Hemodialysis-related changes in phenotypical features of monocytes

Abstract

Hemodialysis (HD) patients exhibit chronic inflammation and leukocyte activation. We investigated the surface-marker profile of monocytes by flow cytometry to assess the chronic effect of uremia and the acute effect of dialysis on their phenotypical and functional features in 16 healthy controls (CON) and 15 HD patients before and after a polysulfone-based dialysis session. Median fluorescence intensities were analyzed indicating expression of CD14, CD16, integrins (CD11b, CD18), chemokine receptors (CCR2, CX3CR1), scavenger receptors (CD36, CD163) and Toll-like receptor-2 (TLR2). Before and after dialysis, HD patients harbour 0.9-fold less CD14⁺⁺CD16^- (Mo1), 1.8-fold more CD14⁺⁺CD16⁺ (Mo2) and CD14⁺CD16⁺⁺ (Mo3) monocytes than CON. HD patients' Mo1 showed elevated expression of CD11b (1.7-fold), CD18 (1.2-fold) and CD36 (2.1-fold), whereas CD163 expression was reduced in Mo1 and Mo2 (0.6-fold) compared to CON. These markers remained unaffected by dialysis. CX3CR1 expression on Mo2 and Mo3 was lower in HD patients before (0.8-fold) and further diminished after dialysis (0.6-fold). Stimulation of monocytes resulted in diminished responses in HD patients compared to CON. In conclusion, a systematic analysis of the expression of particular surface markers on distinct monocyte subsets may help to distinguish between uremia and/or dialysis induced effects and to evaluate the functionality of monocytes and biocompatibility of HD.

Figure 1

Comparison of percentages of monocyte subpopulations and expression of surface markers on monocytes from healthy donors and dialysis patients. Blood from healthy donors (CON) and hemodialysis patients (HD) before (pre) and after (post) a dialysis session was stained with fluorochrome labeled antibodies to gate for monocyte subsets as outlined in supplementary Fig. 1. The scatter plots show the percentages of Mo1, Mo2 and Mo3 of total monocytes (A), the expression of CD11b (B), CD18 (C), CD36 (D), CD163 (E), TLR2 (F), CCR2 (G) and CX3CR1 (H) from 16 healthy controls (blank circles) and 15 HD patients before (HD pre, black circles) and after (HD post, black diamonds) a dialysis session. P-values comparing CON with HD pre or HD post were calculated with Mann-Whitney U-test, p-values comparing HD pre and HD post with Wilcoxon matched-pairs signed rank test. MFI: median fluorescence intensity. n.s.: not significant.

Figure 2

Personalized expression of CX3CR1 and TLR2 before and after dialysis. The graphs show the median fluorescence intensity (MFI) of CX3CR1 (A) or TLR2 (B) on Mo1, Mo2 and Mo3 monocytes from 15 individual patients before (pre) and after (post) hemodialysis (HD).

Figure 3

Summary of the findings. The table in style of a heatmap summarizes the changes of percentage or marker expression in the indicated monocyte subpopulation, whereby values obtained from hemodialysis patients (HD) before (pre) and after (post) dialysis were related to healthy volunteers (CON). Asterisks indicate the power of significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); MFI: median fluorescence intensity.

Figure 4

Uremic conditions strongly influence CX3CR1 expression on monocytes. Whole blood from healthy donors (blood group 0) was diluted 1:1 with self-serum (reference), non-uremic control or HD patient serum. (A) The diagram shows the percentual change of CX3CR1 surface expression on Mo1, Mo2 and Mo3 monocytes after 4 h incubation at 37 °C, 5% CO₂ with the indicated foreign serum compared to incubation with self-serum (100%, indicated by a dotted horizontal line). P-values were calculated using the Mann-Whitney U-test.

Figure 5

Adhesion capacity of leukocytes before and after dialysis Whole blood from healthy donors (n = 3) and HD patients (n = 5, collected either before or after a single dialysis session) was incubated for 30 min in cell culture plates. (A) Characteristic microscopic pictures taken with 5x magnification and enlargements of the framed region in each picture show adherent leukocytes of healthy volunteers (left), HD patients before (center) and after (right) a single dialysis session. The scale bar shows 100 µm. (B) The graph shows the number of adherent leukocytes from healthy volunteers (white bar), or HD patients before (grey bar) and after (black bar) a single dialysis session. P-values were calculated using the unpaired or paired t test (for CON/HD post or HD pre/HD post, respectively).

Figure 6

Response of healthy controls’ or HD patients’ monocytes upon LPS stimulation Whole blood from healthy donors (CON, n = 7) and HD patients (HD, n = 9, collected both before, pre, and after, post, a single dialysis session) was incubated for 30 min with 10 ng/ml LPS or left untreated. The graphs show the percentual change of TLR2 (A), TLR4 (B), CD18 (C) or CD11b activated (D) expression on all three monocyte subpopulations in healthy volunteers (white bars), or HD patients before (grey bars) and after (black bars) a single dialysis session. P-values comparing CON with HD pre or HD post were calculated with two-tailed Mann-Whitney U-test, p-values comparing HD pre and HD post with Wilcoxon matched-pairs signed rank test. MFI: median fluorescence intensity.