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Abnormalities in primary granule exocytosis in neutrophils from Type I diabetic patients with nephropathy
Nicholas J M Fardon1, Robert Wilkinson, Trevor H Thomas
1Department of Medicine (Nephrology), Medical School, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, U.K.
Abstract:
Microalbuminuria in Type I diabetes involves a cell membrane abnormality and is associated with a large increase in cardiovascular risk. The hypothesis that the membrane abnormality alters granule exocytosis in neutrophils, which could contribute to the increased incidence of cardiovascular disease, was investigated. PMA-stimulated expression of CD11b and CD69 on neutrophils from normal controls (NC), long-term uncomplicated Type I diabetic control patients (DC) and diabetic nephropathy patients (DN) was determined by fluorescence activated cell scanning. Neutrophils from DN were faster than neutrophils from either NC or DC to exocytose primary granules with CD69 following initial expression of the adhesion molecule CD11b. However, a larger proportion of neutrophils from DN failed to withdraw CD11b from the cell membrane after 90 min incubation. The protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM), showed that a larger proportion of neutrophils from DN, compared with DC or NC, exocytosed primary granules independent of PKC. The calpain inhibitor, E64d, showed that a larger proportion of neutrophils from both groups of diabetic patients, compared with NC, exocytosed primary granules independent of calpain. Cytoskeletal disruption with cytochalasin D had an effect on CD11b and CD69 exocytosis similar to that of BIM and E64d. The pathways controlling granule exocytosis in neutrophils from diabetic patients are abnormal. A change characteristic of DN causes rapid exocytosis of primary granules, and also causes the adhesion molecule CD11b to persist on an increased proportion of neutrophils. This will make an important contribution to increased vascular damage in these patients.
Insights
Diabetic nephropathy alters neutrophil function, causing faster granule release and prolonged CD11b expression. These changes in cell membrane abnormalities contribute to increased cardiovascular risk in Type I diabetes.
Area of Science:
- Immunology
- Diabetology
- Cardiovascular Research
Background:
- Microalbuminuria in Type I diabetes is linked to cell membrane abnormalities and elevated cardiovascular risk.
- Neutrophil granule exocytosis may be altered by these membrane abnormalities, potentially contributing to cardiovascular disease incidence.
Purpose of the Study:
- To investigate if cell membrane abnormalities in Type I diabetes alter neutrophil granule exocytosis.
- To determine the role of protein kinase C (PKC) and calpain pathways in these altered exocytosis processes.
Main Methods:
- Fluorescence activated cell scanning was used to measure PMA-stimulated CD11b and CD69 expression on neutrophils from normal controls (NC), diabetic controls (DC), and diabetic nephropathy patients (DN).
- Inhibitors of PKC (bisindolylmaleimide, BIM) and calpain (E64d), as well as cytochalasin D, were used to probe the exocytosis pathways.
Main Results:
- Neutrophils from DN patients showed faster primary granule exocytosis (CD69 expression) and prolonged CD11b expression compared to NC and DC.
- A significant proportion of DN neutrophils exhibited PKC- and calpain-independent granule exocytosis.
- Cytoskeletal disruption mimicked the effects of BIM and E64d on CD11b and CD69 exocytosis.
Conclusions:
- The pathways controlling neutrophil granule exocytosis are abnormal in diabetic patients, particularly in those with nephropathy.
- Diabetic nephropathy is characterized by rapid primary granule exocytosis and persistent CD11b expression, contributing to vascular damage.