Medical Department IV, Nephrology, University of the Saarland, Homburg, Germany
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Abstract |
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Methods. We determined the amount of CD14+CD16+ monocytes in HD patients and characterized their fate during HD treatment. In 34 HD patients and 17 healthy controls, the distinct cell populations were determined by differential blood counts and flow cytometry. Cells from 14 HD patients were analysed at the start, 10, 30 and 120 min thereafter, and at the end of HD treatment.
Results. Before HD, patients show a monocytosis with a strongly increased CD14+CD16+ subpopulation. Early during HD treatment, circulating leukocyte numbers decrease, with monocytes being most profoundly influenced. Interestingly, among them, sequestration is most pronounced in the CD14+ CD16+ subpopulation. After 30 min, 83±9% of CD14+CD16+ cells are removed from circulation. This sequestration does not differ between patients treated with polyamide or haemophan membranes. The sequestration is a short-lived temporary effect and cell numbers are replenished within 120 min of treatment for the entire monocyte population. Beyond that time point, cellular activation by the dialyser membrane becomes visible. Reappearence kinetics of CD14+CD16+ monocytes is slower; however, initial numbers are reached by the end of treatment.
Conclusion. Haemodiaysis leads to temporary removal of monocytes from the bloodstream followed by the reappearance of activated cells. This might contribute to the state of chronic microinflammation, which is reflected by high levels of CD14+CD16+ monocytes.
Keywords: CD14+CD16+ monocytes; flow cytometry; haemodialysis; inflammation
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Introduction |
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Recent studies indicated a significant heterogeneity of circulating monocytes [15]. Two subpopulations of monocytes have been characterized, which can be identified based on the expression of CD16 (also known as FcRIII). In healthy subjects,
90% of monocytes are negative for CD16 and exhibit a strong expression of CD14 (CD14++CD16-), whereas a smaller subpopulation is CD16 positive and shows a normal or low expression level of CD14 (CD14+CD16+). The latter represent more mature monocytes with proinflammatory capacities [16] and their relative amount is increased during inflammation or physical stress [15]. A recent study showed that the number of CD14+CD16+ cells is increased in HD patients suffering from acute or chronic infection [17].
Studies of monocyte subpopulations have to take the temporal changes of cell numbers during HD treatment into account. Bloodmembrane contact leads to an increased cellular activation and sequestration into the capillary bed of the lung [18]. The influence of the sequestration on the number of mature monocytes was studied by analysing the fate of monocytes and in particular of the CD14+CD16+ subpopulation before and during HD treatment. This was done to find out if the more mature CD14+CD16+ monocytes are spared from sequestration, which might explain their elevated levels during the dialysis-free interval. Alternatively, based on the assumption that sequestration is only a temporary and reversible effect, the repeated cellular activation that causes adhesion of monocytes to the endothelium might also be responsible for the proinflammatory changes and increased generation of CD14+CD16+ cells.
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Subjects and methods |
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Among HD patients, six individuals on GFS plus 20, and five individuals on Polyflux 21S were analysed further to characterize the fate of CD14+CD16+ monocytes (Figure 3), leukocyte populations (Figure 1
), and the expression of different surface markers (Table 2
) during a single HD session. Furthermore, the relative number of CD14+CD16+ monocytes during dialysis in these six and additional three patients on GFS plus 20 was compared with respective numbers during an in vitro dialysis of heparinized blood from healthy blood donors (Figure 2
). In all settings, blood was drawn prior to HD treatment, 10 min, 30 min and 2 h thereafter, and at the end of the treatment session.
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In vitro dialysis
The in vitro dialysis is based on a standard dialysis machine (AK100; Gambro) and was designed as a closed loop circuit system without endothelial contact. We used conventional arterial and venous lines (children size). In vitro dialysis was performed with haemophan dialysers (GFS plus 11; Gambro).
