Dendritic cells in patients with type I Gaucher disease are decreased in number but functionally normal

Blood Cells, Molecules, and Diseases 36 (2006) 298 – 307 www.elsevier.com/locate/ybcmd

Dendritic cells in patients with type I Gaucher disease are decreased in number but functionally normal I. Micheva a , T. Marinakis c , C. Repa d , A. Kouraklis-Symeonidis a , V. Vlacha b , N. Anagnostopoulos c , N. Zoumbos a , A. Symeonidis a,⁎ a

Hematology Division, Department of Internal Medicine, University of Patras Medical School, Rion, 261.10, Patras, Greece Pediatric Hematology-Oncology Division, Department of Pediatrics, University of Patras Medical School, Patras, Greece c Department of Hematology, “G. Gennimatas” General Hospital of Athens, Athens, Greece d Department of Hematology, 3rd Hospital of IKA, Athens, Greece

b

Submitted 31 October 2005; revised 15 December 2005 (Communicated by E. Beutler, M.D., 15 December 2005)

Abstract Gaucher disease is a lysosomal storage disorder, in which undigested glucosylceramide is deposited in the cytoplasm of mature macrophages, which accumulate in the bone marrow and the reticuloendothelial system. Dendritic cells are bone marrow-derived cells, specialized for the uptake, processing, transport and presentation of antigens to T-lymphocytes. We investigated peripheral blood dendritic cell-precursors, as well as the potential of peripheral blood monocytes and bone marrow-derived progenitor cells, to differentiate into mature dendritic cells in 12 patients with type I Gaucher disease. Results of the 10 adult patients were compared with those of 10 healthy volunteers, matched for age and sex. Six patients were anemic and 9 were thrombocytopenic, but none had severe bone disease. Both myeloid and plasmacytoid dendritic cells of patients with Gaucher disease, as well as the yield of the monocyte-derived dendritic cells, obtained after GM-CSF and IL-4 stimulation, were found significantly decreased, when compared to controls (myeloid dendritic cells: 0.19 ± 0.07% vs. 0.34 ± 0.10%, P = 0.009, plasmacytoid dendritic cells: 0.17 ± 0.12% vs. 0.39 ± 0.13%, P = 0.004, monocyte-derived dendritic cells: 4.8 ± 3.5% vs. 8.3 ± 3.2%, P = 0.036). However, the immunophenotypic profile of dendritic cells, estimated by CD1a, CD40, CD54, CD80, CD83 and HLA-DR expression, the endocytic and allostimulatory capacity of the immature, as well as of the TNF-α- or lipopolysaccharite-stimulated mature monocyte-derived dendritic cells, was similar to those obtained by healthy controls. In addition, bone marrow-derived CD34+ cells differentiated in the presence of GM-CSF, SCF, TNFα and IL-4 into mature dendritic cells that did not differ in number, phenotype and allo-stimulatory activity from those of controls. Our findings suggest that patients with Gaucher disease exhibit mainly quantitative defects of their dendritic cells' system, demonstrated by decreased circulating dendritic cell precursors of both myeloid and plasmacytoid type. This finding may contribute to the poor immune response against infectious agents and an impaired immune surveillance, associated with an increased risk of developing a neoplastic disease. © 2006 Elsevier Inc. All rights reserved. Keywords: Gaucher disease; Dendritic cells; Immune regulation; Monocyte differentiation

Introduction Gaucher disease, the prototype inherited lysosomal storage disorder, is the result of glucocerebrosidase deficiency, a human lysosomal enzyme, which catabolizes the lipid substrate glucosylceramide [1]. Impaired-inadequate catabolism of glucosylceramide leads to the accumulation of this substrate within

⁎ Corresponding author. Fax: +30 2620 993950. E-mail address: [email protected] (A. Symeonidis). 1079-9796/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.12.029

the macrophages, which in turn accumulate and infiltrate the organs of the reticuloendothelial system and the bone marrow. Three major clinical types of Gaucher disease have been described types I, II and III. Hepatosplenomegaly, variable degree of peripheral blood cytopenias, mainly thrombocytopenia and bone disease, but absence of neurological defects, is the most common clinical manifestations of type I Gaucher disease [2]. In addition to the classical manifestations of this disease, many patients exhibit increased susceptibility to systemic bacterial and non-bacterial infections, which occasionally, but not always, could be attributed to the decreased neutrophil

