Monocyte CD163 and CD36 Expression in Human Whole Blood and Isolated Mononuclear Cell Samples: Influence of Different Anticoagulants

Abstract

We investigated whether the choice of anticoagulant or the application of density gradient mononuclear cell isolation may account for conflicting published data regarding the levels of the scavenger receptors' expression in healthy individuals. We demonstrate that the detection of CD163, but not CD36, differs dramatically among the methods.CD163 is a member of the scavenger receptor cysteine-rich family of proteins, accounting for the clearance of hemoglobinhaptoglobin complexes, which in turn fuel an anti-inflammatory response mediated by heme metabolites (6, 10). CD163 is also an attractive candidate for potential diagnostic use as a marker of monocyte/macrophage activity in inflammatory diseases (1,3,8,11). However, it is still unclear how many circulating monocytes in normal subjects express the CD163 molecule on their surface. Previous studies with Mac 2-48, RM3/1, Ber-Mac3, and other monoclonal antibodies indicated that 0 to 100% of CD14 ϩ monocytes were positive for CD163, as detected by flow cytometry (2,4,11,12,13). Possibly due to different protocols used for CD163 identification in various studies, some discrepancies were found even when the same clone of monoclonal antibody was used for different studies.In the current study, we analyzed the influence of three commonly used anticoagulants and mononuclear cell isolation on the level of CD163 expression evaluated with the use of commercially available, fluorochrome-conjugated GHI/61 monoclonal antibody. For comparison, the same kind of analysis was performed for another scavenger receptor, CD36, which is also expressed on monocytes and is involved mainly in phagocytosis of apoptotic neutrophils and uptake of modified low-density lipoproteins.Forty-eight individuals (20 female and 28 male) had blood drawn between 7 and 9 a.m. The donors were healthy volunteers aged 18 to 56 years and selected in accordance with the guidelines of the Bialystok Local Ethics Committee (no. 8-I-003/132/2004). Blood samples were divided into tubes with EDTA, citrate, or heparin and immediately processed.Whole-blood samples were incubated with CD14 fluorescein isothiocyanate (clone MP9), CD163 phycoerythrin (clone GHI/61), CD36 allophycocyanin (clone CB38) (all obtained from Becton Dickinson, San Jose, CA), and fluorochromeconjugated isotype controls for 30 min at room temperature. Red blood cells were lysed for 10 min with fluorescence-activated cell sorter lysing solution (Becton Dickinson, San Jose, CA). The remaining white blood cells were washed twice with phosphate-buffered saline and fixed with 400 l of 2% paraformaldehyde (5).Portions of each whole-blood sample were also used for isolation of mononuclear cells. Mononuclear cells were isolated using Histopaque (density, 1.077 g/ml; Sigma-Aldrich) density gradient centrifugation as described previously (9). The isolated cells were washed twice with phosphate-buffered saline, and the cell concentration was adjusted to 1 ϫ 10 6 cells/ ml. Mononuclear cells were stained with 20 l of fluorochrome-conjugated monoclonal antib...

Influence of different anticoagulants on monocyte procoagulant functions and monocyte‐platelet aggregates formation

“…Anticoagulation is extremely effective at preventing recurrent DVT and therefore continued anticoagulation reduces PTS by preventing ipsilateral recurrent DVT, which is a strong risk factor for PTS. However, duration of a finite period of anticoagulation with a vitamin K antagonist (VKA) has no effect on PTS arising as a result of an initial DVT [1,2]. In contrast there is strong evidence that subtherapeutic anticoagulation is associated with the development of PTS [3,4], with an odds ratio of 2.7 associated with more than 50% of the time spent with an international normalized ratio <2.0 [3].…”

“…Previous concerns regarding hampered cellular reactivity and thrombogenicity of monocytes in whole blood anticoagulated with EDTA and citrate [12], along with differential expression of monocyte surface CD14 [13] and CD163 [1], but not CD36 [1], in PBMCs isolated from different anticoagulants, urged us to study whether this observation also holds true for monocyte procoagulant functions in isolated PBMCs. Our findings, however, indicate that the three anticoagulants tested had a similar influence on monocyte procoagulant activity, but EDTA provided the highest yield of PBMCs, highest proportion of monocytes within isolated PBMCs, and less formation of MPA.…”

“…Influence of different anticoagulants on monocyte procoagulant functions and monocyte-platelet aggregates formation Human whole blood and isolated peripheral blood mononuclear cells (PBMCs) are most convenient ex vivo models to study leukocyte receptor expression [1] and their interaction with other blood cells [2]. Monocytes are potent regulators of blood thrombogenicity through expression of cell surface tissue factor (TF) upon activation, and constitutive expression of tissue factor pathway inhibitor (TFPI) [3].…”

CD163 and its role in inflammation

“…Original reports using Ber-Mac3 or RM3/1 antibodies demonstrated expression of CD163 on 5% or 30% of freshly isolated monocytes, respectively [14,15]. More recent reports, however, have demonstrated CD163 expression on a majority of peripheral blood monocytes [16,17]. The discrepancies in reported expression of CD163 on freshly isolated peripheral blood monocytes may be explained by the application of different antibodies and by different techniques used for monocyte isolation.…”

“…Cellular expression of CD163 is restricted to monocytes and macrophages [8,12]. Freshly isolated peripheral blood monocytes express a relatively low level of CD163, but this expression increases with differentiation of such cells into macrophages [14][15][16][17]. Original reports using Ber-Mac3 or RM3/1 antibodies demonstrated expression of CD163 on 5% or 30% of freshly isolated monocytes, respectively [14,15].…”