Heparinised blood (120 ml at 20 U/ml) from healthy blood donors was diluted 1 : 1 with an isotonic solution (Eufusol; Schiwa, Glandorf, Germany) and kept in a reservoir at a constant temperature of 37°C. Blood flow was adapted to the circulating blood volume (15 ml/min). We used bicarbonate dialysate with a flow rate of 500 ml/min according to our dialysis standards. Samples were drawn from the arterial line at the given time points.
Antibodies
Monoclonal antibodies conjugated to fluorescent dyes were used to detect the following cell surface antigens: CD14 (clone RM052; Coulter Immunotech, Hamburg, Germany), CD16 (clone DJ130c; Dako, Hamburg, Germany), HLA-DR (clone B8.12.2 Coulter Immunotech), CD86 (clone IT2.2; Pharmingen, Hamburg, Germany), CD11b (clone 2LPM19c; Dako) and CD11c (clone KB90; Dako).
Cell counts
Leukocyte counts were performed using an automatic cell counter. Differential cell counts were done by microscopically counting cells stained from smears of blood samples anticoagulated with EDTA or by flow cytometry.
Flow cytometry analysis
Whole blood (100 µl) was incubated with saturating amounts of antibodies for 15 min at room temperature in the dark. Subsequently, red blood cells were lysed and white blood cells were fixed by the addition of 1 ml of fluorescence activated cell sorting (FACS) lysing solution (Becton Dickinson, Heidelberg, Germany) for 5 min at room temperature. After centrifugation, cells were washed with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin, 1 mM CaCl2 and MgCl2, resuspended in 100 µl of PBS containing 1% paraformaldehyde and analysed on a FACScan flow cytometer using the Cellquest software system (Becton Dickinson). Further analysis of data was done on a personal computer using the WinMDI 2.8 system provided by J. Trotter (Scripps Research Institute, La Jolla, CA, USA). Monocytes were gated in a SSC/CD14 dotplot and a gate was set on all CD14 positive cells, excluding cells with granulocyte scatter properties. Mean fluorescence intensity of respective surface molecules was calculated from these monocytes.
Statistical analysis
For data management and statistical analysis we used the Prism V3.0 statistical software package (Graphpad, San Diego, CA, USA). All data are given as means±SD or, if indicated, the standard error of the mean (SE). Significance of differences between the two groups was calculated using the t test for unpaired values. Significance of differences during HD treatment was calculated using repeated measures analysis of variance with Bonferroni's post test.
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Results |
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The monocytic cell populations were characterized further by flow cytometry. CD14+CD16- monocytes and the more mature population of CD14+CD16+ monocytes were clearly distinguishable. Granulocytes and natural killer cells were most appropriately excluded by gating in a side scatter (SSC)/CD14 dotplot. Subsequently, the respective percentage of monocyte subpopulations in this region could be quantified. While CD14+CD16+ cells account for 11.2±3.9% of monocytes in healthy controls, this population was significantly enhanced in HD patients (Table 1, last row: 16.3±4.7%; P<0.001).
Sequestration during the early period of HD treatment
In order to characterize the influence of haemodialysis on leukocyte subpopulations, we performed differential blood counts before dialysis, 10, 30 and 120 min after dialysis, and at the end of dialysis (Figure 1). Total circulating leukocyte numbers strongly decreased during the first 1020 min of a treatment session. Within the subsequent 120 min of treatment, cells regained normal levels (Figure 1A
). Interestingly, the sequestration primarily affected neutrophils and monocytes, with the most striking effect on the latter (Figure 1B
and C
). Lymphocyte numbers were hardly influenced by dialysis (Figure 1D
). All cell populations were replenished by the end of the treatment session.
Preferential depletion of CD14+CD16+ monocytes during haemodialysis
The most mature subpopulation of monocytes defined by the expression of CD14+CD16+ was preferentially depleted from circulation (Figure 2B). While the entire monocyte population reaches the nadir at 47.1±22.7% of initial cell numbers (Figure 1C
), the drop of the CD14+CD16+ subpopulation after the start of dialysis was much more striking, and was detectable in all patients tested (n=9, Figure 2B
). The proportion of these cells among all monocytes decreased from 20.8±9.4% to 7.9±5.3% after 10 min (P<0.01), and to 6.0±4.6% after 30 min (P<0.01). This represents a decrease to 28.0±12.2% of initial values. At 120 min and at the end of dialysis, cells reappeared in circulation.
