Differential expression of TNFR1 (CD120a) and TNFR2 (CD120b) on subpopulations of human monocytes

Abstract

Background: Three subpopulations of monocytes can be distinguished in human blood: classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++). CD16 expressing monocytes are expanded in patients with sarcoidosis and in various other inflammatory diseases. In sarcoidosis, it is unclear whether either intermediate, nonclassical or both CD16 expressing monocytes are responsible for this increase. Data relating to the monocyte subpopulations is receiving increasing attention, but the expression of TNF receptors on these subpopulations has not been studied thus far. The aim of this study was to determine frequencies of monocyte subpopulations and their expression of TNFR1 and TNFR2 in both sarcoidosis patients and healthy controls.

Methods: Peripheral blood cells of sarcoidosis patients and healthy controls were stained for the markers HLA-DR, CD14, CD16, CD120a and CD120b. Cells were measured on a FACSCalibur and analyzed with FlowJo. We used Student's t-test and a parametric One-way ANOVA for statistical analysis.

Results: Sarcoidosis patients had a significant higher frequency of intermediate monocytes than healthy controls. Significant differences in TNF receptor expression were found between the monocyte subpopulations, both in sarcoidosis patients as well as in healthy controls: intermediates expressed more TNFR1 than classicals and nonclassicals and nonclassicals expressed more TNFR2 than intermediates, whereas intermediates showed higher expression than classicals.

Conclusions: In both sarcoidosis patients and healthy controls intermediate monocytes show the highest expression level of TNFR1 among monocyte subpopulations and nonclassical monocytes show the highest expression level of TNFR2. These findings, as wells as the higher frequency of intermediate monocytes in sarcoidosis patients, provide evidence for the existence of two functionally-distinct CD16 expressing monocyte subpopulations.

Figure 1

Sarcoidosis patients show a higher frequency of intermediate monocytes than healthy controls. Percentage of intermediates within the monocyte population of patients with sarcoidosis (n = 38) and healthy controls (n = 13). Data are presented as box plots, where the boxes represent the 25^th to 75^th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10^th and 90^th percentiles.

Figure 2

Differential expression of HLA-DR, CD14, CD16 on monocyte subpopulations in sarcoidosis patients and healthy controls. Median fluorescence intensity (MFI) of HLA-DR (A,B), CD14 (C) and CD16 (D) on classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺) and nonclassical (CD14⁺CD16⁺⁺) monocytes in sarcoidosis patients (n = 38) and in healthy controls (n = 13). Please note that, for sake of clarity, the data in panel A are regrouped in panel B to allow better comparison between the monocyte subpopulations. Values are expressed as mean ± SEM. All three markers were significantly different between the three monocyte subpopulations. Between sarcoidosis patients and healthy controls, nonclassical monocytes showed significant different levels of HLA-DR expression and classical monocytes showed significant different levels of CD14 expression. Comparison of three groups of data (subpopulations of monocytes) was performed by a parametric One-way ANOVA and comparison of two groups of data (patients versus controls) was performed by a two-tailed Student’s t-test.

Figure 3

Differential expression of TNFR1 and TNFR2 on monocyte subpopulations in sarcoidosis patients and healthy controls. Median fluorescence intensity (MFI) of TNFR1 (A) and TNFR2 (B) on classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺) and nonclassical (CD14⁺CD16⁺⁺) monocytes in sarcoidosis patients (TNFR1 n = 18, TNFR2 n = 38) and in healthy controls (TNFR1 n = 5, TNFR2 n = 13) minus the MFI of the fluorescence minus one (FMO) control. Values are expressed as mean ± SEM. Both markers were significantly different between the three monocyte subpopulations. All monocytes expressed TNFR1, but intermediates had a higher expression of TNFR1 than classicals and nonclassicals. All monocytes also expressed TNFR2, but nonclassicals had a higher expression of TNFR2 than intermediates and a much higher expression than classicals. Comparison of the three subpopulations of monocytes was performed by a parametric One-way ANOVA.