Alineamientos de Banda del Material de Banda Intermedia

In an attempt to further understand the mechanism of altered neutrophil function in sickle cell disease (SCD) the data presented in this chapter test whether the various com plications associated with SCD could account for the prim ing defect o f neutrophils.

There is evidence for low neutrophil counts among blacks in comparison to whites (Freedman et a l, 1997) and because SCD patients in this study were of Afro- Caribbean background, I decided to investigate w hether defective prim ing of neutrophils could be explained in terms of racial differences. A group of healthy Afro- Caribbean individuals were tested and the data obtained excluded the racial differences as a mechanism as no defects in neutrophil H2O2 production were seen in healthy Afro-Caribbean subjects.

Another feature common to patients with SCD is hyposplenism. As this organ is inactive in SCD patients, the neutrophil H2O2 production was tested in a group of non-SCD patients who received splenectomies previously. The data showed that there was no defects in neutrophils H2O2 production in asplenic patients.

The effects observed in SCD were also not reproduced in a group o f patients with active rheumatoid arthritis. This study appears to be the first to test neutrophil function in whole blood from patients with arthritis, whereas previous studies using purified cells or diluted whole blood have produced contradictory results, some showing evidence of activated (Laurindo et a i, 1995; Eggleton et a i , 1995; Miesel et a i , 1996) or defective (Al-Balla et a i, 1990; Mur et a i , 1997) neutrophils in the circulation with reduced responses after cytokine-mediated priming (Eggleton et a i ,

1995; Al-Balla et a i , 1990). However, Kowanko (1996), found no differences between patients with RA and healthy individuals with respect to their T N F a priming of neutophils respiratory burst. It has been shown that high levels of platelet activating factor (PAF) may also be a contributing factor to the inflammatory state of SCD (Oh et al., 1997). These findings with rheumatoid arthritis patients em phasize that the defective priming seen in SCD is likely to be specifically associated with events in the circulation rather than being a general feature of inflammatory disorders.

It was further investigated whether the priming defect could be attributed to the marked anaemia of the patients with SCD, who had haemoglobin levels of 80-90 g/L. However, this study showed no evidence for reduced H2O2 production in a series of patients with iron-deficiency anaemia whose mean haemoglobin was 76 ± 5 g/L (n=6). Finally, one other factor associated with SCD is the premature destruction of sickled red blood cells thus giving rise to increased plasma haemoglobin levels. There is no previous evidence to suggest a correlation between the haemolytic events of SCD

and defective priming o f neutrophils, therefore prim ing o f H2O2 production in neutrophils was measured in patients with P-thalassaemia intermedia, a group of diseases resulting from decreased beta-globin mRNA expression and characterised by ineffective erythropoiesis and excessive haemolysis. The data from patients with p- thalassaemia showed reduced neutrophil H2O2 production when cells were primed by either GM -CSF or T N F a to 62% and 59% respectively. This suggets that the intravascular haemolysis may be a common feature in each condition accounting for defective priming of their neutrophils with cytokines. However, these data were not statistically significant and this needs to be verified by a larger study population. One other feature of P-thalassaemia is an elevated level of bilirubin which has also been shown in the plasma of patients with SCD which reduces the antioxidant capacity of plasma (Dailly et al., 1998) which may possibily increase membrane damage, and hence increase adherence. If haemolysis is one of the contributing factors in defective priming of neutrophils in SCD, the replacement of sickle cell plasma with plasma from healthy individuals should ameliorate this defect. This hypothesis was tested in patients with SCD either in steady state or during crisis. Those in steady state of disease showed 20% increase in H2O2 production when neutrophils were primed with TNF (500 U/ml) and 2) those during crisis showed the same pattern. As this abnormality was only partially reversed by the addition of normal plasma, it suggests that the priming defect was of cellular origin which was partially corrected by normal plasma.

The effect of plasma was further studied when normal cells were suspended in plasma from SCD patients in steady state which showed reduction of H2O2 production to 38% of the control and with diluted plasma from patients in crises the reduction was 15% of the same day control. These data support the possibility of soluble inhibitory factor(s) in plasma from SCD patients that partially reduced H2O2 production in neutrophils. The effect of plasma factors on priming of neutrophils in SCD patients has not been investigated previously, and the nature of plasma inhibitors were not identified.

However, plasma factors such as C-reactive protein (CRP) which is mainly synthsised in the liver has been reported to be moderately increased in SCD patients during asymptomatic steady state and significantly increased during painful crisis (Akinola el al., 1992; Stuart et a l , 1994), production of these proteins could be triggered by inflammatory cytokines such as, IL-6, IL-1, TGF(3-1 and T N F a (Croizat

et al., 1994). There is also evidence to show an increased thrombotic risk in these patients which could be due to increased thrombin and fibrin formation (Leslie et al.,

1975; Francis, 1989) with reduced levels of heparin cofactor II (Porter et al., 1993),

antithrombin III, protein C and protein S (Caccioloa et al., 1989; Karayalcin et a i ,

1989; Francis, 1988).

H2O2 production in unprimed neutrophils stimulated by FMLP or PMA showed no significant differencefrom contols indicating that the defect observed in priming was not due to defect of NADPH oxidase itself. Taken together these findings imply both cellular and plasma defects associated with SCD.

CHAPTER 5

MITOGEN-ACTIVATED PROTEIN KINASE