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      Hemodialysis-Induced Degranulation of Polymorphonuclear Cells: No Correlation between Membrane Markers and Degranulation Products


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          Background/Aims: Degranulation of polymorphonuclear leukocytes (PMN) during hemodialysis (HD) is usually assessed by measuring degranulation products. However, this process might also be estimated by the assessment of cell surface markers. In this study, the relationship between the expression of PMN degranulation markers (CD63 and CD66b) and the release of degranulation products [myeloperoxidase (MPO) and lactoferrin (LF)] was investigated during clinical HD in order to evaluate cell surface markers as a useful index of PMN degranulation. Methods: The expression of CD63 and CD66b on PMN and the release of MPO and LF were investigated in 10 chronic HD patients, during both heparin (HD<sub>hep</sub>) and trisodium citrate anticoagulation (HD<sub>cit</sub>), in a randomized order. Samples were drawn from both the efferent and afferent lines of the dialyzer at 0, 7.5, and 180 min. Results: During HD<sub>hep</sub> at first passage, a major increase in MPO (from 158 ± 32 to 448 ± 177 µg/l, p = 0.001) and LF (from 134 ± 52 to 260 ± 120 µg/l, p = 0.01) was found across the dialyzer, whereas marked changes were not observed during HD<sub>cit</sub>. The expression of CD63 and CD66b increased across the dialyzer during both anticoagulation modalities, but was only significant in the case of HD<sub>hep</sub> (CD63: mean fluorescence intensity from 247 ± 61 to 331 ± 118, p < 0.01; CD66b: mean fluorescence intensity from 340 ± 76 to 434 ± 103, p = 0.01). During HD<sub>hep</sub> a correlation was noted between the degranulation products and markers of both azurophilic and specific granules (MPO and CD63: r = 0.35; p < 0.01; LF and CD66b: r = 0.39, p < 0.01). Significant differences in the expression of CD63 and CD66b between HD<sub>hep</sub> and HD<sub>cit</sub> were not observed. When analyzing the combined data for both HD<sub>hep</sub> and HD<sub>cit</sub>, no correlation was observed between degranulation products and markers. Conclusion: Our data suggest that the measurements of cell surface markers may not be a reliable indicator of the degree of HD-induced PMN degranulation.

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          Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils.

          We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compares favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells) to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca2+ concentration of 1 microM, a value of the cytosolic free Ca2+ concentration occurring in formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-gamma-S besides 1 microM Ca2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.

            Author and article information

            S. Karger AG
            July 2000
            21 June 2000
            : 85
            : 3
            : 267-274
            aMedical Center Alkmaar, bUniversity Hospital VU Amsterdam, cDiakonessen Hospital Utrecht, and dSt. Maartens Hospital Nijmegen, The Netherlands
            45671 Nephron 2000;85:267–274
            © 2000 S. Karger AG, Basel

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            Page count
            Figures: 3, Tables: 1, References: 29, Pages: 8
            Self URI (application/pdf): https://www.karger.com/Article/Pdf/45671
            Self URI (text/html): https://www.karger.com/Article/FullText/45671
            Self URI (journal page): https://www.karger.com/SubjectArea/Nephrology
            Technical Note

            Cardiovascular Medicine,Nephrology
            CD63,CD66b,Hemodialysis,Heparin,Citrate,Polymorphonuclear leukocytes, degranulation


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