Characterization of buffy coat-derived granulocytes for clinical use: a comparison with granulocyte colony-stimulating factor/dexamethasone-pretreated donor-derived products.

BACKGROUND AND OBJECTIVES: Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte colony-stimulating factor (G-CSF). The latter is - dependent on the local and national settings - obtained following a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. MATERIALS AND METHODS: We pooled ten buffy coats following the production of platelet concentrates. Saline 0·9% was added to decrease the viscosity and the product was split into plasma, red cells and a 'super' buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. RESULTS: Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in vitro killing of Staphylococcus aureus, Escherichia coli and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal; especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily available in the regular blood production process and would take away the concerns around the apheresis product. CONCLUSION: The product described appears a promising alternative for transfusion purposes.

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils.

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS: Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis.

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.