**2.2.3 Apoptosis and cell counts**

There was no significant difference in the total number of leukocytes at the inflammatory sites between patients on high-flux hemodialysis/hemodiafiltration and healthy subjects.

In our study of leukocytes from patients on high-flux hemodialysis/hemodiafiltration, leukocytes were studied at their actual site of action, namely after *in vivo* extravasation. This is advantageous, since leukocyte function in patients with chronic kidney disease or on dialysis has previously almost exclusively been studied on cells collected from the peripheral circulation.

In both the neutrophil and monocyte populations, we observed no significant differences in the percentage of apoptotic cells (Annexin V+ and Annexin V+ PI+) in the peripheral circulation or at the sites of interstitial inflammation between patients on high-flux hemodialysis/hemodiafiltration and healthy subjects.

Chemokines in skin blister fluids and serum from the peripheral circulation were analyzed with commercially available immunoassays (Quantikine®, R&D Systems Inc. Minneapolis, MN, USA). All immunoassays were used in accordance with the manufacturer's instructions. For further details, please review publications (Olsson et al. 2007 & Olsson et al.

There was a similar expression of CD11b on monocytes and neutrophils in patients on highflux hemodialysis/hemodiafiltration and healthy subjects, both in the peripheral circulation and at the three sites of interstitial inflammation. *In vitro* activation with fMLP induced a significant increase in the expression of CD11b on monocytes and neutrophils in the peripheral circulation and at the sites of interstitial inflammation, both in patients on high-

The preserved capacity of both monocytes and neutrophils to express CD11b at the sites of interstitial inflammation in patients on high-flux hemodialysis/hemodiafiltration, as shown by our findings, may have important biological consequences in terms of an adequate performance of leukocyte functions in which the CD11b molecule plays a key role (Thylen et al. 1997; Moshfegh et al. 2002). Extravasated neutrophils and monocytes from patients on high-flux hemodialysis/hemodiafiltration showed a maintained response to fMLP as a

The mechanism behind this preserved leukocyte function in patients on high-flux biocompatible hemodialysis/hemodiafiltration could be the removal of small and middlesized leukocyte inhibitory molecules by high-flux hemodialysis/hemodiafiltration (Vanholder et al. 1994a), but membrane compatibility could also play an important role.

Results for hydrogen peroxide production in neutrophils and monocytes are displayed in Figures 3-6. The findings indicate the presence of a dose-response phenomenon in terms of leukocyte function at the site of interstitial inflammation in patients on high-flux hemodialysis/hemodiafiltration, which could be due to leukocyte refractoriness when encountered with an intense inflammatory stimulus. Refractoriness of leukocytes could be caused by previous priming, giving rise to an impaired response to a second activating

There was no significant difference in the total number of leukocytes at the inflammatory sites between patients on high-flux hemodialysis/hemodiafiltration and healthy subjects. In our study of leukocytes from patients on high-flux hemodialysis/hemodiafiltration, leukocytes were studied at their actual site of action, namely after *in vivo* extravasation. This is advantageous, since leukocyte function in patients with chronic kidney disease or on dialysis has previously almost exclusively been studied on cells collected from the

In both the neutrophil and monocyte populations, we observed no significant differences in the percentage of apoptotic cells (Annexin V+ and Annexin V+ PI+) in the peripheral circulation or at the sites of interstitial inflammation between patients on high-flux

flux hemodialysis/hemodiafiltration and healthy subjects.

second inflammatory stimulus after extravasation.

**2.2.2 Hydrogen peroxide formation** 

**2.2.3 Apoptosis and cell counts** 

hemodialysis/hemodiafiltration and healthy subjects.

peripheral circulation.

2009).

**2.2 Results 2.2.1 CD11b** 

stimulus.

Fig. 3. Respiratory burst in neutrophils at the site of an intermediate interstitial inflammation expressed as mean fluorescence intensity (MFI). P is indicated where a significant difference is present.

 Median 25%-75%

Patients Non-Outlier Range

Healthy PMA

Patients PMA

Healthy

inflammation. No significant difference for any comparison.

