Alterations in monocyte CD16 in association with diabetes complications

Abstract

Monocytes express many cell surface markers indicative of their inflammatory and activation status. Whether these markers are affected by diabetes and its complications is not known and was investigated in this study. Blood was obtained from 22 nondiabetic and 43 diabetic subjects with a duration of diabetes >10 years, including 25 without and 18 with clinically significant complications. The number of CD45(+)CD14(+) monocytes and the percentage expressing the proinflammatory marker CD16 were determined by flow cytometry. Other markers of monocyte activation and expression of chemokine receptors were also examined. The relationship between monocyte CD16 and clinical data, selected cytokines, and chemokines was also investigated. Diabetes had no effect on total white cell number but increased monocyte number. Diabetes also significantly decreased the number of CD16(+) monocytes but only in those with diabetic complications. Other markers of monocyte activation status and chemokine receptors were not affected by diabetes or complications status. Diabetes induced plasma proinflammatory cytokines and they were lower in diabetic subjects with complications compared to those without complications. These results suggest that the circulating monocyte phenotype is altered by diabetic complications status. These changes may be causally related to and could potentially be used to predict susceptibility to diabetic complications.

Figure 1

Flow cytometry gating strategy. Flow cytometry data obtained from a representative sample is shown. (a) Peripheral blood cells were first gated based on forward scatter (FSC-A)/side scatter (SSC-A), with exclusion of dead cells. (b) The events were then visualized using FSC-A/FSC-H dot plot, and the singlets (single cells) were gated. (c) Leukocytes were identified by their positive staining with CD45. (d) Monocytes were then defined as the CD14⁺ cells within the CD45⁺ leukocyte population. (e) The final gating was based on the CD marker of interest within the CD45⁺CD14⁺ cell population. (f) Necrotic cells in the whole blood sample were detected by Propidium Iodide (PI) staining.

Figure 2

The effect of diabetes on circulating monocytes. Leukocytes and monocytes were identified by flow cytometry. Shown in control and diabetic subjects are (A) the CD45⁺ leukocyte population and (B) the CD45⁺CD14⁺ monocyte population. (C) Representative forward scatter and side scatter plots from a control and diabetic subject showing CD45⁺CD14⁺ monocytes (red dots). The three subsets of monocytes grouped according to size and granularity are shown in the diabetic sample as (a), (b), and (c). *P < 0.05 different between groups.

Figure 3

The effect of diabetes and its complications on percentage of CD16 monocytes. (A) Representative forward scatter and side scatter plots data from a control and diabetic subject showing the CD16⁺ cells (red dots) within the CD45⁺CD14⁺ monocyte population. (B) The percentage of CD16⁺ monocytes in control and diabetic subjects with and without complications. (C) The percentage of CD16⁺ monocytes in each monocyte subset grouped according to size and granularity. *P < 0.05 different between groups.

Figure 4

The relationship between CD16⁺ monocyte and other CD markers. The relationship between the CD16⁺ monocyte and (a) macrophage marker CD68⁺ and (b) 27E10⁺, a marker of acute inflammatory response.