated at room temperature in a dark chamber for 3 min. The color reaction was stopped with 5 µl 8 N N 2 SO 4. Optical densities (OD) were measured by Microelisa Reader 21 (Organon Teknika, Belgium) at a wavelength of 492 nm. Final OD values were determined by the mean of triplicate readings. Relative IgG subclass levels were expressed as the ratio of OD yield obtained with subclass-specific reagents and OD obtained with anti- IgG (gamma-specific) reagents. For ACIV IgG subclasses the test values were considered positive if they had an OD >mean + 2SD of the mean OD value of normal healthy control values, which were used to establish the normal range for the assay. Ophthalmoscopy through dilated pupils was carried out in all diabetic patients to assess the presence of retinopathy, with all patients studied by the same ophthalmologist. Glycated hemoglobin, total cholesterol and triglyceride concentrations, arterial blood pressure, AER and antibodies to advanced glycated endproducts (anti-age) were measured as previously described (5). Statistical analysis All values are expressed as mean ± SD. Statistical analysis was done using computer programs Excel and Statgraphics Plus for Windows. Student s t-test, χ 2 of Pearson and ANOVA were used to assess differences between study groups. The correlation analysis was also used. The level of significance was set at p<.5. RESULTS ACIV IgG subclasses were detected in all tested sera of diabetic and healthy children. The upper normal limits (OD >mean + 2SD of the mean OD value of normal healthy control values) were as follows:.395 for IgG1,.31 for IgG2,.223 for IgG3, and.155 for IgG4. Table 1. IgG subclasses to human collagen type IV in sera of children with type 1 diabetes mellitus with or without vascular complications (VC): frequency of patients positive for each IgG subclass according to patient groups Group Sample size (n) Antibodies to collagen type IV Positive for antibodies to collagen type IV subclasses N egative (n) Positive (n) IgG1 IgG2 IgG3 IgG4 Normal /17 3/17 9/17 with VC 22 / 3/ 8/ without VC / 6 1/ 6 134

3 DIABETIC MICROVASCULAR COMPLICATIO Table 1 shows the frequency of patients with a raised level of IgG subclasses to human CIV. There was no healthy person with a raised level of ACIV IgG subclasses and no diabetic patient with a raised level of ACIV IgG4. Seventeen diabetic patients were positive for ACIV IgG subclasses, having been diabetics for more than 5 years. Twenty-six percent (17/65) of diabetic children were positive for ACIV IgG1, 6% (3/65) for IgG2 and 14% (9/65) for IgG3. Thirty-four percent (22/65) of the patients had diabetic vascular complications, i.e. 1 retinopathy and 12 microalbuminuria, all of whom had diabetes duration for more than 5 years. Fifty percent (/22) of the patients with vascular complications were positive for ACIV IgG subclasses: 5% (/22) for IgG1, 13% (3/22) for IgG2 and 36% (8/22) for IgG3 (Table 1). Positive for ACIV IgG subclasses were three patients with retinopathy and eight patients with microalbuminuria. Six of them were positive for anti-age antibodies. Fourteen percent (6/43) of the patients without vascular complications were positive for ACIV IgG subclasses, i.e. 14% for IgG1 and 2% for IgG3. Table 2 shows the frequency of patients with a raised level of IgG subclasses to human CIV in each group of vascular complications. Patients with retinopathy had a predominant restriction of IgG1 to CIV (3%). Raised levels of IgG2 and IgG3 were detected at a lower frequency (1% and 2%, respectively). The distribution among patients with microalbuminuria was 75% for ACIV IgG1, 17% for ACIV IgG2 and 5% for ACIV IgG3. Table 3 summarizes data comparing ACIV IgG positive and negative patients with respect to several risk factors for the development of vascular complications. The two groups were similar, as none of the variables showed a significant difference between the groups (p>.5). Table 3 also summarizes clinical data on diabetic children with and without vascular complications, showing the two groups to be similar for all variables (p>.5). Table 2. IgG subclasses to human collagen type IV in sera of children with type 1 diabetes mellitus with vascular complications (VC): frequency of patients positive for each IgG subclass according to VC groups Antibodies to collagen Group Sample size (n) type IV IgG1 IgG2 IgG3 IgG4 Normal Retinopathy Microalbuminuria Table 3. Clinical data of diabetic patients positive and negative for IgG subclasses to human collagen type IV (ACIV IgG) and of patients with or without vascular complications (VC) Clinical data Mean (yrs) Mean (yrs) age diabetes duration Mean glycated hemoglobin (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Triglycerides (mmol/l) Total cholesterol (mmol/l) ACIV IgG positive (n=17) ACIV IgG negative ( n=48) p with VC (n=22) without VC ( n=43) p.6± ± ± ± ± ± ± ± ± ± ± ± 1. 6 ± 15 1 (6%) 73± 12 1 (6%) 1.15 ±.63 3 (18%) 17 ± 14 1 (2%) 71± (%) 1.4±.43 6 (12%) 4.97± ±.85 4 (24%) 9 (19%) 5± 1 2 (9%) 72 ± 12 1 (4.5%) 1. ±.67 4 (18%) 17± 14 ( %) 71± 1 (%) 1.4 ±.61 5 (12%) 4.92 ± ±.96 5 (23%) 8 (19%) Diabetologia Croatica -3,