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count, or to impaired microbicidal capacity of the mononuclear phagocytes [3]. Moreover, the course of Gaucher disease has been associated with the occurrence of various lymphoid malignancies, such as multiple myeloma, chronic lymphocytic leukemia and childhood acute lymphoblastic leukemia as well as solid tumors [4–8]. For the explanation of these clinical manifestations, immunological abnormalities, such as decreased numbers of natural killer cells, CD4+ and CD8+ Tlymphocytes [9,10], as well as abnormalities of the antigen presenting cells have been postulated as possible implicating mechanisms. Dendritic cells are the most potent antigen presenting cells, specialized for the uptake, processing, transport and presentation of antigens to T-lymphocytes [11]. Dendritic cells originate from the bone marrow pluripotent hematopoietic stem cell, via different developmental pathways [12]. Variations among the tissue distribution, as well as differences in their phenotype and function, indicate the existence of a heterogeneous dendritic cell population, which in the peripheral blood is represented by the myeloid and the plasmacytoid dendritic cells [13]. Currently, no data exist about the number and the functional capacity of the dendritic cell subsets in patients with Gaucher disease. Some years ago, Guy et al. working in a transgenic mouse model have found that murine dendritic cells, as well as all the other cells belonging to the mononuclear phagocyte system, did expressed a human β-glucosylcerebrosidase transgene, whose expression was controlled by the MHC class II locus control region [14]. In view of the possibility that dendritic cells are mainly monocytederived phagocytyzing cells and their function might be influenced and compromised by the undigested lipid substrate of Gaucher disease, we aimed to study the quantitative and the qualitative properties of these cells in a rather homogeneous group of mainly untreated patients with type I Gaucher disease. We investigated the status of the blood dendritic cell precursor populations detecting the percentage of the circulating myeloid and plasmacytoid dendritic cells in the peripheral blood. We also studied the potential of peripheral blood monocytes, as dendritic cell precursors, and of bone marrow CD34 + progenitors to differentiate into functionally active dendritic cells in vitro.

299

spleen enlargement. No patient exhibited neurologic manifestations or any other organ involvement, besides mild to moderate bone abnormalities. The bone disease was identified in 3 adults and one pediatric patient and consisted of mild pain, not necessitating major pharmaceutical intervention, and moderate radiological or MRI findings. Two patients had common hemorrhagic manifestations and one had experienced recurrent infectious episodes, mainly localized on the upper respiratory tract. To evaluate the disease severity in patients with Gaucher disease, we have used the Zimran Severity Score Index [15]. According to this index, only one adult patient was classified to the intermediate severity category, whereas the remaining 9 adults and both pediatric patients were in the low-severity category. Nine patients had not received any kind of treatment until the time they were studied, while the remaining adult patient as well as one pediatric patient had been treated with imiglucerase the last 18 and 12 months, respectively. Ten healthy volunteers matched for age and sex (5 females and 5 males, median age 35.5 years, age range 24–55 years) served as controls. Eight of the controls were donating bone marrow for allogeneic hematopoietic stem-cell transplantation, and they were informed that a small quantity of their marrow would be used for research purposes and that if something would be found of clinical relevance for them, they would be informed accordingly. The remaining 2 controls were lab personnel and donated peripheral blood only. Therefore, the complete set of tests was performed in 8 controls, while two controls were tested upon blood tests only. Informed consent was obtained from all subjects participating in the study, patients and controls. The study protocol has been approved by the Ethical and Scientific Committee of the University Hospital of Patras. The demographic data of patients and controls are presented in Table 1, while the main clinical and laboratory features of the patients are shown in Table 2. Cell separation and culture Samples of heparinized whole blood and 3 ml of total bone marrow were received during diagnostic work up from the patients and during the harvest procedure from the donors, under sterile conditions. Peripheral blood mononuclear cells

