**1.4 Respiratory burst**

The enzyme complex NADPH oxidase promotes the generation of reactive oxygen species (e.g. superoxide anions) in leukocytes, in a process referred to as the respiratory burst. Respiratory burst is a central mechanism for the leukocyte function of phagocytosis and elimination of invading microorganisms (Babior 1999). Superoxide anions are converted to hydrogen peroxide in the phagolysosome by the action of superoxide dismutase. In the absence of superoxide dismutase, superoxide anions can form the highly aggressive oxidative substance peroxynitrite (by reacting with nitric oxide) and hydroxyl radicals (Dahlgren & Karlsson 1999; Johnson & Giulivi 2005).

### **1.5 Apoptosis**

In early apoptosis, there is a reconformation of the cell membrane, with phosphatidyl serine (PS) translocated from the inner surface to the outer leaflet of the cell membrane. Fluorescein-conjugated Annexin V binds to PS with high affinity and identifies early apoptotic cells. Propidium iodide enters through damaged cell membranes after loss of membrane integrity and stains DNA, identifying late stages of apoptosis and secondary necrotic cells. PS is identified by phagocytes in the extracellular milieu in order to remove the dying cells by phagocytosis.

CXC (α-chemokines) and CC (β-chemokines) depending on the positions of two cysteine residues (C) relative to other amino acids (X) (Charo & Ransohoff 2006). Chemokines are produced by inflammatory cells after stimulation with proinflammatory cytokines or bacterial products, and there are both soluble and membrane-bound chemokines, with various functions (Parkin & Cohen 2001). Some of the chemokines and cytokines analyzed

Leukocyte binding to endothelial cells induces production of signaling molecules in the endothelial cells and activation of NADPH oxidase in leukocytes. NADPH oxidase promotes production of reactive oxygen species that break down the barrier to leukocyte passage between the endothelial cells and through the basement membrane (van Buul & Hordijk 2004). PECAM-1 plays an important role in transendothelial migration of leukocytes, by inducing phosphorylation of tyrosine in junctional proteins which leads to

When neutrophils extravasate, they produce enzymes (i.e. elastase and other proteases such as matrix metalloproteinase-9, MMP-9) that break down extracellular matrix proteins and in this way promote leukocyte migration through the interstitium (Hermant et al. 2003). The final step of the transmigration is the chemokine concentration gradient, which guides leukocytes through the interstitium and towards the inflammatory site. CXCL8 (IL-8) and CCL2 (MCP-1) act as chemotactic factors for neutrophils and monocytes, respectively. They bind to proteoglycans in the extracellular matrix and to similar molecules on the leukocytes

Neutrophils and monocytes in blood normally express a low amount of CD11b on their surface. Following chemokine-mediated activation of the cells, CD11b is mobilized on the cell surface and the molecules are activated in order to display their functions (Adams & Shaw 1994; Albelda et al. 1994; Adams & Lloyd 1997; Gonzalez-Amaro & Sanchez-Madrid 1999). Mobilization of CD11b is important in the process of leukocyte transmigration, phagocytosis and complement activation as a response to inflammation/infection (Bainton

The enzyme complex NADPH oxidase promotes the generation of reactive oxygen species (e.g. superoxide anions) in leukocytes, in a process referred to as the respiratory burst. Respiratory burst is a central mechanism for the leukocyte function of phagocytosis and elimination of invading microorganisms (Babior 1999). Superoxide anions are converted to hydrogen peroxide in the phagolysosome by the action of superoxide dismutase. In the absence of superoxide dismutase, superoxide anions can form the highly aggressive oxidative substance peroxynitrite (by reacting with nitric oxide) and hydroxyl radicals

In early apoptosis, there is a reconformation of the cell membrane, with phosphatidyl serine (PS) translocated from the inner surface to the outer leaflet of the cell membrane. Fluorescein-conjugated Annexin V binds to PS with high affinity and identifies early apoptotic cells. Propidium iodide enters through damaged cell membranes after loss of membrane integrity and stains DNA, identifying late stages of apoptosis and secondary necrotic cells. PS is identified by phagocytes in the extracellular milieu in order to remove

in our study, and their respective functions, are listed in Table 1 and Table 2.

loss of cell-cell adhesion (van Buul & Hordijk 2004).

et al. 1987; Borregaard et al. 1987; Miller et al. 1987).

