Abstract

Bacterial infections are a serious complication in the beta-thalassemia syndrome, but the mechanisms underlying the increased susceptibility to these infections are not fully understood. Factors, likely to be involved are anemia, splenectomy, iron-overload, alterations in immune responses and defective phagocytic cells. Polymorphonuclear neutrophils (PMN) destroy microbial organisms by a burst of reactive oxygen species (ROS) in response to bacterial components as well as to phorbol myristate acetate (PMA). The respiratory burst is mediated by a multi-component enzyme - NADPH oxidase. We previously showed that in thalassemia, RBC and platelets are under oxidative stress, which may affect their functionality and integrity, leading to premature hemolysis and platelet activation.

In the present study, we utilized flow cytometric techniques to compare ROS generation in normal (n=18) and in beta-thalassemic (n=10) PMN and to assess respiratory burst in response to stimulation by PMA. Peripheral blood samples were mixed with 3% gelatin, and following 30 min incubation the upper layer, containing PMN, platelets and some RBC, was collected. ROS generation was measured by FACS analysis following staining of the cells with 2′-7′-dichlorofluorescein (DCF). PMN were “gated” according to granularity and size. The identity of the cells in the PMN gate was verified by immuno-staining for CD11b and CD15, specific PMN antigens. The intensity of the resultant ROS-mediated cell fluorescence was linearly related to the height (intensity) of the respiratory burst.

The average mean fluorescence channel (MFC) of DCF-stained PMN of thalassemia patients and of normal donors at basal level was 39 and 10, respectively. Upon activation with 100 ng/ml PMA for 15 min. at 37°C, while the average MFC of the normal PMN increased by 36-fold, the thalassemic PMN increased by only two and a half fold.

These results indicate high basal ROS generation by thalassemic PMN, but a reduced response to PMA. To determine whether this abnormal behavior of thalassemic PMN was due to oxidative stress, normal PMN were treated with various oxidants followed by DCF staining. Pre-treatment with hydrogen peroxide (1 mM), butyl-hydroxyperoxide (0.6 mM), hemin (0.1 mM) or ferric ammonium citrate (0.1 mM) increased the basal level of ROS generation (5 to 22-fold, but decreased the PMA response by 20–30%. In contrast, overnight treatment of thalassemic PMN with antioxidants such as N-acetyl-L-cysteine and vitamin C (both at 0.05 mM) reduced the basal ROS level and enhanced the PMN response to PMA.

Our findings suggest that the oxidative status of PMN plays an important role in the defense mechanism against bacterial infections. PMN that are under chronic oxidative stress, such as in thalassemia (e.g., as a result of iron-overload) have a reduced capacity to elicit a respiratory burst. This may explain the increased susceptibility of thalassemic patients to bacterial infections. Consequently, these findings raise the possibility of using antioxidants in thalassemia and other conditions associated with recurrent bacterial infections.

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