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To the Editor: Neutrophil dysfunction contributes to the pathogenesis of the vascular complications of Type 2 diabetes. Adhesion of neutrophils to the vascular endothelium is mediated by surface exposure of the β2 integrin CD11b/CD18, which is closely associated with the actin cytoskeleton [1]. We have shown that neutrophils respond to activation with phorbol ester by rapidly increasing surface expression of CD11b, followed by loss of surface CD11b in a proportion of cells [2]. Loss of surface CD11b in response to phorbol ester is associated with actin polymerisation and leads to loss of neutrophil adherence [3]. In Type 2 diabetes both the proportion of neutrophils polymerising actin and losing CD11b and the proportion of cells exocytosing primary granules (an important step in microbial killing and identified as surface exposure of the antigen CD69) is reduced [2].
Several aspects of neutrophil dysfunction in Type 2 diabetes have been attributed to the metabolic consequences of hyperglycaemia and normalisation of glycaemia has been associated with an overall improvement in neutrophil function [4]. However, it is unclear whether impaired neutrophil-cytoskeletal remodelling and abnormal trafficking of cell surface antigens in Type 2 diabetes is due to altered metabolism. Therefore, we studied the effect of phorbol ester on neutrophil actin polymerisation and antigen exposure in samples from non-diabetic first-degree relatives of patients with Type 2 diabetes to test the hypothesis that these aspects of neutrophil function are familial and unaffected by the degree of glycaemia.
Venous blood samples were obtained from 20 first-degree relatives (FDR) of patients with Type 2 diabetes and 17 normal control subjects (NC). All subjects gave written informed consent and ethics approval was granted by the local Joint Ethics Committee. FDR were the siblings, children or parents of patients with Type 2 diabetes, NC were recruited from the local population as well as hospital and university staff. All NC subjects had fasting venous plasma glucose concentrations of less than 6.1 mmol/l and had no personal history of diabetes, gestational diabetes, hypertension, dyslipidaemia or cardiovascular disease. Data are presented as mean ± SEM. A p value of less than 0.5 was considered statistically significant. FDR were the same age as NC (age in years: NC 36.7±3.1, FDR 33.9±2.8) but had a higher BMI and diastolic blood pressure than NC [BMI (kg/m2) FDR 26.7±1.1, NC 22.0±0.3, p<0.01; diastolic BP (mmHg) FDR 84 (74–104), NC 73 (60–82), p<0.01] and lower HDL than NC (HDL mmol/l FDR 1.3±0.1, NC 1.6±0.1, p<0.01). There was no difference in systolic blood pressure, fasting venous plasma glucose concentrations (NC 4.9±0.1 mmol/l, FDR 5.2±0.1 mmol/l), fasting insulin, serum creatinine, total cholesterol and serum triglyceride concentrations between subject groups and all values were within normal range.
Venous blood samples from each subject were mixed with an equal volume of phosphate buffered saline (PBS) containing 5 mmol/l glucose and 5 mmol/l glutamine and incubated with 162 nmol/l phorbol 12 myristate 13 acetate (PMA) for 30 min at 37°C. Aliquots of 100 µl were then either: stained with 1 µl anti-CD69-PE and 1 µl anti-CD45-FITC on ice for 30 min in the dark, mixed with a tenfold excess of Erythrolyse (Serotec, Abingdon, UK) and washed in PBS; or stained with 1 µl anti-CD45-TRIC and 1 µl anti-CD11b-PE, fixed in 100 µl Leucoperm reagent A (Serotec, Abingdon, UK), stained with 10 µl phalloidin-FITC (which binds polymerised F-actin filaments) in 100 µl Leucoperm reagent B and washed in PBS. Cellular fluorescence was measured by flow cytometry. Neutrophils were identified from a mixed leukocyte population according to their light-scatter properties and their surface expression of CD45 as described previously [5]. Flow cytometry data were analysed using WinMDI version 2.8.