Materials and methods Patient characteristics Twelve patients in total, six male and six female with a median age of 32 years and an age range between 8 and 66 years, diagnosed with type I Gaucher disease were included in the study. Two patients were in the pediatric range of age 8 and 12 years old, and their results are presented separately. The 10 adult patients were 5 male and 5 female with a median age of 34 years and an age range of 20–66 years. No patient had been splenectomized and no underlying co-morbidity was reported by any of them, except of mild hypertension and a past history of uncomplicated nephrolithiasis in one patient. Eight of the 10 adult patients had substantial splenomegaly (spleen tip palpable ≥10 cm below left costal margin), while both children had mild

Table 1 Demographic data of patients and controls

Total studied Adults Children Male/female M/F ratio Median age (adults only) Age range (adults only) Cigarette smokers Co-morbid conditions Hypertension Nephrolithiasis Mild iron deficiency

Patients

Controls

12 10 2 6/6 1 34 20–66 3/12

10 10 – 5/5 1 35.5 17–55 4/10

1/12 1/12 –

1/10 – 1/10

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Table 2 Clinical and laboratory features of the patients Clinical and laboratory parameters

Palpable spleen size (cm) Palpable liver size (cm) Hemoglobin (g/dl) White blood cells (x 109/l) Platelets (x 109/l) Serum β-glucosidase (nmol/mg Pr/h) Serum chitotriosidase (nmol/ml/h Serum acid phosphatase (IU/l) Serum SACE (IU/l) Abnormal liver biochemistry CNS or other organ involvement Evidence of bone disease Zimran severity score index

Adult patients, N = 10

Pediatric patients, N = 2

Median

Range

Median

Range

12 2 12.2 4.3 88 0.75 15,498 22.5 206 3/10 0/10 3/10 7

7–21 0–4 9.7–14.8 3.6–5.5 52–112 0.25–1.3 6372–30,743 15.2–28.8 137–288 – – – 1–12

3.5 2.5 12.2 5.9 189 1.65 4630 10 144 1/2 0/2 1/2 3

3–4 2–3 11.6–12.8 5.1–6.7 142–236 1.6–1.7 4335–4924 8.8–11.2 118–170 – – – 2–4

and bone marrow mononuclear cells were isolated by density gradient centrifugation over Ficoll–Hypaque (Biochrom AG, Berlin, Germany). Bone marrow CD34+ cells were positively selected using Magnetically Activated Cell Sorting (MACS) CD34 isolation kit and MS MACS column (Miltenyi Biotec, Gladbach, Germany). The purity of CD34+ cells was 75–95%. CD3+ T-cells were separated from peripheral blood mononuclear cells from healthy adults using CD3 antibody-coated immunomagnetic beads (Miltenyi Biotec) and frozen in fetal bovine serum (Biochrom), 10% dimethylsulfoxide (Sigma Chemical Co, St Louis, MO, USA) until further use. Peripheral blood mononuclear cells were cultured at a concentration of 106 cells/ml in a culture medium composed of RPMI-1640, 10% fetal bovine serum, 200 mmol/l L-glutamine, 50 μg/ml streptomycin and 50 U/ml penicillin (all from Biochrom) for 2 h at 37°C in a humidified atmosphere of 5% CO2 in 25 cm2 tissue culture flasks. The non-adherent cells were removed, the flasks were washed and the adherent cells were cultured in a culture medium, containing 100 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF) and 10 ng/ml interleukin-4 (IL-4) (Biosource, Nivelles, Belgium) for 5 days, to obtain immature dendritic cells. Monocyte-derived dendritic cells were further cultured for 2 additional days in a plain culture medium or in the presence of 10 ng/ml tumor necrosis factor alpha (TNF-α) (Biosource) or 0.1 μg/ml Lipopolysaccharide (LPS, Escherichia coli, serotype 055:B6, Sigma). The percentage cell yield was calculated as cell number obtained in culture over the total cell number initially put in the culture × 100. CD34+ cells were cultured in 6-well plates (Costar, NY, Cambridge, MA, USA) at a concentration of 105 cells/5 ml in culture medium supplemented with the following recombinant human cytokines: GM-CSF, 100 ng/ml; Stem cell factor (SCF), 20 ng/ml; TNF-α, 10 ng/ml; IL-4, 10 ng/ml (all cytokines were obtained from Biosource). IL-4 was applied on day 7 of culture. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium and the cytokines were freshly replaced every 3–5 days. Proliferating crowded cultures were split and transferred onto new tissue culture plates. The

