CD14+CD16+ and CD14+CD163+ monocyte subpopulations in kidney allograft transplantation

Abstract

Background: Monocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation.

Results: The phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes.

Conclusions: We assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used.

Figure 1

Percentage of peripheral blood CD14+CD16+ monocytes after kidney allograft transplantation. The co-expression of CD14 and CD16 antigens on peripheral blood monocytes was evaluated by flow cytometry in three different groups of patients undergoing kidney allograft transplantation (a-without induction, b-thymoglobulin, c-basiliximab). In patients without the induction therapy (n = 25 ) and those treated with thymoglobulin (n = 28 ), the percentage of CD14+CD16+ monocytes decreased during the first two weeks after the transplantation as compared to only moderate changes observed in subjects treated with basiliximab (n = 18). Analysis of data from flow cytometry was performed by ANOVA with repeated measures and a comparison within groups between two time points by contrasts. *Statistically significant differences.

Figure 2

Downregulation of CD14+CD16+ monocyte subpopulation after the kidney transplantation. The flow cytometry plot expresses the data of one representative patient without the induction therapy. The number of CD14+CD16+ were strongly downregulated during the first week after the kidney transplantation.

Figure 3

Percentage of peripheral blood CD14+CD163+ monocytes after kidney allograft transplantation. The presence of CD14+ CD163+ monocytes in peripheral blood was evaluated by flow cytometry during the first year after the kidney allograft transplantation. In patients without the induction therapy a (n = 25 ) and those treated with basiliximab c (n = 18 ), the percentage of CD14+CD163+ monocytes dramatically increased during the first week after the transplantation in contrast to subjects treated with thymoglobuline b (n = 24). Analysis of data from flow cytometry was performed by ANOVA with repeated measures and a comparison within groups between two time points by contrasts. *Statistically significant differences.

Figure 4

Expansion of CD14+CD163+ monocytes following kidney transplantation. The flow cytometry plot expresses the data of one patient with basiliximab induction therapy. The percentage of CD14+CD163+ monocytes was highly upregulated during the first week after the transplantation.

Figure 5

Proportional changes of CD14+CD163+ subpopulation as compared to the whole population of peripheral blood monocytes. The absolute numbers of peripheral blood monocytes and their CD14+CD163 subpopulation were monitored during the first year following kidney allograft transplantation (n = 71). The early massive upregulation of CD14+CD163+ subpopulation is in a contrast with only moderate changes observed in whole population of peripheral blood monocytes (absolute numbers of monocyte subpopulations are calculated from the total number of leukocytes and are expressed as number of cells *10⁶/l).

Figure 6

Case reports of two patients with complicated outcome of kidney allograft transplantation associated with changes of CD14+CD16+ subpopulation. Patient No.1 is a 56-year old male with delayed graft function and diagnosed acute cellular rejection IIB in early phase following kidney allograft transplantation, treated successfully with thymoglobulin. Patient N. 2 is a 51-year old male with C4d negative humoral rejection (FACSXM positive, presence of MICA antibodies) diagnosed one month after the transplantation. After changing of immunosuppression and subsequent IVIG therapy, the kidney functions became stable. In both cases, the clinical status of patients correlated with proportions of CD14+CD16+ monocytes. TCMR = T cell-mediated rejection, ATN = acute tubular necrosis (absolute numbers of monocyte subpopulations are calculated from the total number of leukocytes and are expressed as number of cells *10⁶/l).

Figure 7

In vitro effect of immunosuppressives on the percentage of CD14+CD16+ monocytes peripheral blood monocytes collected from a healthy donor were cultured in the presence of rATG, basiliximab and methylprednisolone. Changes of CD16 expression on the surface CD14-positive monocytes were measured at following time points: 0, 1, 3, 6, 24, 48 and 72 h. Incubation with rATG (a) downregulated the proportion of CD14+CD16+ monocytes already after one hour. Basiliximab (b) had no effect. Methylprednisolone (c) upregulated the percentage of CD14+CD16+ subpopulation. Dynamics of the parameters was fitted by quadratic regression and the estimated coefficients were compared by z-statistcs.

Figure 8

In vitro effect of immunosuppressives on the percentage of CD14+CD163+ monocytes peripheral blood monocytes collected from a healthy donor were cultured in the presence of rATG, basiliximab and methylprednisolone. Membrane expression of CD163 on cultured monocytes was measured at following time points: 0, 1, 3, 6, 24, 48 and 72 h. Both the rATG (a) and methylprednisolone (b) upregulated the proportion of CD14+CD163+ monocytes while basiliximab (c) had no effect. Dynamics of the parameters was fitted by quadratic regression and the estimated coefficients were compared by z-statistcs.