**3. Neutrophil activation**

210 Chronic Kidney Disease

Erythrocyte membrane proteins can be classified into three categories, according to their functional properties in the membrane struture (An & Mohandas, 2008; Mohandas & Gallagher, 2008). The first includes cytoskeletal proteins, as spectrin (α and β chains), protein 4.1, and actin; the second includes integral/transmembrane proteins of which the representative proteins are band 3 and glycophorins; the third includes anchoring/linker proteins, namely, ankyrin (also known as band 2.1) and protein 4.2. The anchoring/linker membrane proteins mediate the attachment of cytoskeletal proteins to integral proteins (Fig.

Fig. 1. Schematic representation of red blood cell membrane, showing the topographical localization of proteins and their interactions. The membrane is a complex structure in which a plasma membrane envelope composed of amphiphilic lipid molecules is anchored

(transmembrane proteins) embedded in the lipid bilayer. Adapted from An & Mohandas,

The cytoskeleton is a 3-dimensional network of proteins that covers the cytoplasmatic surface of the erythrocyte membrane and is responsible for its biconcave shape and the properties of elasticity and flexibility. It comprises approximately half the membrane protein mass and is primarily composed of spectrin, protein 4.2 and actin. Spectrin is the major protein of the cytoskeleton, and, therefore, the primary cause of erythrocyte shape, integrity and deformability. It is linked to the lipid bilayer, by vertical protein interactions with the transmembrane proteins, band 3 and glicophorin A (Lucchi, 2000). In the vertical protein interaction of spectrin with band 3 there are also ankyrin (also known as band 2.1) and protein 4.2 involved. A normal linkage of spectrin with the other proteins of the cytoskeleton assures normal horizontal protein interactions. The vertical and horizontal interactions between membrane constituents account for the integrity, strength, and deformability of the cell (An & Mohandas, 2008; Mohandas & Gallagher, 2008). Disruption of vertical interactions because of membrane protein deficiencies favours membrane vesiculation with loss of surface area and development of spherocytic cells, with increasing

to a two dimensional elastic network of skeletal proteins through tethering sites

2008.

**2. Erythrocyte membrane protein composition**

1) (Lucchi, 2000; Gallagher, 2005; Mohandas & Gallagher, 2008).

Leukocytosis and recruitment of circulating leukocytes into the affected areas are hallmarks of inflammation. Leukocytes are chimio-attracted to inflammatory regions and their transmigration from blood to the injured tissue is primarily mediated by the expression of cell-adhesion molecules in the endothelium, which interact with surface receptors on leukocytes (Muller, 1999; Sullivan, 2000). This leukocyte-endothelial interaction is regulated by a cascade of molecular steps that lead to the morphological changes that accompany adhesion. At the inflammatory site, leukocytes release their granular content and may exert their phagocytic capacities.

In acute inflammation, the leukocyte infiltration is predominantly of neutrophils, whereas in chronic inflammation an infiltration predominantly of macrophages and lymphocytes is observed. Leukocyte-endothelial cell interactions are important for leukocyte transmigration and trafficking in physiological conditions. There is increasing evidences that changes in those leukocyte-endothelial interactions, due to endothelium damage or dysfunction, might be implicated in the pathogenesis of diseases, such as inflammatory diseases (Harlan, 1985; Ley, 2007).

Leukocytosis is essential as the primary host defence, and neutrophils, the major leukocyte population of blood in adults, play a primordial role. It is well known that neutrophils have mechanisms that are used to destroy invading microorganisms. These cells use an extraordinary array of oxygen-dependent and oxygen-independent microbicidal weapons to destroy and remove infectious agents (Witko-Sarsat, 2000). Oxygen-dependent mechanisms involve the production of reactive oxygen species (ROS), which can be microbicidal (Roos, 2003), and lead to the development of oxidative stress. Oxygen-independent mechanisms include chemotaxis, phagocytosis and degranulation. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex, known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which catalyzes the formation of superoxide anion (O2 ·–). Activated neutrophils also undergo degranulation, with the release of several components, namely, proteases (such as elastase) and cationic proteins (such as lactoferrin) (Saito, 1993; Brinkmann, 2004).

