LRP1 Expression in Sickle Cell Disorders: Insights into the Heterogeneity of Monocytic Response to Heme

Introduction: The importance of heme and its main scavenger hemopexin in the pathophysiology of sickle cell disease (SCD) has been demonstrated in animal models of SCD vaso-occlusion. Increase in monocyte-derived inflammatory cytokines in SCD can be attributed to heme-mediated activation of TLR4 signalling to produce TNF-alpha, and amplification of IL-6 production through a mechanism dependent on heme iron. This suggests that monocytes not only respond to heme but also have receptors to acquire it. We hypothesized that varying levels of heme and hemopexin in SCD could affect how circulating monocytes express LDL receptor related protein (LRP1), the receptor that targets the heme/hemopexin complex to degradation, which can be cleaved to produce a soluble form (s-LRP1).

Methods: Peripheral venous blood samples from 119 subjects (39 healthy volunteers - AA, 44 homozygous sickle cell anemia patients - SS, 8 not on hydroxyurea treatment, and 36 hemoglobin SC disease patients - SC) were collected upon signed informed consent approved by the local ethics board. Automated complete blood counts and serum lactate dehydrogenase (LDH), bilirubin levels, and heme and hemopexin concentrations were determined. LRP1 expression was measured by mean fluorescence intensity through conventional flow cytometry (FACS Calibur and FACS DIVA software) in different monocyte subsets, classified according to their surface markers into classical (CD14⁺⁺ CD16^-), intermediate (CD14⁺ CD16⁺) and nonclassical (CD14^dim CD16⁺) monocytes.

Results and Discussion: As expected, reticulocyte count, LDH, bilirubin, and heme were significantly more elevated in SS than in SC patients when compared to AA subjects, and hemopexin was reduced in both patient groups (P <0.0001). Monocyte subsets differed significantly among the groups: SS and SC patients had more circulating intermediate and nonclassical monocytes than AA subjects (P <0.0001 and P=0.0002, respectively), which is compatible with the systemic inflammation that characterizes SCD. Noticeably, expression of membrane-bound LRP1 was significantly higher in monocytes from SS patients than in those from SC or AA. We also found that LRP1 expression was higher in intermediate and classical monocytes, which are characterized by higher CD14 expression, than in nonclassical CD14^dim CD16⁺ cells. LRP1 expression correlated with hemopexin (r_S=0.58, 95%CI 0.26-0.79, P=0.0012) and heme levels (r_S=-0.39, 95%CI -0.58 to -0.16, P=0.0012) in SS, but not in SC patients. To address whether monocytic LRP1 expression was associated with circulating LRP1 levels, we measured s-LRP1 and found higher mean s-LRP1 levels in SS than in SC patients (5.52±2.32ug/ml vs. 4.23±2.08ug/mL, P=0.01), but AA subjects showed intermediate s-LRP1 levels (4.70±1.67ug/ml). In SS patients, s-LRP1 correlated with LDH (r_S=0.45, 95% CI 0.22-0.64, P=0.0002), but not with all biomarkers of hemolysis, suggesting that s-LRP1 production may also be influenced by mechanisms other than hemolysis. Our data show that more severe hemolysis in SS patients (higher LDH, heme, and lower hemopexin levels) is associated with elevated expression of monocyte LRP1 and its circulating form s-LRP1. Our finding that LRP1 expression in intermediate monocytes correlates with heme and hemopexin levels, particularly in SS patients, suggests this cell type responds to excessive heme. Along with previous publications that this monocyte subtype upregulates heme oxygenase gene HMOX, this supports that monocytes equipped with LRP1 can capture heme/hemopexin complexes and detoxify heme. In addition, higher LRP1 expression in CD14⁺ circulating monocytes corroborates that these cells can respond simultaneously to free heme with TLR4/CD14-dependent TNF-alpha production and to hemopexin-bound heme captured by LRP1 to foster iron-mediated augmentation of IL-6 production (Dagur et al. 2017).

Conclusions: This study highlights that some monocyte subtypes are more prone to capture heme/hemopexin complexes and may react to both free and hemopexin-bound heme, adding to the complexity of the pathophysiology of SCD. Modulation of monocyte differentiation towards subsets with lower responsiveness to heme may be further explored in SCD and other hemolytic diseases in the future.

References: Dagur et al. Br J Haematol 2017 doi: 10.1111/bjh.14663.