High glucose concentrations induce TNF-α production through the down-regulation of CD33 in primary human monocytes

Abstract

Background: CD33 is a membrane receptor containing a lectin domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. CD33 is expressed by monocytes, and reduced expression of CD33 correlates with augmented production of inflammatory cytokines, such as IL-1β, TNF-α, and IL-8. However, the role of CD33 in the inflammation associated with hyperglycemia and diabetes is unknown. Therefore, we studied CD33 expression and inflammatory cytokine secretion in freshly isolated monocytes from patients with type 2 diabetes. To evaluate the effects of hyperglycemia, monocytes from healthy donors were cultured with different glucose concentrations (15-50 mmol/l D-glucose), and CD33 expression and inflammatory cytokine production were assessed. The expression of suppressor of cytokine signaling protein-3 (SOCS-3) and the generation of reactive oxygen species (ROS) were also evaluated to address the cellular mechanisms involved in the down-regulation of CD33.

Results: CD33 expression was significantly decreased in monocytes from patients with type 2 diabetes, and higher levels of TNF-α, IL-8 and IL-12p70 were detected in the plasma of patients compared to healthy donors. Under high glucose conditions, CD33 protein and mRNA expression was significantly decreased, whereas spontaneous TNF-α secretion and SOCS-3 mRNA expression were increased in monocytes from healthy donors. Furthermore, the down-regulation of CD33 and increase in TNF-α production were prevented when monocytes were treated with the antioxidant α-tocopherol and cultured under high glucose conditions.

Conclusion: Our results suggest that hyperglycemia down-regulates CD33 expression and triggers the spontaneous secretion of TNF-α by peripheral monocytes. This phenomenon involves the generation of ROS and the up-regulation of SOCS-3. These observations support the importance of blood glucose control for maintaining innate immune function and suggest the participation of CD33 in the inflammatory profile associated with type 2 diabetes.

Figure 1

CD33 expression in human monocytes from type 2 diabetes patients. (A) Monocytes were stained with anti-CD3, CD14 and CD33 mAbs. At least 50,000 events were acquired for the flow cytometry analysis. CD33 expression is shown after gating for the CD3-CD14+ cells, and a histogram of CD33 expression was plotted for type 2 diabetes patients (tinted histogram) and healthy subjects (open histogram). (B) A bar graph showing the mean intensity fluorescence (MFI) data for CD33 expression in freshly isolated monocytes from patients with type 2 diabetes (n = 10) and healthy donors (n = 10). The data are presented as the mean ± SD. * P < 0.05 compared to healthy donors. (C) Monocytes from type 2 diabetes patients (n = 9) and healthy donors (n = 8) were evaluated using Taqman gene expression analysis for CD33 mRNA expression. The results were analyzed according to the ΔΔCt method, and the data are presented as the mean ± SD. * P < 0.05 compared to healthy donors.

Figure 2

Pro-inflammatory cytokine production in type 2 diabetes patients. Plasma from type 2 diabetes patients and healthy controls was tested for the presence of pro-inflammatory cytokines using the CBA kit for IL-8, IL-1β, IL-6, IL-10, IL-12p70 and TNF-α. (A) The bar graphs show the quantification of these cytokines for type 2 diabetes patients (n = 14) and healthy controls (n = 10). All data are presented as the mean ± SD. * P < 0.05 compared to healthy donors. (B) The expression of TNF-α mRNA and 18 S ribosomal RNA was analyzed for monocytes collected from type 2 diabetes patients (n = 7) and healthy subjects (n = 9). The results are expressed according to the ΔΔCt method, and the data are presented as the mean ± SD. * P < 0.05 compared to healthy donors.

Figure 3

High glucose concentrations down-regulate CD33 expression in human monocytes. Monocytes from healthy donors were cultured in the presence of D-glucose (5.5, 15, 30, or 50 mmol/l) for 7 days. (A) Representative histograms show the expression of CD33 in monocytes cultured with 5.5 mmol/l (gray), 15, 30, or 50 mmol/l (open histogram) D-glucose. (B) Bar graphs show the MFI of CD33 in monocytes cultured under high glucose compared to those cultured in normal medium. The data are presented as the mean ± SD (n = 6). (C) Taqman gene expression mRNA analysis of CD33 and 18 S ribosomal RNA expression. The results were analyzed according to the ΔΔCt method, and all data are presented as the mean ± SD (n = 3). * P < 0.05 compared to 5.5 mmol/l D-glucose.

Figure 4

Effect of high glucose concentrations on pro-inflammatory cytokine production. Supernatants from monocytes cultured in the presence of 5.5 and 50 mmol/l D-glucose for 7 days were used for the quantification of IL-8, IL-1β, IL-6, IL-10, IL-12p70 and TNF-α protein levels. (A) The bar graphs represent the quantification of cytokine production (n = 3). (B) The expression of TNF-α mRNA from monocytes cultured in high glucose. TNF-α mRNA and 18 S ribosomal RNA expression was analyzed by qPCR, and the results are expressed according to the ΔΔCt method. The data are presented as the mean ± SD for all graphics (n = 3). * P < 0.05 compared to treatment with 5.5 mmol/l D-glucose.

Figure 5

TNF-α production in CD33^lowand CD33^highmonocytes. Monocytes from healthy donors were cultured in the presence of glucose for 7 days and were then stained with anti-human CD33 and anti-human TNF-α antibodies. (A) Representative dot-plots show the percentages of TNF-α-producing CD33^low and CD33^high monocytes that were cultured with either 5.5 mmol/l (left) or 50 mmol/l (right) D-glucose (B) The bar graph summarizes the levels of TNF-α production by CD33^low (white) and CD33^high (black) monocytes cultured with 50 mmol/l D-glucose (n = 4). The data are expressed as the mean ± SD. * P < 0.05 as compared to the TNF-α production by CD33^low and CD33^high

Figure 6

The effect of α-tocopherol treatment on monocyte CD33 expression andTNF-α production. Monocytes from healthy donors were cultured in 50 mmol/l D-glucose either with or without α-tocopherol for 7 days. (A) Representative histogram of ROS generation from monocytes cultured with 5.5 mmol/l (gray) or 50 mmol/l D-glucose (open histogram) without α-tocopherol (left) or with α-tocopherol (right). The bar graph shows relative ROS generation (n = 6). (B) A representative histogram showing CD33 expression from monocytes cultured without (left) or with α-tocopherol (right). The bar graph shows the relative CD33 expression levels (n = 3). (C) Left panel, the production of TNF-α, as measured by flow cytometry. Right panel, relative fold change of TNF-α mRNA expression with and without α-tocopherol treatment (n = 3). All data are expressed as the mean ± SD. * P < 0.05 compared to treatment with 5.5 mmol/l D-glucose.

Figure 7

Expression of SOCS3 mRNA in monocytes. Monocytes from healthy donors were cultured in 5.5 or 50 mmol/l D-glucose for 2, 24, or 48 hours or 7 days, and the expression levels of SOCS3 mRNA and 18 S ribosomal RNA were evaluated using the Taqman gene assay. Gene expression was normalized to that of the housekeeping gene, and the results are expressed according the ΔΔCt method as the relative fold-change with respect to cells treated with 5.5 mmol/l D-glucose. The data are presented as the mean ± SD (n = 3). * P < 0.05.