Sequestration is absent in in vitro dialysis
The observed decrease of CD14+CD16+ monocytes could be due to shedding of the CD16 molecule during dialysis or it could be the result of a physical removal of monocytes by adherence to the dialysis system or endothelium. To distinguish between these possibilities, we performed dialysis experiments in vitro. Dotplots of a representative in vitro experiment are depicted in Figure 2A (lower panels). As opposed to the situation in vivo (Figure 2A
, upper panel, and B), monocyte counts and distribution of subpopulations did not change (Figure 2C
). While the drop in these mature monocytes was clearly detectable in all in vivo observations, the cell numbers did not change in any of the three in vitro experiments. This indicates that the pronounced drop in CD14+CD16+ monocytes in vivo is due to a temporal removal from circulation and not due to dialysis-induced downregulation of CD16 expression. Furthermore, the difference between the fate of the CD14+CD16+ monocytes in the two systems emphasizes the importance of the endothelium for monocyte sequestration during dialysis in vivo.
Haemophan and polyamide dialysers equally deplete CD14+CD16+ monocytes
Cellular sequestration during dialysis follows complement activation by the dialyser surface. The extent of complement activation is considered as a parameter for the biocompatibility of a dialyser membrane. To test whether currently used biocompatible membranes differ in their effect on the sequestration of monocyte subpopulations, temporal monocytic changes were comparatively analysed during dialysis with haemophan and polyamide (Figure 3). Surprisingly, both haemophan and the less complement activating polyamide membranes [20,21] led to a nearly complete removal of CD14+CD16+ cells from circulation after 10 and 30 min. The drop in the first 10 min was very uniform and sharp in all patients, and was independent from membrane type. Compared with the total number of monocytes (Figure 3A
), the reduction of CD14+CD16+ cells was more pronounced and changes were more uniform in all individuals tested (Figure 3B
). The reappearance of CD14+CD16+ monocytes was slower than the reappearance of the total monocyte population. While the latter reached initial values at 120 min, a complete replenishment of the CD14+ CD16+ subpopulation was only observed at the end of dialysis.
Dialysis sequestration is temporary and followed by rebound of activated monocytes
To clarify the apparent contradiction between depletion of mature monocytes during dialysis and the presence of high numbers of mature, proinflammatory monocytes in the interdialytic interval (Table 1), the fate of these cells was followed using the expression density of different types of functional surface markers (Table 2
). We chose molecules that are associated with complement-induced cell adhesion to the endothelium (CD11b and CD11c), the endotoxin receptor CD14 and markers for activation or maturation such as CD86, HLA-DR and CD16. During HD, the expression of the endotoxin receptor CD14 slightly increased; however, the changes were not statistically significant. We observed a constant increase in the expression of CD11b. This indicates monocyte activation, which is further supported by an increased expression of CD11c and CD86 after 120 min and beyond. The expression of both HLA-DR and CD16 rapidly decreased after 10 and 30 min, respectively. The reappearance of high expression levels of CD16 as well as HLA-DR indicates that the depletion of mature cells after activation is only temporary and does not have a sustained effect during the interdialytic interval.
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Discussion |
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Monocytosis in these patients results from elevated absolute cell numbers, which on average lay above the normal range. This indicates that the phenomenon is independent of the well-known lymphopenia of end-stage renal disease. High monocyte numbers before dialysis are in obvious contradiction to the fact that this cell type is preferentially sequestrated and removed from circulation during treatment. We show here that the mature and proinflammatory subpopulation of CD14+CD16+ cells does not only participate in sequestration but is preferentially removed. These findings are in line with a preferential depletion of IL-6-producing monocytes [22].