Elastase is a member of the chymotrypsin superfamily of serine proteinases, expressed in monocytes and mast cells, but mainly expressed by neutrophils, where it is compartmentalized in the primary azurophil granules. The intracellular function of this enzyme is the degradation of foreign microorganisms that are phagocytosed by the neutrophil (Brinkmann, 2004). Elastase can also degrade local extracellular matrix proteins (such as elastin), remodel damaged tissue, and facilitate neutrophil migration into or through tissues. Moreover, elastase also modulates cytokine expression at epithelial and endothelial surfaces, up-regulating the production of cytokines, such as IL-6, IL-8, transforming growth factor β (TGF-β) and granulocyte-macrophage colony-stimulating factor (GM-CSF); it also promotes the degradation of cytokines, such as IL-1, TNF-α and IL-2. There is evidence in literature that high levels of elastase are one of the major pathological factors in the development of several chronic inflammatory lung conditions (Fitch, 2006).

Neutrophil Activation and Erythrocyte Membrane

protein. Adapted from Costa, 2008a.

Protein Composition in Stage 5 Chronic Kidney Disease Patients 213

Hb (g/dL) 13.90 (13.2-15.00) 10.90 (10.30-12.30)\* White cell counts (x 109/L) 5.78 ± 1.59 6.23 ± 2.10 Lymphocytes (x 109/L) 2.35 ± 0.75 1.47 ± 0.60\* Monocytes (x 109/L) 0.25 ± 0.08 0.38 ± 0.16\* Neutrophils (x 109/L) 3.03 ± 1.02 4.14 ± 1.79\* Albumin (g/dL) NM 3.8 0.4 CRP (mg/dL) 1.75 (0.76-4.70) 5.75 (1.90-14.01)\* Elastase (μg/L) 28.29 (26.03-34.74) 36.11 (29.69-50.65)\* Elastase/Neutrophil ratio 10.86 (7.44-12.12) 8.91 (7.43-13.78) Lactoferrin (μg/L) 236.56 (193.56-295.03) 239.35 (165.64-332.60) Lactoferrin/Neutrophil ratio 72.11 (55.52-111.83) 60.32 (42.82-99.45) Table 1. Haematological data and neutrophil activation markers, for controls and for stage 5 CKD patients.\* *p*<0.05, *vs* controls. NM: not measured. Results are presented as mean ± standard deviation or as median (interquartile ranges). Hb: Haemoglobin; CRP: C-reactive

The haemodialysis procedure, itself, seems to lead to neutrophil activation (Costa, 2008a). By evaluating CKD patients before and after haemodialysis procedure (Costa, 2008b), we found a higher haemoglobin concentration and erythrocyte count after haemodialysis (Table 2). This increase in circulating erythrocytes, has been associated (Dasselaar, 2007) to a

Hb (g/dL) 12.10 (10.95-12.80) 13.20 (11.15-14.60)\* White cell counts (x 109/L) 5.86 1.5 1 5.93 2.19 Neutrophils (x 109/L) 3.82 1.24 3.97 1.77 Monocytes (x 109/L) 0.24 0.38 0.17 0.12 Lymphocytes (x 109/L) 1.64 0.69 1.66 0.64 Elastase (μg/L) 36.16 (29.71-47.13) 51.69 (40.08-71.68)\* Elastase/Neutrophil ratio 10.66 (7.32-13.54) 14.66 (13.34-18.95)\* Lactoferrin (μg/L) 198.61 (137.81-216.97) 236.56 (171.28-363.63)\* Lactoferrin/Neutrophil ratio 48.33 (33.88-64.31) 60.72 (51.81-94.81)\* CRP (mg/dL) 3.06 (1.39-5.22) 3.53 (1.54-5.56) Table 2. Hematological data and neutrophil activation markers for stage 5 CKD patients, before and after haemodialysis procedure. *\*p*<0.05, *vs* before haemodialysis. Results are presented as mean ± standard deviation or as median (interquartile ranges). Hb:

haemoglobin; CRP: C-reactive protein. Adapted from Costa, 2008b.

**Stage 5 CKD Patients (n=20) Before After** 

**Controls (n=26)**

**All patients (n=63)**

Plasma lactoferrin is predominantly neutrophil derived and its presence in the specific granules is often used to identify these types of granules. Lactoferrin is also found in other granules, in the tertiary granules, though in lower concentrations (Olofsson, 1977; Baynes 1986; Halliwell Gutteridge, 1990; Saito, 1993). Lactoferrin is a multifunctional iron glycoprotein, which is known to exert a broad-spectrum primary defence activity against bacteria, fungi, protozoa and viruses. It can bind to large amounts of free iron. The ironbound lactoferrin is taken up by activated macrophages, which express specific lactoferrin receptors. During inflammation, this contributes to iron deprivation of the erythroid precursors, which do not express lactoferrin receptors (Bárány, 2001). Other mechanisms in which lactoferrin is implicated include a growth regulatory function in normal cells, coagulation, and perhaps cellular adhesion modulation (Levay and Viljoen, 1995).

