Modification of the content of plasma protein carbonyl groups in donors after granulocyte colony stimulating factor-induced stem cell mobilization

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Abstract

Granulocyte-colony stimulating factor (G-CSF), the most established agent for the mobilization of stem cells in current clinical practice, could induce a condition of oxidative stress. Herein plasmatic levels of protein carbonyl groups (a biomarker of oxidative stress) were measured in a group of donors given recombinant human G-CSF (rHuG-CSF) at different times: (1) before starting rHuG-CSF administration, (2) on day 5 of rHuG-CSF administration, (3) on the same day immediately after the end of the first leukapheresis procedure and (4) one week after rHuG-CSF withdrawal. Plasma levels of protein carbonyl groups enhanced significantly in donors after 5 days of rHuG-CSF treatment and appeared further slightly increased following leukapheresis procedure; 7 days following rHuG-CSF withdrawal, they showed a tendency to normalization. These findings may contribute to better understand the oxidative reactions following rHuG-CSF treatment and leukapheresis.

Introduction

Mobilized cells following treatment with cytokines are currently the preferable and major source of stem and progenitor cells harvested for autologous and allogeneic transplantations because of the higher yield of these cells, leading to faster engrafment and decreased procedural risks compared with harvested bone marrow cells [1]. The mobilization process mimics the physiological release of stem cells and their progenitors from the bone marrow in response to stress signals occurring naturally during inflammation and injury. The mobilization of stem cells in the bone marrow is determined by the local microenvironment, consisting of fibroblasts, osteoblasts and endothelial cells, and is mediated by proteinases such as elastase, cathepsin G and matrix metalloproteinases [2]. In particular, granulocyte-colony stimulating factor (G-CSF), that today is the standard mobilizing agent, releases the proteinases elastase and cathepsin G from neutrophils [3]. G-CSF is the most established agent for the mobilization of stem cells in current clinical practice because it has been proven to be superior to any other agent tested to date not only in terms of mobilization capacity [4], [5], [6], but also of tolerance. In fact, the acute G-CSF-associated side effects (bone and muscle pain, a slight spleen enlargement and hypercoagulable state) are generally mild and severe allergic reactions are rare. However, the long-term side-effects of G-CSF need to be further studied [7], [8].

When stimulated, neutrophils undergo an oxidative burst resulting in the generation of large amounts of superoxide, which is further metabolized to H2O2; they also release the heme enzyme myeloperoxidase (MPO), which utilizes H2O2 to convert chloride to hypoclorous acid (HOCl) [9]. Sustained levels of this oxidant are associated with side effects of inflammation, such as cell injury and tissue damage [10]. HOCl is known to oxidize a wide variety of substrates including proteins, DNA, lipids and cholesterol; however, proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl [11].

Taking into account these data, may be interesting to investigate if the administration of G-CSF followed by the leukapheresis procedure could induce a condition of oxidative stress in stem cell donors. The aim of this study was to assess the in vivo time course of plasmatic levels of protein carbonyl groups in a group of stem cell donors given recombinant human G-CSF (rHuG-CSF). In fact, the action of reactive oxygen species (ROS) on proteins has been widely demonstrated to result in the formation of carbonyl groups [12], [13]. Carbonyl stress may be also due to toxic effects of various mono-dicarbonyls (such as malonyldialdehyde and 4-hydroxy-2,3-nonenal) and α-dicarbonyls. The level of these modified molecules can be quantified by measurement of the protein carbonyl content, which have been shown to increase in a variety of diseases and processes [13]. Since the circulating proteins destroyed by oxidation have a quite long half-life period, the evaluation of carbonyl group content in blood proteins is considered a useful biomarker of oxidation induced by ROS, including HOCl [14].

In our study, plasmatic levels of protein carbonyl groups were measured at different intervals: (1) before starting rHuG-CSF administration, (2) on day 5 of rHuG-CSF administration, (3) on the same day soon after the end of the first leukapheresis procedure (postapheresis) and (4) one week after rHuG-CSF withdrawal.

Section snippets

Donors

Twenty-consecutive normal donors (sex, 14 male and 6 female; median age, 46 years; age range, 16–56 years) were seen at our institution 2–3 weeks before starting mobilization. The workup included a detailed history, clinical examination, abdomen ultrasound and ECG. Laboratory evaluation included blood counts and differentials, clinical chemistry, infectious diseases markers, and β-HCG in female donors to exclude pregnancy. rHuG-CSF (Neupogen, Dompé Biotec, Milan, Italy) was administered

Results

When compared with baseline values, plasma levels of protein carbonyl groups enhanced significantly in donors after 5 days of rHuG-CSF treatment and appeared further increased (but not in a statistically significant way) when measured in the same day immediately following the leukapheresis procedure (Fig. 1). Furthermore, as measured in 9 subjects, plasma protein carbonyl groups showed a tendency to return to baseline values, although the reduction was not statistically significant, 7 days

Discussion

The results of our study show a significant increase in the levels of plasma protein carbonyl groups, a biomarker of oxidative stress, following rHuG-CSF administration. The formation of protein carbonyl groups is a common phenomenon during oxidation, so that their quantification can be used to measure the extent of oxidative modification. In many pathological conditions, the elevation in protein carbonyl groups correlates well with the progression and severity of the disease, although it is

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