The paradoxon of monocytosis and selective sequestration of monocytes can be understood against the background of current knowledge of the mechanism of cellular sequestration and our actual findings. At the bloodmembrane contact, several activation processes occur. Complement activation and formation of anaphylatoxins such as C3a and C5a strongly influence monocytes [21]. Furthermore, direct cellular activation at the artificial surface may occur [14]. In addition, bacterial endotoxins from contaminated dialysate fluids may pass the dialyser membrane to the blood compartment [23,24]. Together, these processes lead to an upregulation and temporal conformational changes in surface adhesion molecules such as CD11b or CD11a on circulating cells [25], which mediate subsequent adhesion to the endothelium. Early studies showed that this sequestration process takes place mainly in the capillary bed of the lung [18,26], obviously because it represents the first large low-flow vasculature reached from the venous side of the extracorporal equipment. These sequestration processes may also be relevant for CD14+CD16+ cells, since we found a strong depletion of this cell type in vivo, which was not observed during dialysis in vitro. In line with these results, unphysiological stress situations such as ultramarathon similarly lead to a depletion of CD14+CD16+ monocytes [27]. Furthermore, our findings exclude the possibility that CD16 is shedded or internalized and they emphasize the importance of the endothelium for the adherence of CD14+CD16+ cells.
In order to bind tightly to, and subsequently transmigrate through, the endothelial layer after adhesion, not only monocytes but also endothelial cells themselves have to be activated to express further adhesion molecules and chemokines such as ICAM-1 or IL-8. Without this second adhesion synapse, adherent cells progressively detach from the endothelium and recirculate [28]. During a local inflammatory process, activation of monocytes and endothelial cells occur at the same time and in the same region. During dialysis, however, monocyte activation occurs unphysiologically separated from the activation of the endothelium. Thus, initial adhesion of circulating cells without local tissue inflammation might not lead to tight binding and transmigration. Instead, monocytes detach again and return to circulation. These cells most likely detach from the marginal pool of endothelia from tissues such as the lung, liver, spleen and gut. One could speculate that the individual velocity of cell reappearance in circulation is determined by the individual level of endothelial activation. On the other hand, mature monocytes that are known to express higher levels of adhesion molecules [27,29] bind more tightly and reappear in circulation later than the less mature cells. This leads to the nearly complete removal of CD14+CD16+ cells and the slower kinetics of replenishment after the start of dialysis treatment (Figure 3). It seems unlikely that reappearing cells represent cells coming directly from the bone marrow since we did not observe any increase in expression of markers for immature monocytes, such as CD34, during dialysis (data not shown).
This model explains the temporary nature of sequestration. Removal of cells from circulation hardly lasts longer than the complement activation. Our data show that initial monocyte levels are reached again after 120 min. Therefore, this short-lived effect hardly influences the systemic microinflammatory status of HD patients. In contrast, the activation process that leads to short term sequestration of cells leaves its traces on those monocytes that return to circulation later on. Expression levels of cellular activation markers such as CD11b, CD11c and CD86 are higher at the end of the dialysis session. This may give a clue as to the pathogenetic contribution of dialysis-induced monocyte activation to the systemic inflammatory process.
Modern synthetic dialysers claim higher biocompatibility than cellulose-based membranes. Complement activation and cytokine induction are lower in patients treated with synthetic materials [20,21]. Since sequestration of CD14+CD16+ cells is a highly sensitive marker for the influence of the extracorporal procedure on monocytes, it seemed rational to test it as a parameter to evaluate differences in biocompatibility between membranes. However, even polyamide dialysers, which are among the membranes with the lowest complement activation currently available, still induce reductions in the number of CD14+CD16+ cells as severe as haemophan dialysers. Given the fact that CD14+CD16+ monocytes represent a sensitive marker for inflammation or cellular activation, depletion of these cells may offer an easily accessible parameter that is more sensitive than complement activation for biocompatibility studies on forthcoming improved dialyser membranes.
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Acknowledgments |
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Notes |
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References |
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