(Dahlgren & Karlsson 1999; Johnson & Giulivi 2005).

(Janeway & Travers 2005).

**1.4 Respiratory burst** 

**1.5 Apoptosis** 

the dying cells by phagocytosis.


Table 1. Chemokines analyzed in our study and their respective functions.

as angiogenin and complement factor D), κ- and λ-light chains and chemotaxis inhibitory protein (Vanholder et al. 1994b; Haag-Weber & Horl 1996a; Kaysen 2001; Horl 2002; Kaysen

Historically, dialysis has contributed to saving many lives over the years. Without dialysis, a uremic patient unavoidably goes towards death. However, the life quality of patients on dialysis still has to be improved to develop an optimal treatment. In spite of the process in the last years to strive towards more biocompatible materials and methods, including highflux dialysis treatment, patients on hemodialysis still display a high morbidity and mortality in infections (Bloembergen & Port 1996; Powe et al. 1999; Graff et al. 2002). Neutrophil dysfunction in dialysis patients is manifested by reduced chemotaxis, adherence, respiratory burst and glucose consumption in response to an inflammatory stimulus (Vanholder et al.

The dysfunctional state of inflammatory activation seen in dialysis patients could be caused by several different factors (Cheung et al. 1989; Haag-Weber et al. 1991; Descamps-Latscha 1993; Schindler et al. 2001; Carracedo et al. 2002; Horl 2002; Raj et al. 2002; Kosch et al. 2003; Koller et al. 2004). Fragments of bacterial products can be present in small amounts in the dialysate and enter the circulation by diffusion through the dialysis membrane (Horl 2002). These bacterial fragments activate proinflammatory cytokines such as IL-6, TNF-α and IL-1. There is also direct activation of complement factors and of leukocytes by contact with the dialysis membrane. Another aspect is the removal of cytokines and other inflammatory markers (lipopolysaccharide fragments, granulocyte inhibitory proteins 1 and 2, IL-1, TNFα) and complement factors (C3a, C5a) by the hemodialysis procedure as well as the adsorption of substances to the hydrophobic high-flux membrane (e.g. factor D) (Clark et al. 1999; Schindler et al. 2006). Dialysis can reduce leukocyte-endothelial interactions and impair transmigration (Thylen et al. 1997). In patients on hemodialysis with cuprophane or polysulfone membranes, a significantly higher serum level of MCP-1 is seen compared with healthy subjects both before and after the hemodialysis session, independent of the

Biocompatibility of dialysis membranes probably plays an important role in determining leukocyte function in patients on hemodialysis (Himmelfarb et al. 1991; Himmelfarb et al. 1993; Hernandez et al. 2004; Schindler et al. 2006). High serum levels of cytokines and chemokines have been observed in patients on hemodialysis with modified cellulose membranes (Descamps-Latscha 1993; Pawlak et al. 2004; Muniz-Junqueira et al. 2005). Highflux hemodialysis causes lower levels of IL-6 and IL-1β than low-flux hemodialysis or dialysis with cuprophane membranes (Schindler et al. 2006). Our group has previously demonstrated that neutrophils and monocytes recruited to an induced interstitial inflammatory site in patients treated with low-flux bioincompatible hemodialysis have an impaired capacity of mobilizing CD11b in response to the induced inflammation, compared with the corresponding cells from healthy subjects (Thylen et al. 2000; Jacobson et al. 2002). Chronic kidney disease is a state that induces apoptosis, but this is normalized with continuous and high-flux hemodialysis modalities (D'Intini et al. 2004; Bordoni et al. 2006). This is in accordance with studies showing that dialysis membrane characteristics affect leukocyte cell apoptosis (Martin-Malo et al. 2000; Sela et al. 2005; Sardenberg et al. 2006). The degree of spontaneous apoptosis of leukocytes is higher when bioincompatible membranes

& Kumar 2003; Cohen & Horl 2009b, 2009a).

membrane used (Jacobson et al. 2000; Thylen et al. 2000).

**1.6.2 Patients on hemodialysis** 

1993b; Vanholder et al. 1993a).


Table 2. Cytokines analyzed in our study and their respective functions.