In a recent study of our group (Pereira, 2010), we found that stage 5 CKD patients present a decreased expression of the CXCR1 neutrophil surface marker, which plays an important role in neutrophil migration (Fig. 2); a higher elastase plasma levels was also found, as compared to a control group (table 1).

Fig. 2. Decreased expression of the CXCR1 neutrophil surface markers in stage 5 CKD patients. A – Control; B – Stage 5 CKD patient. Cells were stained with allophycocyanin (APC) conjugated anti-CD11B and phycoerythrin (PE) conjugated anti-CXCR1.

CXCR1 is a receptor that recognizes CXC chemokines, particularly, the pro-inflammatory IL-8 (Pay, 2006; Sherry, 2008). The decreased expression of this receptor in neutrophil surface is associated to the release of components of neutrophil granules and is correlated with the need for inotropic support. Recently, it was reported that the levels of the neutrophil chemoattractant receptor, CXCR1, is mildly diminished in CKD pediatric patients, as a consequence of the end stage renal disease itself, and that the recurrent serial bacterial infections they suffered was markedly exacerbated by CXCR1 neutrophil loss (Sherry, 2008). This loss of CXCR1 on neutrophils might be due to the uremic state, to changes in leukocyte adhesion molecule expression or membrane microvilli and/or to crossdesensitization of this receptor, due to prior exposure to several unrelated chemoattractants, including N-formylated peptides and the complement cleavage product C5a (Sherry, 2008). Chronic exposure of circulating inflammatory cells to these mediators may lead to loss of chemokine receptor expression and/or function via cross-desensitization.

Plasma lactoferrin is predominantly neutrophil derived and its presence in the specific granules is often used to identify these types of granules. Lactoferrin is also found in other granules, in the tertiary granules, though in lower concentrations (Olofsson, 1977; Baynes 1986; Halliwell Gutteridge, 1990; Saito, 1993). Lactoferrin is a multifunctional iron glycoprotein, which is known to exert a broad-spectrum primary defence activity against bacteria, fungi, protozoa and viruses. It can bind to large amounts of free iron. The ironbound lactoferrin is taken up by activated macrophages, which express specific lactoferrin receptors. During inflammation, this contributes to iron deprivation of the erythroid precursors, which do not express lactoferrin receptors (Bárány, 2001). Other mechanisms in which lactoferrin is implicated include a growth regulatory function in normal cells,

coagulation, and perhaps cellular adhesion modulation (Levay and Viljoen, 1995).

Fig. 2. Decreased expression of the CXCR1 neutrophil surface markers in stage 5 CKD patients. A – Control; B – Stage 5 CKD patient. Cells were stained with allophycocyanin

CXCR1 is a receptor that recognizes CXC chemokines, particularly, the pro-inflammatory IL-8 (Pay, 2006; Sherry, 2008). The decreased expression of this receptor in neutrophil surface is associated to the release of components of neutrophil granules and is correlated with the need for inotropic support. Recently, it was reported that the levels of the neutrophil chemoattractant receptor, CXCR1, is mildly diminished in CKD pediatric patients, as a consequence of the end stage renal disease itself, and that the recurrent serial bacterial infections they suffered was markedly exacerbated by CXCR1 neutrophil loss (Sherry, 2008). This loss of CXCR1 on neutrophils might be due to the uremic state, to changes in leukocyte adhesion molecule expression or membrane microvilli and/or to crossdesensitization of this receptor, due to prior exposure to several unrelated chemoattractants, including N-formylated peptides and the complement cleavage product C5a (Sherry, 2008). Chronic exposure of circulating inflammatory cells to these mediators may lead to loss of

(APC) conjugated anti-CD11B and phycoerythrin (PE) conjugated anti-CXCR1.

chemokine receptor expression and/or function via cross-desensitization.

compared to a control group (table 1).

In a recent study of our group (Pereira, 2010), we found that stage 5 CKD patients present a decreased expression of the CXCR1 neutrophil surface marker, which plays an important role in neutrophil migration (Fig. 2); a higher elastase plasma levels was also found, as


Table 1. Haematological data and neutrophil activation markers, for controls and for stage 5 CKD patients.\* *p*<0.05, *vs* controls. NM: not measured. Results are presented as mean ± standard deviation or as median (interquartile ranges). Hb: Haemoglobin; CRP: C-reactive protein. Adapted from Costa, 2008a.