Normal range – 0–1 12–17 3.8–10.5 140–345 6–23 0–150 0–5 0–48 – – – –

generation of dendritic cells was monitored by inverted microscopy. After 12–14 days of culture, the cells were harvested and counted using hemocytometer. Flow cytometric analysis and sorting Peripheral blood dendritic cells were identified by 3-color staining, performed on peripheral blood mononuclear cells, using the following monoclonal antibodies: ILT-3-PC5 (Immunotech, Villepinte, France), CD11c-PE (PharMingen, BD, Erembodegen, Belgium) and FITC-labeled monoclonal antibodies against lineage markers CD14, CD16, CD19 and CD56 (Immunotech). Cells which did not label with these lineage markers were qualified as lin−. In order to determine the phenotypic profile of the cultured dendritic cells, the following monoclonal antibodies directed against dendritic cell-surface markers were used: CD1a-PE and-FITC, CD80-FITC, CD83-PE, CD54-PE, CD40-FITC, CD14-FITC, CD34-PE, HLA-DR-PE, CD11c-PE (all from PharMingen, BD) and appropriate isotype-matched negative controls. Non-viable cells were excluded from the analysis by gating, based on PI. All samples were analyzed on EPICSXL (Coulter, Miami, FL, USA) flow cytometer. Dendritic cells were separated using FACSorting (FACS Vantage, BD, Erembodegen, Belgium). CD34+-derived dendritic cells were first sorted, according to their expression of CD1a, and then they were used as stimulants in mixed lymphocyte reaction. Mannose receptor-mediated endocytosis Monocyte-derived dendritic cells were incubated in a culture medium with dextran-FITC (MW 40000, Sigma) at a concentration of 1 mg/ml for 30 min at 37°C, and also at 4°C in order to determine the background uptake. Dendritic cells were then washed four times with ice-cold phosphate-buffersaline in a refrigerated centrifuge and analyzed immediately by flow cytometry. Dextran uptake was evaluated by the mean fluorescence intensity (MFI) of the FITC channel and calculated in arbitrary units as (MFI37°C − MFI4°C) / MFI4°C.

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Mixed lymphocyte reaction FACS sorted CD1a+ CD34-derived dendritic cells and monocyte-derived dendritic cells were treated with Mitomycin C (Sigma) (50 mg/ml) and cultured in 24-well microplate (Costar) with allogeneic normal CD3+ cells for 5 days as previously described [16]. Bromodeoxyuridin (BrdU) 10 μM was added for the last 16 h. T-cell proliferation was assessed by the BrdU incorporation using three-color staining flow cytometry assay (PharMingen, BD) with anti-BrdU-FITC, CD3-PE monoclonal antibodies and 7-amino-actinomycin-D (7AAD) following the manufactures' procedure. Statistical analysis Kolmogorov–Smirnov test was initially applied to determine whether the distribution of each set of values under comparison could be considered normal. Student's t test or Mann–Whitney U test was then used as appropriate. All data sets were compared by independent two-tailed t tests. P values lower than 0.05 were considered as statistically significant. All comparisons between patients and controls were performed between adult patients only. To test the effect of age on the results, we first compared the parameters of dendritic cells obtained in this study between patients (adult and pediatric taken together) older and younger than the median age, and second, estimated the correlation coefficient r between each parameter and age. Moreover, to estimate the existence of possible sex differences in the parameters of dendritic cell function, we compared the results of male and female patients in both groups studied, patients and controls. Results Reduced number of peripheral blood dendritic cells in patients with Gaucher disease Circulating peripheral blood dendritic cell subsets were analyzed on peripheral blood mononuclear cells using lineage markers, CD11c and ILT3. According to the expression of CD11c, two populations of lin−/ILT3+ cells were observed: lin− /ILT3+/CD11c+ myeloid dendritic cells and lin−/ILT3+/ CD11c− plasmacytoid dendritic cells (Fig. 1A). In the group of patients with Gaucher disease, the percentages of both dendritic cell subsets were significantly reduced as compared to controls (myeloid dendritic cells 0.19% ± 0.072% vs. 0.34% ± 0.10%, P = 0.009 and plasmacytoid dendritic cells 0.16% ± 0.14% vs. 0.39% ± 0.13%, P = 0.004) (Fig. 1B). Interestingly, in the two pediatric patients tested, both peripheral blood dendritic cell subsets were numerically almost in the range of normal controls. Differentiation of peripheral blood monocytes into dendritic cells after cytokine stimulation in culture Following 7 days of culture of adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4,