0

10

20

30

40

50

60

70

Patients fMLP

Healthy fMLP

Fig. 5. Respiratory burst (MFI) in monocytes at the site of intermediate interstitial

Fig. 4. Respiratory burst (MFI) in neutrophils at the site of intense interstitial inflammation.

p < 0.001

 Median 25%-75%

Patients Non-Outlier Range

Healthy PMA

Patients PMA

Healthy

p < 0.05

0

20

40

60

80

100

120

140

160

180

200

220

240

Patients fMLP

p < 0.01

Healthy fMLP

Fig. 4. Respiratory burst (MFI) in neutrophils at the site of intense interstitial inflammation.

Fig. 5. Respiratory burst (MFI) in monocytes at the site of intermediate interstitial inflammation. No significant difference for any comparison.

*In vivo* extravasated monocytes and neutrophils from patients on high-flux hemodialysis/hemodiafiltration have a preserved capacity to mobilize CD11b, compared with the corresponding cells from healthy subjects (Olsson et al. 2007). Furthermore, monocytes and neutrophils were able to respond to a second signal (fMLP) at the site of interstitial inflammation, indicating an adequate response to bacterial peptides (Olsson et al. 2007). After the most potent stimulation, both monocytes and neutrophils that had extravasated *in vivo* and been recruited to the site of intense inflammation showed a lower capacity to produce hydrogen peroxide in response to activation, compared with the corresponding cells from healthy individuals (Olsson et al. 2007). The apoptotic rates of neutrophils and monocytes were similar in patients and in healthy subjects (Olsson et al. 2007). Clearance of leukocytes from the site of infection via apoptosis is essential for the coordinated resolution of inflammation. The balance between pro-apoptotic and antiapoptotic factors is necessary for the maintenance of an effective immune response without

The higher concentration of MCP-1 and equal concentration of IL-8, MMP-9/NGAL and MIP-1α at the sites of intermediate and intense inflammation in patients on high-flux hemodialysis/hemodiafiltration (Olsson et al. 2009) could be of importance for the maintained capacity of leukocytes to extravasate and mobilize CD11b compared with healthy subjects (Olsson et al. 2007). These data contrast with our previous studies on patients with chronic kidney disease or patients on peritoneal dialysis, in which the concentrations of MCP-1 and IL-8 are significantly lower, coupled with an impaired capacity to up-regulate CD11b on neutrophils at sites of interstitial inflammation (Dadfar et

The results of our study support a preserved neutrophil and monocyte function in terms of extravasation and activation at the inflammatory focus. One possible explanation for the preserved capacity of monocytes and neutrophils to express CD11b in response to an interstitial inflammation in patients on high-flux hemodialysis/hemodiafiltration may be that the cells extravasate into a milieu which contains equal or higher concentrations of factors involved in transmigration and CD11b expression (MCP-1, IL-8, MIP-1α and MMP-9/NGAL) compared with healthy subjects. The maintained capacity to produce chemokines in the interstitium in patients on high-flux hemodialysis/hemodiafiltration may be due to an increased intradialytic removal of uremic substances that inhibit leukocyte function.

I want to thank my co-investigators and co-authors Stefan H Jacobson, Joachim Lundahl, Ali Moshfegh, Josefin Paulsson and Elham Dadfar. I also want to thank Titti Nieminen and Anette Bygdén-Nylander for skilful technical support. These studies were made possible by an unrestricted grant from Karolinska Institutet, Stockholm, Sweden and Terumo Europe N.V. *dedicated to well being*. The author also received financial support from the Swedish

Adams DH, Shaw S (1994) Leucocyte-endothelial interactions and regulation of leucocyte

the harmful side effects of an excessive neutrophil activation.

al. 2004c; Dadfar et al. 2004b, 2004a).

**4. Acknowledgement** 

Society of Nephrology.

migration. *Lancet* 343(8901): 831-836.

**5. References** 

**3. Conclusion** 

Fig. 6. Respiratory burst (MFI) in monocytes at the site of intense interstitial inflammation.