The haemodialysis procedure, itself, seems to lead to neutrophil activation (Costa, 2008a). By evaluating CKD patients before and after haemodialysis procedure (Costa, 2008b), we found a higher haemoglobin concentration and erythrocyte count after haemodialysis (Table 2). This increase in circulating erythrocytes, has been associated (Dasselaar, 2007) to a


Table 2. Hematological data and neutrophil activation markers for stage 5 CKD patients, before and after haemodialysis procedure. *\*p*<0.05, *vs* before haemodialysis. Results are presented as mean ± standard deviation or as median (interquartile ranges). Hb: haemoglobin; CRP: C-reactive protein. Adapted from Costa, 2008b.

Neutrophil Activation and Erythrocyte Membrane

shortened erythrocyte survival.

band 3 profile.

Protein Composition in Stage 5 Chronic Kidney Disease Patients 215

Leukocyte activation is part of an inflammatory response, and is an important source of ROS and proteases, both of which may impose oxidative and proteolytic damages to erythrocyte and plasma constituents. Actually, oxidative stress has been reported to occur in stage 5 CKD patients and has been proposed as a significant factor in haemodialysis-related

In literature, there are few reports about the effect of CKD and haemodialysis procedure in erythrocyte membrane protein composition (Matos, 1997; Wu, 1998; Ibrahim, 2002). Studies performed in erythrocytes from stage 5 CKD patients, using cuprophane and polyacrylonitrile dialysis membranes, showed some changes in the membrane proteins, namely, a reduction in spectrin and band 3, and an isolated reduction in band 3, respectively (Sevilhano, 1990; Delmas-Beauvieux, 1995). Wu et al (Wu, 1998) and Ibrahim et al (2002) showed that stage 5 CKD patients presented a median osmotic fragility higher than the

Recently, we reported for the first time, changes in the erythrocyte membrane band 3 profile in stage 5 CKD patients. These patients presented a decrease in HMWAg and in HMWAg/band 3 monomer ratio (Fig. 3 and table 3). These changes seem to reflect a younger erythrocyte population; however, CKD presented also a decrease in Pfrag and in Pfrag/band 3 monomer ratio, both suggesting a rise in erythrocyte damage. Thus, it seems that the band 3 profile observed in CKD patients is associated both to an increase in younger erythrocytes and to an increase in damaged erythrocytes (Costa, 2008c). This study also showed that the haemodialysis procedure *per se* does not lead to an increase in the studied markers of erythrocyte damage. Actually, no differences were found after haemodialysis, in

> **Controls (n=26)**

HMWAg (%) 19.90 (15.42-21.12) 15.23 (13.38-19.40)\*

Band 3 monomer (%) 55.28 (53.39-57.41) 61.84 (56.87-64.41)\*

Pfrag (%) 26.29 ± 4.78 22.70 ± 6.01\*

HMWAg/ Band 3 monomer 0.33 ± 0.07 0.27 ± 0.07\*

Pfrag/ Band 3 monomer 0.48 ± 0.11 0.38 ± 0.13\*

are presented as mean ± standard deviation or as median (interquartile ranges).

Table 3. Band 3 profile for controls and stage 5 CKD patients.

\* p<0.05 *v*s controls. HMWAg; high molecular weight aggregates; Pfrag: proteolytic fragments. Results

Some changes in erythrocyte membrane protein composition of stage 5 CKD patients using high-flux polysulfone FX-class dialysers of Fresenius, were also observed (Costa, 2008b; Costa, 2008d). A decrease in spectrin was the most significant change (table 4). This reduction in spectrin may account for a poor linkage of the cytoskeleton to the membrane, favoring membrane vesiculation, and, probably, a reduction in the erythrocyte lifespan of

**Stage 5 CKD patients (n=63)**

controls, and, after the haemodialysis procedure, that osmotic fragility decreased.

translocation of erythrocytes from the splanchnic circulation (and possibly from the splenic circulation) in order to compensate the hypovolemic stress during dialysis ultrafiltration. We also found, after haemodialysis, an increase in mean cell hemoglobin concentration and a decrease in mean cell volume that could be related to erythrocyte membrane protein loss during the hemodialysis procedure (Costa, 2008b). Markers of neutrophil activation were also found to be increased after haemodialysis. In fact, a decrease in CXCR1 neutrophil expression was observed after the haemodialysis procedure [before haemodialysis: 252.25 ± 45.14 MFI (mean fluorescence intensity) vs after haemodialysis: 239.71 ± 47.62 MFI; *p*=0.04], as well as an increase in elastase and lactoferrin plasma levels (Table 2). The enhanced neutrophil activation state after haemodialysis could result from different mechanisms; namely, complement activation, direct interaction with haemodialysis membrane, and from the passage into the blood of bacterial fragments, such as LPS, from contaminated dialysate through the dialyzer membrane.
