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Keywords:

  • flow cytometry;
  • PNH;
  • GPI-linked proteins;
  • PrPc expression

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The cellular isoform of the prion protein (PrPC) is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidyl-inositol (GPI) anchor. PrPC is involved in the pathogenesis of prion diseases and has recently been shown to play a role in haemopoietic cell activation and proliferation. We have used the PrPC-specific monoclonal antibody (mAb) 3F4 in a flow cytometry approach to analyse the constitutive expression of PrPC on human peripheral blood (PB) cell populations from patients with paroxysmal nocturnal haemoglobinuria (PNH), which are characterized by a deficiency of GPI-linked cell surface proteins. Comparable PrPC expression levels (P > 0·05), quantified as mean fluorescent intensity, were measured on lymphocytes isolated from normal donors (n = 10) and patients with PNH (n = 5), whereas PNH PB monocytes and granulocytes exhibited substantially lower PrPC surface immunoreactivity than their normal counterparts (P < 0·05). More detailed histogram analyses of the PNH PB leucocytes revealed that PrPC was absent in PNH granulocytes, but was normally expressed in lymphocytes from four out of five patients. However, in one patient a bimodal distribution of 3F4 mAb staining was observed, indicating the presence of a PrPC-deficient lymphocyte subpopulation. In conclusion, our results show that PNH haemopoietic cells are deficient in cell surface-bound PrPC.

The prion protein (PrPC) is well known for its essential role in the pathogenesis of a group of transmissible brain diseases, including Creutzfeldt–Jakob disease in humans as well as scrapie and bovine spongiform encephalopathy (BSE) in animals (Prusiner et al, 1998). These diseases have been denoted transmissible spongiform encephalopathies (TSE) and are often referred to as prion diseases. While the role played by the pathological isoform of the prion protein (PrPSc) in the pathogenesis of prion diseases has been analysed in great detail, the normal cellular function of PrPC still remains enigmatic. PrPC is a cell surface glycoprotein that is attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (Stahl et al, 1987; Caughey & Raymond, 1991). PrPC was found to be encoded in the genome of all mammals investigated to date and shows a high degree of evolutionary conservation (Schatzl et al, 1995; Krakauer et al, 1998), pointing towards an essential functional role. As far as the expression of PrPC on peripheral blood cells is concerned, it has been demonstrated previously that it is mainly expressed by monocytes/macrophages and lymphocytes, whereas it is virtually undetectable on erythrocytes and granulocytes (Cashman et al, 1990; Dodelet & Cashman, 1998). Recent studies by our own group (Dürig et al, 2000) and colleagues (Holada et al, 1998; Barclay et al, 1999) have shown that PrPC expression on human haemopoietic cells correlates with the activation and developmental status of these cells, suggesting an important functional role for PrPC in the haemopoietic system.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder occurring in totipotent haemopoietic progenitor cells and characterized by intravascular haemolysis, episodes of haemoglobinuria and venous thromboses (Hillmen et al, 1995; Hillmen & Richards, 2000). The underlying genetic defect is a somatic mutation of the PIG-A (phosphatidylinositol-glycan complementation class A) gene located on the X chromosome (Xp22.1; Bessler et al, 1994; Rotoli & Boccuni, 1995), resulting in the synthesis of dysfunctional GPI molecules. Thus, PNH cells are characterized by a partial or complete deficiency of GPI-linked cell surface proteins, more than 20 of which have been described over the past two decades (Hillmen & Richards, 2000). Based on the clinical features and the size of the PNH clones, as determined using multiparameter flow cytometry, the disease can be classified into two different types: (i) haemolytic PNH, characterized by episodes of intravascular haemolysis and typically large PNH clones, and (ii) hypoplastic PNH, with clinical characteristics of cytopenia (neutropenia, thrombocytopenia and/or anaemia) and, usually, small PNH clones (Hillmen & Richards, 2000).

As PrP-deficient human haemopoietic cells may serve as a model system to study the physiological function of PrP in vitro, we used a PrP-specific monoclonal antibody in a flow cytometry approach to investigate whether or not PNH cells exhibit a decreased PrP-surface immunoreactivity.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cells Whole heparinized and EDTA peripheral blood (PB) samples were obtained by venepuncture from 10 healthy volunteer donors (five women, five men, median age: 30 years, range: 27–54 years) and five patients with PNH (Table I). In all patients, the diagnosis was established by a positive Ham's test and/or flow cytometric analysis of CD59 and CD55 expression on erythrocytes and granulocytes. Patients with a previous history of aplastic anaemia (AA) who later developed a positive Ham's test and/or had evidence of defective expression of GPI-linked proteins by flow cytometry were considered to have an AA–PNH syndrome. All samples were obtained with informed consent according to institutional guidelines.

