Up-regulation of CD163 expression in subpopulations of blood monocytes after kidney allograft transplantation

Abstract

M2 macrophages expressing CD163 are known to suppress immune responses but have been also found in biopsies of patients with chronic kidney allograft injury associated with interstitial fibrosis. The aim of our study was to evaluate the expression of CD163 in blood monocytes, precursors of tissue macrophages, in kidney allograft recipients with uncomplicated outcome (n=94) compared with those developing acute rejection (n=44). Blood samples were collected before the transplantation and at 1 week, 1 month and 1 year. The expression of CD163 increased during the first week after the transplantation not only in classical (CD14+CD16-) but also in intermediate (CD14+CD16+) and nonclassical (CD14lowCD16+) monocytes in all patients regardless of their rejection status. In patients developing acute rejection, higher pre-transplant expression of CD163 on blood monocytes was found. In vitro experiments confirmed strong induction of membrane CD163 on monocytes together with CD206 (an alternative marker of M2 macrophages) in response to IL-10. We assume from our data that dramatic upregulation of CD163 by peripheral blood monocytes may have a pathophysiological role in early phases after kidney allograft transplantation and high pre-transplant expression of CD163 on blood monocytes might be involved in events leading to acute rejection.

Fig. 1

The gating strategy to differentiate subpopulations of blood monocytes. Three subpopulations of peripheral blood monocytes were delineated by the expression level of CD14 and CD16 molecules into classical (CD14⁺CD16⁻), intermediate (CD14⁺CD16⁺) and nonclassical (CD14^lowCD16⁺) subsets (Fig. 1a). The expression of CD163 was then measured in the whole population of blood monocytes and selected three subpopulations.

Fig. 2

CD163 expression in subpopulations of blood monocytes from kidney allograft recipients. The expression of CD163 was evaluated in classical (CD14⁺CD16⁻), intermediate (CD14⁺CD16⁺) and nonclassical (CD14^lowCD16⁺) peripheral blood monocytes obtained from patients (n=138) before the kidney transplantation and then one week, one month, and one year after the surgery. The percentage of CD163 positive cells was lower in nonclassical monocytes as compared to classical and intermediate ones (p<0.001) but the expression of the molecule was upregulated at one week after the kidney transplantation in all three subsets (p<0.001) and then decreased after one month (p<0.001). The expression of CD163 after one year did not changed as compared to values at one month and remained higher as compared to pre-transplant percentage of positive cells (p<0.001).

Fig. 3

CD163 expression after the transplantation in patients with acute rejection. The expression of CD163 was evaluated on blood monocytes obtained from patients before the kidney transplantation and one week after the surgery. We compared two groups of patients, those with uncomplicated outcome (n=94) and those developing acute rejection (n=44). Post-transplant CD163 expression was upregulated in both groups of patients but those with acute rejection had higher pre-transplant percentage of CD163 positive monocytes. The initial upregulation of CD163 was followed by a subsequent decrease at one month after the transplantation and did not change at 1 year. * p<0.05; *** p<0.001.

Fig. 4

CD206 expression in peripheral blood monocytes. In kidney transplant recipients, the expression of CD206 was determined on peripheral blood monocytes. The expression of CD206 was relatively low as compared to CD163 and increased during the first week after the transplantation only in patients with acute rejection. Data are expressed as mean ± SEM; * p<0.05.

Fig. 5

Flow cytometry plots showing induction of CD163 and CD206 on cultured monocytes by IL-10. Peripheral blood mono-nuclear cells were cultured in the presence or absence of IL-10 (10 ng/ml) and the expression of CD163 and CD206 was analyzed on day 0, day 1, day 3, and day 6. The expression of CD163 on IL-10 stimulated monocytes increased dramatically on day 3, CD206 expression was upre-gulated by IL-10 from day 1.

Fig. 6

Modulation of two different epitopes of CD163 by IL-10. Peripheral blood mononuclear cells were cultured for 6 days in the presence or absence of IL-10 (10 ng/ml) and the expression of CD163 was measured by two monoclonal antibodies (RM3/1 and GHI/61) recognizing different epitopes. In whole blood samples, only the GHI/61 clone showed clear membrane expression of CD163 in blood monocytes but staining by RM3/1 emerged after gradient centrifugation of mono-nuclear cells or in buffy coats (6a). In tissue cultures of mononuclear cells, both epi-topes of CD163 on monocytes were markedly upregulated by IL-10 on day 3 (6b).