4 DIABETIC MICROVASCULAR COMPLICATIO ACIV IgG1 were independently associated with microalbuminuria (r=.35, p=.2), duration of diabetes (r=.35, p=.2), HbA1c (r=.33, p=.5), anti-age antibodies (r=.88, p=.3) and ACIV IgG3 with microalbuminuria (r=.39, p=.1). DISCUSSION There was a significant association between a raised level of IgG1 and IgG3 to human collagen type IV, and the development of diabetic microalbuminuria in children with type 1 diabetes mellitus. The prevalence of microalbuminuria in type 1 (insulin-dependent) diabetic patients is a sensitive predictor of future development of diabetic nephropathy. Microalbuminuria not only implies the presence of renal involvement in diabetes mellitus, but is also associated with generalized vascular damage as indicated by increased transcapillary leakage of albumin and markers of endothelial lesions. Patients with microalbuminuria had a higher frequency of ACIV IgG1 and ACIV IgG3 than either those with retinopathy or those without vascular complications. It is a suggestion that the raised level of ACIV IgG1 and IgG3 is associated with an increased risk for the development of microalbuminuria. There is evidence that proteins, including immunoglobulins, accumulate in the matrix lining the walls of small blood vessels in diabetic subjects. The amount of IgG cross-linked to the glomerular basement membrane is five times higher in diabetic than in nondiabetic rats (4). IgG binds preferentially to basement membranes, which contain AGE (4). Prolonged hyperglycemia results in irreversible cross-linkage of collagen containing AGE (6). Immunoglobulins bind to collagen - AGE complex, increasing attachment sites for other proteins and expanding the matrix. Macrophages react with new epitopes on CIV molecule (CIV with AGE) and release monokines, tumor necrosis factor and interleukin-1. The latter is an increased nonspecific pathologic stimulus for the production of more ACIV IgG subclasses, which will react with the new epitopes and maybe will cross-react with normal epitopes of CIV molecule. The presence of IgG subclasses in patient sera has implications for the nature of the antigen that has triggered the response. Thus, IgG1 and IgG3 are particularly associated with the immune response to proteins (7,8). In contrast, IgG2 is more frequently seen after immunization with polysaccharides (8,9), and IgG4 has frequently been reported in the sera of patients with hypersensitivities, hemophilias with antibodies to VIII and IX factors, and antithyroglobulin and antimicrosome antibodies to Hashimoto s disease (1-12). Low IgG2 levels were found in patients with juvenile diabetes mellitus (13). In the present study, the prevalence of ACIV IgG1 and IgG3 may have been the result of antigen stimulus from increased level of serum CIV-derived peptides in diabetic children with vascular complications, as previously described (2). Probably, the pattern of the IgG subclasses against self-civ could be due to a new antigen (e.g., CIV plus AGE) that triggers this response. This is supported by the fact that six of ACIV IgG subclasses positive patients with vascular complications were also positive for anti-age antibodies. The increased immune response to CIVderived peptides may result in an increased production of ACIV IgG subclasses and activation of CIV degradation. The prevalence of ACIV IgG1 and ACIV IgG3 demonstrated their potential role in the pathogenesis of the development of diabetic vascular complications, since both subclasses may activate complement system and have the highest binding capacity to Fc receptors of phagocytes. In conclusion, we have identified that ACIV IgG1 and IgG3 are associated with the development of diabetic microalbuminuria in children with type 1 diabetes. Although it has been shown that metabolic control influences the development of microvascular disease, it now appears very probable that an immunologic component is also involved in the pathogenesis of microalbuminuria. Elucidation of the mechanism whereby immunoglobulins influence the development of early diabetic nephropathy will require intensive research. 136

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