Phenotypic commitment of monocytes towards a protective hemoglobin scavenging phenotype (CD14posCD163highHLA-DRlow) following cardiopulmonary bypass (CPB)

Under physiological conditions, extra-corpuscular hemoglobin (Hb) is bound by the acute phase protein haptoglobin (Hp) and the complex cleared via the Hb-scavenging receptor, CD163, on cells of the monocyte-macrophage lineage. Intracellular heme is metabolised by heme-oxygenase-1 (HO-1) into carbon monoxide and biliverdin, which possess potent anti-inflammatory and anti-oxidant properties; and Fe2+ which is sequestered into ferritin stores. CD163 is a marker of monocytes/macrophages, denoting the resolving inflammation phenotype.

Recently, a novel subset of macrophages was identified juxtaposed to areas of hemorrhage in atherosclerotic plaques. They bear the CD68 macrophage lineage marker, high expression of CD163 and low expression of human leukocyte antigen DR (HLA-DR) (CD68posCD163highHLA-DRlow). This distinguishes them from the foam cell macrophages which are in proximity to the lipid core and bear the CD68posCD163lowHLA-DRhigh phenotype. CD163high cells are also functionally distinct and maintain reduced oxygen stress despite being iron laden. The cyto-protective properties of CD163high macrophages in plaques are credited to increased scavenging and processing of free Hb via CD163 and HO-1, thereby bestowing compensatory athero-protection.

Objectives:

The aim of this study was to prove that an equivalent compensatory, anti-oxidant phenotype (CD163highHLA-DRlowintracellular HO-1high) will evolve in circulating CD14pos monocytes under intravascular haemolytic conditions. During the process of cardiopulmonary bypass (CPB), intravascular hemolysis occurs as an inevitable consequence. Because CPB related haemolysis is induced and thereby lends itself to predictability and reproducibility, this condition was chosen as a prototype.

Methods:

Ethical approval was obtained from the Ministry of Health and The University of the West Indies Ethics Review Board. Twelve patients undergoing elective surgery with CPB at the Queen Elizabeth Hospital Barbados were enrolled with informed consent. Whole-blood samples were collected in EDTA at the following time-points: Pre-op, 30mins and 2hours after cross-clamping, 24hours, 48hours, 72hours and 120hours after de-clamping. Whole-blood samples were evaluated by 3-colour flow cytometry for expression of CD14, HLA-DR and CD163; and permeabilized CD14pos cells were analyzed by intracellular flow cytometry to detect cytoplasmic levels of HO-1 protein (n=3). Plasma [Hp-Hb] concentration was determined by ELISA (n=6). Pre-operative and 24hours post-declamping comparisons of CD163 and HLA-DR were performed via the Wilcoxon signed rank test and HO-1 comparisons were made by the paired t-test (GraphPad Prism Software, Inc., San Diego, CA).

Results:

A striking phenotypic switch from CD163lowHLA-DRhigh pre-operatively towards CD163highHLA-DRlow 24hours after de-clamping was observed on circulating CD14pos monocytes (Figure below). This was accompanied by a statistically significant increase in the mean fluorescent intensity (MFI) of CD163 [43.2 ± 1.3 (mean ± S.E.M.) at 24hrs vs. 10.6 ± 1.0 pre-operatively; p<0.006] and decrease in HLA-DR [17.8 ± 1.3 at 24hrs vs. 96.0 ± 12.6 pre-operatively; p=0.002].

A time-course examined the evolution of surface HLA-DR and CD163 in relation to plasma Hb-Hp complex formation. Peak Hb-Hp levels (peak hemolysis) occurred at 2 hours after cross-clamping [68.5 ± 5.2 (mean ± S.E.M) vs. 41.2 ± 6.5 pre-operatively], preceding the 24hour CD163 peak and HLA-DR nadir. The monocyte phenotype was largely reverted by 5 days post surgery. The mean fluorescent intensity of intracellular HO-1 protein was also up-regulated at 24 hours after de-clamping [69.0 ± 4.8 (mean ± S.E.M) at 24hrs vs. 51.0 ± 1.0 pre-operatively; p=0.02 (n=3)]. We have shown for the first time a phenotypic commitment of monocytes towards a protective (CD14posCD163highHLA-DRlowintracellularHO-1high) endpoint occurring in the circulation during the recovery phase of CPB.

Output/Expected Output:

This first clinical research collaboration between the CDRC and the Dept. Cardiothoracic Surgery at the Queen Elizabeth Hospital, Barbados, has produced an Abstract presented at the international ―Outcomes‖ Symposium and a full paper in press.

Impact/Expected Impact:

The CD14posCD163highHLA-DRlow phenotype may provide a useful tool for evaluating the effects of interventions that either limit hemolysis, e.g. minimizing pericardial suction; or attenuate pro-oxidant free Hb, e.g. haptoglobin infusions