Table I.  Patient characteristics.
Patient number Sex Age (years) Time from diagnosis (years)CD59-negative cells (%)CD55-negative cells (%) PNH type/clinical features
ErythrocytesGranulocytesErythrocytesGranulocytes 
  1. PNH, patients presenting with haemolytic anaemia, a positive Ham's test and/or flow cytometric evidence of defective expression of GPI-linked proteins; AA–PNH, patients with a previous history of aplastic anaemia (AA) and flow cytometric evidence of defective expression.

1Male60545·689·710·554·2PNH
2Male301550·740·68·45·6AA–PNH
3Male621734·794·25·766·1PNH
4Male35313·521·32·84·4AA–PNH
5Female48530·775·19·050·5AA–PNH

Monoclonal antibodies (mAbs) and single-colour staining of unseparated whole blood Heparinized PB samples were prepared for flow cytometry by ammonium chloride erythrocyte lysis (Ortho-mune Lysing Reagent, Ortho Diagnostic Systems, Raritan, NJ, USA). For analysis of prion protein expression, the mAb 3F4 was used at a 1:2000 dilution as previously described (Dürig et al, 2000). mAb 3F4 (Kascsak et al, 1987) was purified from cell culture media of clone 3F4 (approx. 1 mg/ml) and kindly provided by Prof. H. Diringer (Robert-Koch-Institut, Berlin, Germany). Binding of the primary antibody was detected using a rabbit anti-mouse Fab fluorescein isothiocyanate (FITC) conjugate (DAKO, Glostrup, Denmark). Isotype-matched irrelevant control mAbs (DAKO) were included in all experiments.

Flow cytometry All samples were processed within 4 h of venepuncture. Analysis was performed on a FACScan flow cytometer (Becton-Dickinson, Heidelberg, Germany) and data acquisition was performed using CellQuest software (Becton-Dickinson). PrPC expression was analysed in uniformly set gates comprising either lymphocytes, monocytes or granulocytes, defined by their side- and forward-scatter characteristics. The relative PrPC density on these cells was quantified by determining the mean fluorescent intensity (MFI) for FITC in identical gates and subtracting MFI values of isotype-matched irrelevant controls. The percentage of CD59-negative erythrocytes and granulocytes was determined in EDTA blood samples using the commercially available CellQuant and RedQuant CD55/CD59 kits (Beckman Coulter, Krefeld, Germany), according to the manufacturer's instructions.

Statistical analysis Data are expressed as mean ± SEM. Statistical significance was determined employing Student's t-test for unpaired samples.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Prion protein expression in normal vs. PNH peripheral blood cells

Human PB leucocytes from normal donors and PNH patients were analysed for PrPC expression by flow cytometry using the 3F4 mAb (Fig 1). On the basis of forward- (FSC) and side-scatter (SSC) criteria (representing measures for size and granularity respectively), three major leucocyte populations could be identified, and 3F4 staining was analysed separately in each of these populations as previously described (Dürig et al, 2000). MFI values for PrPC expression in normal compared with PNH lymphocytes, monocytes and granulocytes are given in Fig 2. Comparable PrPC expression levels were measured on lymphocytes (P > 0·05) from normal donors and patients with PNH, whereas PNH PB monocytes and granulocytes exhibited substantially lower PrPC surface immunoreactivity than their normal counterparts (P < 0·05). More detailed histogram analyses (Fig 1) of the PNH PB leucocytes revealed that PrPC was absent in PNH granulocytes, whereas it was normally expressed in lymphocytes from four out of five patients. However, in one patient (patient 2, Fig 1) a bimodal distribution of 3F4 mAb staining was observed, indicating the presence of a PrPC-deficient lymphocyte subpopulation. Furthermore, in three patients, a complete absence of PrPc was observed in the monocytic cell population, whereas in the remaining two individuals (Fig 1, patients 4 and 5), the presence of PrPC-positive cells indicated the persistence of normal monocytes.

image

Figure 1. Expression of prion protein by human peripheral blood leucocytes. Forward- and side-scatter criteria were used to define three major subpopulations: lymphocytes, monocytes and granulocytes (Dürig et al, 2000). PrPC expression was quantified by gating on each of these populations and determining the mean fluorescent intensity (ΔMFI) using histogram plots (solid lines) and subtracting MFI values obtained from isotype-matched irrelevant controls (represented by the dashed lines).

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image

Figure 2. Comparison of MFI values obtained from analyses of 10 normal donors and five PNH patients. Results of the normal control group have previously been published (Dürig et al, 2000). Data are means ± SEM. Differences in PrPC expression levels between the leucocyte subpopulations were statistically analysed using Student's t-test for unpaired samples. The asterisk denotes statistical difference between normal controls and PNH patients (P < 0·05).

