**5. Other pathologies associated with neutrophil activation and erythrocyte damage**

Several physiological (physical exercise, pregnancy) and pathological (hereditary spherocytosis, cardiovascular disease, preeclampsia, psoriasis) conditions presenting with neutrophilic leukocytosis have been associated to an altered erythrocyte membrane protein composition and to other changes reflecting erythrocyte damage. Moreover, they have been associated to increased neutrophil activation products, suggesting that leukocyte activation may trigger injuries in the neighboring erythrocytes.

In Hereditary Spherocytosis (HS), mutations in genes encoding for some membrane proteins - band 3, spectrin, protein band 4.2 and ankyrin - may result in their partial or inaccurate assembly to the membrane. Deficiencies in one or more of those proteins cause a decrease in membrane stability that, in turn, leads to loss of membrane surface area through membrane vesiculation. By losing membrane vesicles, the cell will become spherocytic and the membrane more rigid, triggering the sequestration of cell in the spleen and, therefore, the reduction of the erythrocyte lifespan and the development of anemia (Mohandas & Gallagher, 2008).

Two distinct pathways lead to the reduction in membrane surface area: i) deficiencies in spectrin, ankyrin, or protein 4.2 reduce the density of the membrane cytoskeleton, causing a weaker linkage to the lipid bilayer, favoring the loss of membrane vesicles containing lipids and band 3; ii) deficiency in band 3 favors the development of band 3 deficient areas in the membrane, with loss of the lipid-stabilizing effect of band 3, and therefore, the release of band 3-free microvesicles, from the membrane (Iolascon, 2003; Perrotta, 2008). In a recent work by our group (Rocha, 2010), studying 160 HS patients, the analysis of erythrocyte membrane protein profile showed that 109 patients presented a primary deficiency in band 3, 35 patients a primary ankyrin deficiency, 14 patients an isolated deficiency in spectrin and 2 patients an isolated deficiency in protein 4.2. Furthermore, severe HS patients presented with higher neutrophil count and higher levels of TNF-α, IFN-, elastase, lactoferrin and ferritin. Our data show HS as a disease linked to enhanced erythropoiesis that is disturbed in the more severe forms, to which inflammation, at least in part, seems to contribute.

Neutrophil Activation and Erythrocyte Membrane

367-375, ISSN 0007-1048

vol. 69, pp.145-151, ISSN 0902- 4441

pp. 971-975, ISSN 0886- 022

pp. 267-273, ISSN 0253-5068

9041

*Journal*, vol. 1, pp. 57-63, ISSN 1874-2416

ISSN 0036-5513

**7. Acknowledgments** 

FEDER-008468).

**8. References**

Protein Composition in Stage 5 Chronic Kidney Disease Patients 221

This work was supported by national funds - "Fundação Portuguesa para a Ciência e Tecnologia" (FCT: PIC/IC/83221/2007) and co-financed by FEDER (FCOMP-01-0124-

An, X.; Mohandas, N. (2008). Disorders of red cell membrane. *Br J Haematol*, vol. 141, pp.

Bárány, P. (2001). Inflammation, serum C-reactive protein, and erythropoietin resistance.

Baynes, R.; Bezwoda, W.; Bothwell, T.; Khan, Q.; Mansoor, N. (1986). The non immune

Belo, L.; Rebelo, I.; Castro, E.M.; Catarino, C.; Pereira-Leite, L.; Quintanilha, A.; Santos-Silva,

Belo, L.; Santos-Silva, A.; Caslake, M.; Cooney, J.; Pereira-Leite, L.; Quintanilha, A.; Rebelo, I.