After incubation with 162 nmol/l PMA for 30 min and staining with anti-CD69-PE, two populations of neutrophil were identified with either high or low concentrations of surface CD69. When cells were stained with anti-CD11b-PE and phalloidin-FITC after PMA, three populations of neutrophil were identified (Fig. 1). The percentage of neutrophils with high surface CD69 exposure after PMA, indicating exocytosis of primary granules, was significantly reduced in samples from FDR compared to NC (% cells CD69hi NC 34.5±2.0, FDR 26.1±2.0, p<0.01). Similarly, the percentage of neutrophils with high F-actin and low surface CD11b after PMA was also reduced in samples from FDR (% cells F-actinhiCD11blo NC 34.8±1.8, FDR 29.3±1.6, p<0.05). These findings are consistent with our previous observations in patients with Type 2 diabetes [2].
First-degree relatives differed from normal control subjects with respect to BMI, diastolic blood pressure and HDL concentrations. To exclude metabolic differences as the cause for altered neutrophil antigen expression in FDR, neutrophil CD69 exposure was determined, after PMA activation, in samples from five control subjects with no family history of Type 2 diabetes and with an increased BMI and diastolic blood pressure. The clinical characteristics of these control subjects (NCb) were: BMI (kg/m2) 26.9±0.7, diastolic BP (mmHg) 86 (80–90), HDL (mmol/l) 1.2±0.4. After PMA activation, the percentage of cells CD69hi was significantly higher in NCb than FDR [%cells CD69hi (NCb) 35.6±4.4, p<0.05]. This suggests that the abnormality observed in FDR is related to the presence of a family history of Type 2 diabetes and not to the altered metabolic state.
In FDR there was an inverse correlation between the age of subjects and the proportion of neutrophils CD69hi (r=−0.64, p<0.01) and F-actinhiCD11blo (r=−0.59, p<0.02). However, in NC there was no decline with age (up to 65 years) in either the percentage of cells CD69hi (p=0.64) or percentage of cells F-actinhiCD11blo (p=0.92) and this was significantly different to the change with age in patients (CD69hi slope NC 0.12±0.16, DM −0.47±0.15, p<0.02; F-actinhiCD11blo slope NC 0.18±0.11, DM −0.42±0.13, p<0.01).
These data show that altered neutrophil actin polymerisation and surface antigen exposure observed in Type 2 diabetes are also present in the non-diabetic first-degree relatives of patients. This strongly supports the hypothesis that this aspect of neutrophil dysfunction is independent of the hyperglycaemic milieu and could indicate the presence of a familial defect that pre-dates the development of Type 2 diabetes. Relatives of patients with Type 2 diabetes are at increased risk of cardiovascular disease independently of other risk factors [6] and it is possible that the basic cellular trafficking defect that leads to altered neutrophil antigen exposure contributes to this added risk.
Chronic disorders such as Type 2 diabetes have been suggested to reflect a condition of abnormal cellular ageing [7]. The present findings support this hypothesis. In the first-degree relatives of patients with Type 2 diabetes neutrophil surface antigen expression becomes more abnormal with age, whereas no association is observed in the control subjects.
In summary, neutrophil antigen exposure is altered with age in the first-degree relatives of patients with Type 2 diabetes. This aspect of neutrophil dysfunction is likely to be important in the pathogenesis of diabetic vascular complications and is familial in origin.
Abbreviations
- PMA:
-
Phorbol 12 myristate 13 acetate
- NC:
-
normal control subjects
- FDR:
-
first-degree relatives of patients with Type 2 diabetes
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Acknowledgements
Dr. A. Advani is a Hypertension Trust Clinical Research Fellow. Dr T.H. Thomas is supported by the Northern Counties Kidney Research Fund. The authors are indebted to Mrs V. Mott and Mrs A. Taylor for their technical assistance.
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Rawling, L.D., Advani, A., Marshall, S.M. et al. Neutrophil antigen exposure is altered with age in relatives of patients with Type 2 diabetes. Diabetologia 47, 353–355 (2004). https://doi.org/10.1007/s00125-003-1290-6
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DOI: https://doi.org/10.1007/s00125-003-1290-6