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In three patients, a two-colour flow cytometric approach was employed to analyse PrP cell surface immunoreactivity in relation to the expression of CD59 [membrane inhibitor of reactive lysis (MIRL)] on the same leucocyte subpopulation (Fig 3 and Table II). CD59 is a GPI-anchored molecule expressed on human haemopoietic cells of all lineages that is widely used in flow cytometric diagnostic assays for PNH (Hillmen & Richards, 2000). Two-parameter dot plot analysis (Fig 3) allowed for the identification of four different subpopulations in lymphocytes, monocytes and granulocytes respectively (Table II). Notably, the proportion of PrPC-deficient monocytes was significantly higher than that of CD59-negative monocytes (Table II, P = 0·02), suggesting that a substantial number of PrPC-deficient monocytes express residual amounts of CD59.

image

Figure 3. Comparison of prion protein and CD59 expression on human peripheral blood leucocyte subsets using two-colour flow cytometry. Whole blood samples were stained for PrPC (FITC) followed by anti-CD59 (PE) and analysed as detailed in Patients and methods. Numbers are percentages of all gated cells. One representative experiment (patient 2) of three performed (Table II) is shown. Because of rounding, not all percentages add to 100.

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Table II.  Two-colour flow cytometric analysis of PrPc and CD59 expression in PNH PB leucocytes.
Patient numberLymphocytesMonocytesGranulocytes
PrPc+/ CD59+PrPc+/ CD59–PrPc–/ CD59+PrPc–/ CD59–PrPc+/ CD59+PrPc+/ CD59–PrPc–/ CD59+PrPc–/ CD59–PrPc+/ CD59+PrPc+/ CD59–PrPc–/ CD59+PrPc–/ CD59–
  1. Two parameter dot plot analysis as shown in Fig 3 allowed for the identification of four different populations in the lymphocyte, monocyte and granulocyte gate respectively. Because of rounding, not all percentages add to 100.

167·726·52·80·32·60·939·157·50·60·139·060·3
275·22·37·614·85·20·333·461·20·70·331·667·5
369·524·24·12·39·92·653·534·10·80·169·030·1
Mean ± SEM70·8 ± 2·317·7 ± 7·74·8 ± 1·45·8 ± 4·55·9 ± 2·11·3 ± 0·742·0 ± 6·050·9 ± 8·50·7 ± 0·10·2 ± 0·146·5 ± 11·452·6 ± 11·5

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We have previously shown that the normal cellular isoform of the prion protein, PrPC, is differentially expressed on peripheral blood leucocytes of healthy human donors depending on the activation and developmental status of these cells (Dürig et al, 2000). In the present study, we demonstrate that PB leucocytes from patients with PNH exhibit reduced PrPC surface expression levels compared with normal controls. Leucocyte subset analysis revealed significantly reduced PrPC expression in the monocyte and granulocyte populations of all patients analysed (n = 5), whereas only one patient showed a PrPC-deficient lymphocyte subpopulation. This latter observation was consistent with previous reports demonstrating highly variable but mostly small GPI-deficient lymphocyte clones in the majority of PNH patients (Richards et al, 1999). Thus, it appears possible that a more detailed lymphocyte subset analysis in our patients may reveal further abnormalities in terms of PrPC expression currently not being detected by gating on all lymphocytes. The larger proportion of PNH cells observed in the granulomonocytic and erythroid lineages compared with the lymhoid/natural killer cell compartment is thought to reflect differences in the proliferation and development of these cells in the context of the bone marrow and thymic microenvironments (Richards et al, 1999). Whether PB granulocytes express PrPC has not been unambiguously demonstrated to date. The degree of expression that we (Dürig et al, 2000) and colleagues (Dodelet & Cashman, 1998; Barclay et al, 1999) found using flow cytometry was so low that its interpretation remained conjectural. Our current experiments demonstrating significantly reduced PrPC staining in PNH granulocytes compared with their normal counterparts now unequivocally show that normal PB granulocytes constitutively express low levels of PrPC.

It is well established that the somatic mutations of the PIG-A gene are varied and have differential effects on the GPI biosynthetic pathway, some of which result in the complete absence of GPI-anchored molecules while others may allow a small quantity of GPI-anchor to be produced (Hillmen & Richards, 2000). This may also explain the commonly observed variability in the expression of different GPI-linked antigens on a particular PNH cell (Piedras & Lopez-Karpovitch, 2000). For instance, in the present study, we found that CD55 antigen expression did not delineate abnormal erythrocytes and granulocytes as well as anti-CD59 did (P≤ 0·05; Table I), confirming a recent study by Piedras & Lopez-Karpovitch (2000). To further analyse PrPC expression in PNH leucocyte populations compared with that of other GPI-linked cell surface molecules, we performed two-colour flow cytometry using the PrP specific antibody 3F4 in conjunction with a mAb directed against CD59. Not surprisingly, the two antibodies identified overlapping but not completely identical PNH cell populations (Table II).

In conclusion, our data provide indirect evidence that PrPC is indeed linked via a GPI-anchor to the plasma membrane of blood cells, confirming previous studies showing that PrPC surface expression on platelets can be reduced by treatment of cells with phosphatidylinositol-specific phospholipase C (Barclay et al, 1999).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We gratefully acknowledge the excellent technical assistance of Ms. K. Halfmeyer and Ms. M. Skubatz. We thank Prof. H. Diringer (Robert-Koch-Institut, Berlin, Germany) for providing mAb 3F4.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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