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dendritic cells were found in the supernatant of the culture as non-adherent or loosely adherent cells. Although no difference was found in the percentage of monocytes initiating the culture, estimated as CD14+ cells, between patients with Gaucher disease and normal controls (10.5% ± 6.6% vs. 9.7% ± 3.6%, P: n.s.), the yield of monocyte-derived dendritic cells was significantly lower in patients as compared to controls. At the end of the culture period, 8.3 ± 3.2% of the peripheral blood mononuclear cells obtained from the controls, but only 4.8 ± 3.5% of the peripheral blood mononuclear cells obtained from the patients with Gaucher disease differentiated into mature monocyte-derived dendritic cells (P = 0.043, Fig. 2A). Moreover, in patients with Gaucher disease, the percentage of mature monocyte-derived dendritic cells yielded from the culture and expressing CD1a antigen following LPS stimulation was significantly lower compared to the corresponding percentage of controls (35.4% ± 12.6% vs. 80% ± 15.8%, P = 0.0088, Fig. 2B). However, this was the only significant difference in the immunophenotypic profile between patients and controls, and the difference in CD1a expression on dendritic cells, following TNF-α simulation (53.6% ± 28.4% vs. 79.5% ± 10.5%, P = 0.193), as well as all the rest antigenic expression tested was not statistically significant (Table 3). Immunophenotype of monocyte-derived dendritic cells obtained from the culture Immunophenotypic studies were performed on both the immature monocyte-derived dendritic cells obtained in culture with GM-CSF and IL-4 and on the mature ones obtained after stimulation with TNF-α or lipopolysaccharide. The percentage of the viable cells, represented by the PI negative dendritic cell population, was higher than 90%; their myeloid origin was confirmed by the expression of myeloid dendritic cell marker CD11c. The expression of the dendritic cell-associated surface molecules (CD80, CD83, CD40, HLA-DR and CD54) was similar, when compared between patients with Gaucher disease and healthy controls. In both patients and controls, culture of immature monocyte-derived dendritic cells in the presence of lipopolysaccharide and TNF-α resulted in a significant upregulation of the dendritic cell-surface marker CD83, costimulatory molecule CD80, adhesion molecule CD54 and HLA-DR, indicating an efficient maturation of monocytederived dendritic cells (Table 3). The endocytic capacity of immature monocyte-derived dendritic cells of patients with Gaucher disease, assessed by their ability to endocytose Dextran-FITC, did not differ significantly from that of the monocyte-derived dendritic cells of the controls (3.0 ± 1.0 vs. 3.5 ± 1.0 arbitrary units, P = 0.298) (Fig. 3). We then investigated the ability of monocyte-derived dendritic cells at different stages of differentiation to induce proliferation of normal T-lymphocytes in an allogeneic mixed-lymphocyte reaction model. The optimal Dendritic cell:T-cell ratio was 1:10. Immature and TNF-α-

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Fig. 1. Examples of the flow cytometry analysis of the circulating DC subsets in a healthy volunteer (a) and patients with Gaucher disease (b, c, d). Blood DCs were identified by 3-color staining performed on PBMCs using the following monoclonal antibodies: ILT-3-PC5, CD11c-PE and FITC-labeled mAbs against lineage markers CD14, CD16, CD19 and CD56. According to the expression of CD11c, two populations of lin−/ILT3+ cells were observed: lin−/ILT3+/CD11c+ MDCs and lin−/ILT3+/CD11c− PDCs (A). Comparison of PBDCs percentage in healthy subjects and patients (B).

or lipopolysaccharide-stimulated monocyte-derived dendritic cells from both patients with Gaucher disease and controls did not differ in their ability to induce a proliferative response of allogeneic T-cells (Table 2 and Fig. 4).