Brinkmann, V.; Reichard, U.; Goosmann, C.; Fauler, B.; Uhlemann, Y.; Weiss, D.S.;

Costa, E.; Rocha, S.; Rocha-Pereira, P.; Nascimento, H.; Castro, E.; Miranda, V.; Faria, M.S.;

hemodialysis patients. *Am J Nephrol*, vol. 28, pp. 935-940, ISSN 1046-6673 Costa, E.; Rocha, S.; Rocha-Pereira, P.; Castro, E.; Miranda, V.; Sameiro-Faria, M.; Loureiro,

Costa, E.; Rocha, S.; Rocha-Pereira, P.; Castro, E.; Miranda, V.; Sameiro-Faria, M.; Loureiro,

Costa, E.; Rocha, S.; Rocha-Pereira, P.; Castro, E.; Miranda, V.; Sameiro-Faria, M.; Loureiro,

Dasselaar, J.J.; Hooge, M.N..; Pruim, J.; Nijnuis, H.; Wiersum, A.; Jong, P.E.; Huisman, R.M.;

inflammatory response: serial changes in plasma iron, iron binding capacity, lactoferrin, ferritin and C reactive protein. *Scan J Clin Lab Invest,* vol. 46, pp. 695-704,

A. (2002). Band 3 as a marker of erythrocyte changes in pregnancy. *Eur J Haematol*,

(2003). Neutrophil activation and C-reactive protein concentration in preeclampsia.

Weinrauch, Y. & Zychlinsky, A. (2004). Neutrophil extracellular traps kill bacteria.

Loureiro, A.; Quintanilha, A.; Belo, L. & Santos-Silva, A. (2008a). Neutrophil activation and resistance to recombinant human erythropoietin therapy in

A.; Quintanilha, A.; Belo, L. & Santos-Silva, A. (2008b). Changes in red blood cells membrane protein composition during hemodialysis procedure. Ren Fail, vol. 30,

A.; Quintanilha, A.; Belo, L. & Santos-Silva, A. (2008c). Band profile as a marker of erythrocyte changes in chronic kidney disease patients. *The Open Clinical Chemistry* 

A.; Quintanilha, A.; Belo, L. & Santos-Silva, A. (2008d). Alterated erythrocyte membrane protein composition in chronic kidney disease stage 5 patients under haemodialysis and recombinant human erythropoietin therapy. *Blood Purif*, vol. 26,

Franssen, C.F.M. (2007). Relative blood volume changes underestimated total blood volume changes during hemodialysis. *Clin J Am Soc Nephrol* 2:669-674, ISSN 1555-

*Nephrol Dial transplant*, vol. 16, pp. 224-227, ISSN 0931- 0509

*Hypertens Pregnancy*, vol. 22, pp.129-141, ISSN 1064-1955

*Science*, vol. 303, pp.1532-1535, ISSN 0036-8075

Patients with cardiovascular disease, namely, with recent myocardial infarction (within the last 48 h), survivors for at least 3 months of myocardial infarction and hypertensive individuals, presented besides a neutrophilic leukocytosis a different band 3 profile, with higher values of HMWAg and lower values of band 3 monomer and of Pfrag (Santos-Silva, 1995). Ischemic stroke patients presented the same altered band 3 profile, associated with increased plasma levels of leukocyte activation products - elastase and lactoferrin - when compared with controls (Santos-Silva, 1998).

Band 3 profile in normal pregnancy in the first trimester of pregnancy, when compared with healthy controls, presented significantly reduced HMWAg and increased Pfrag. Comparing the third with the first trimester, a significant reduction in band 3 and a significant rise in Pfrag was also described. These results suggest band 3 profile as a marker of erythrocyte changes in normal pregnancy, which are independent of the 'physiological anemia' of pregnancy. These changes suggest an increase in damaged erythrocytes, but also an increase in younger erythrocytes in the maternal circulation. We also found alterations in the markers of erythrocyte damage in preeclampsia, in both umbilical cord blood and maternal circulation. In preeclamptic pregnancies in the third trimester of gestation, a significantly higher level of elastase and a significantly higher elastase to neutrophil ratio was also described, suggesting an increased neutrophil activation in these patients (Belo, 2002; Belo, 2003).

Psoriasis was also associated with plasma neutrophil activation, showing increased plasma levels of elastase and lactoferrin, associated with alterations in band 3 profile (Rocha-Pereira, 2004).
