Erythrocyte deformability and eryptosis during inflammation,and impaired blood rheology

1 Introduction

Systemic inflammation is characterised by the presence of various dysregulated circulating cytokines and other inflammatory molecules [1–8]. These inflammatory molecules have an adverse effect on the cells of the haematological system, which include the presence of a hypercoagulable state, effects of blood viscosity, hyperreactive platelets and pathologic deformability and elasticity that eventually results in eryptotic erythrocytes (RBCs) [4, 10]. All of these pathologies cause alterations in blood rheology in inflammatory conditions.

RBCs and the extent of their interactions with each other, circulating proteins and with platelets make them good health indicators, specifically when hemorheological changes happen due to and during inflammation. Inflammation causes RBC pathology, platelet hyper-reactivity and anomalous blood clotting of plasma molecules [11, 12]. The following changes are noted in these blood components:

RBCs: Over the past years, RBC pathology was mostly studied by looking at the phosphatidylserine flip, Ca^2 + leakage into the cell, changes to ceramide and cell shrinkage and the term ‘eryptosis” was coined for RBC cell death [13–20]. RBCs have a highly specialized and organized membrane structure, which interacts with, and reacts to inflammatory molecule insults, and result in either pathological deformability that eventually cell death, similar to apoptosis, known as eryptosis [7, 21]. The presence of enhanced eryptosis and the biochemical changes and processes leading up to it, has a negative effect on blood rheology [22, 23] and also on platelet and plasma protein interactions with RBCs.

The following paragraphs provide a brief overview of the processes that lead to a changed RBC deformability, that eventually result in eryptosis. We show pathological changes found in RBCs during various inflammatory diseases and discuss how biophysical and ultrastructural changes in RBCs may provide an essential in vivo cell model system.

2 Eryptosis in RBCs

Eryptosis is the final process of RBC cell death after a prolonged exposure to pro-inflammatory molecules. It happens because of exposure to circulating inflammatory molecules and oxidative stress; see Fig. 1 and various references that discuss this process in detail [13–15, 33]. Particularly the entrance of Ca^2 + and the exit of KCl from RBCs are key processes in eryptosis. These biochemical changes cause pathology in RBC membranes, affecting the deformability of the cells and ultimately result in eryptosis. Deformability and elasticity are of fundamental importance in hemorheology and if it is affected, the optimal functioning of the haematological system is at risk.

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Fig.1

The process of eryptosis in RBCs (adjusted from [33]).

There are three distinct physiological processes in the phases leading up to eryptosis, they are: cell membrane scrambling, cell shrinkage and membrane blebbing. These processes are visible as ultrastructural changes and can be visualized using scanning electron microscopy. See Fig. 2 for a visual representation of the sequence during the process, that ultimately ends in eryptosis.

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Fig.2

The sequence of eryptosis from (A) healthy to irreversible eryptosis (B to D).

What does a changed RBC deformability during inflammation actually mean, and how does it affect hemorheology? In healthy conditions, RBCs should deform significantly to fit through the tiny capillaries. Deformability is therefore an intrinsic characteristic of RBC function. For this deformation to happen, the RBC membrane needs to be elastic to elongate sufficiently to squeeze through small perimeters. However, of greater importance is that the cell must return to its original biconcave shape after exiting the small diameter capillaries. In our research we have noted that in (systemic) inflammatory conditions like type 2 diabetes, Rheumatoid Arthritis, Hereditary Hemochromatosis, Parkinson’s disease and Alzheimer’s disease, RBCs lose their ability to retain their biconcave structure after deformation [33–38]. These structural changes are also seen when blood smears from these conditions are studied using light microscopy (see Fig. 3 (unpublished micrograph data from [34]). This loss of ability to return to a biconcave shape is likely to be the real dilemma in hemorheology and not necessary the ability to deform in the first place.

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Fig.3

Light microscopy of whole blood from 2 hereditary hemochromatosis individuals (100x magnification of a blood smear showing eryptosis). Arrows show RBCs that do not return to their biconcave shape; and boxes show RBCs that already have become eryptotic (unpublished micrograph data from [34]).

The loss of the ability to retain a biconcave shape is an important dilemma during clot formation. Hypercoagulation is an important hallmark of inflammation and abnormal clot structure involves, not only the presence of hyper-coagulated plasma proteins (particularly fibrin(ogen)), but also the pathological interactions of RBCs with anomalous blood clotting during inflammation. During hypercoagulation, RBCs that are not able to retain their biconcave shape, are trapped tightly in clots (see Fig. 4; unpublished micrograph data from [34, 38]).

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Fig.4

(A) A reprehensive example of trapped RBCs in fibrin fibres of type II diabetes and (B) Hereditary Hemochromatosis. Unpublished data from [34, 38]).

During inflammation, platelets are hyperactivated, with increased pseudopodia formation with increased adherence to RBC membranes, aggregation and spreading. Such interactions affect RBC deformability and impair blood rheology. See Fig. 5 for examples of platelet structure in healthy blood before and after exposure to the inflammatory cytokine, Interleukin-8 (unpublished data from [4].

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Fig.5

(A) RBCs and platelets in a healthy blood smear; (B) after exposure to interleukin 8 (unpublished raw data from [4]).

Hemorheology and RBC deformability are important parameters in hypercoagulation found during inflammation. These parameters may of great importance clinically, to both identify the presence and extent of inflammation, and to follow changes in these parameters during intervention therapies. RBC rheology and deformability may be used successfully as cell models for pharmaceutical testing. Although such models were popular a few decades ago, it seems as if it has lost its attractiveness. However, due to new and state-of-the-art equipment like scanning electron microscopy, super-resolution confocal microscopy and rheometers, the role of RBCs, platelets and plasma proteins, in hemorheology and coagulation, need to be revisited.

Conflict of interest

There are no conflicts of interest to declare by the author.