Induction of dendritic cells production from CD34+ bone marrow progenitors To evaluate the effectiveness of dendritic cells production from bone marrow progenitor cells in patients with Gaucher

Fig. 2. Mean DC yield (A) and CD1a expression (B) of MoDCs generated in the presence of GM-CSF/IL-4 for patients with Gaucher disease and controls.

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Table 3 Surface antigen expression and allogeneic stimulatory activity (MLR% BrdU+ T-cells) of monocyte-derived DCs from adult patients with Gaucher disease (Gaucher) and from healthy subjects (Controls) Immature dendritic cells

HLA-DR (MFI) a CD1α (%) CD14 (%) CD40 (MFI) a CD54 (MFI) a CD80 (MFI) a CD83 (%) MLR BrdU (%) a

TNF-α stimulation

LPS stimulation

Controls

Gaucher

P

Controls

Gaucher

P

Controls

Gaucher

P

44.9 ± 21.9 66.5 ± 16.1 2.76 ± 0.89 4.43 ± 1.33 54.1 ± 15.7 1.90 ± 0.79 9.73 ± 7.48 29.8 ± 6.27

34.1 ± 11.1 70.3 ± 24.3 2.35 ± 1.91 3.59 ± 1.53 57.6 ± 13.6 1.46 ± 0.44 15.3 ± 6.41 26.0 ± 6.62

0.559 0.811 0.718 0.338 0.727 0.281 0.249 0.219

111.7 ± 62.7 76.5 ± 10.5 6.93 ± 2.14 12.2 ± 4.36 103.1 ± 21.0 3.02 ± 1.61 58.7 ± 20.2 46.7 ± 8.24

97.1 ± 28.9 53.6 ± 28.4 4.69 ± 4.35 9.74 ± 3.45 94.2 ± 19.4 2.07 ± 0.53 49.3 ± 13.4 40.9 ± 9.6

0.644 0.193 0.421 0.326 0.537 0.193 0.417 0.195

180.6 ± 32.6 80.0 ± 15.8 7.58 ± 2.35 15.7 ± 4.28 88.3 ± 9.01 5.81 ± 4.10 75.8 ± 25.2 50.1 ± 8.10

194.6 ± 70.6 35.3 ± 12.6 4.78 ± 4.46 11.1 ± 3.89 109.5 ± 55.4 4.19 ± 2.42 69.2 ± 16.1 42.7 ± 9.30

0.831 0.009 0.373 0.174 0.230 0.580 0.719 0.099

Mean fluorescence intensity.

disease, we generated dendritic cells from purified bone marrow CD34+ cells in a liquid culture system, supplemented with GMCSF, SCF, TNF-α and IL-4. MACSorted bone marrow CD34+ cells were cultured for 14 days. After 12–14 days of culture, supernatants were harvested and the cells were counted using a hemocytometer. Cytospins were stained with May–Grunwald– Giemsa and showed that almost half of the cells had typical dendritic cell morphology, both in patients with Gaucher disease and in controls. Furthermore, CD34 + cell-derived dendritic cells were phenotypically defined. Dendritic cells generated from CD34+ progenitors from patients with Gaucher disease and from healthy controls exhibited similar expression of CD1a, HLADR, CD54, CD83 and of co-stimulatory molecules (CD80, CD40) (Fig. 5A). Moreover, the T-lymphocyte response in allogeneic mixed lymphocyte reaction revealed that CD34derived dendritic cells obtained either from patients or from healthy controls did not differ in their allo-stimulatory capacity (35.5% ± 17.5% vs. 31.8% ± 15.1%, P = 0.7) (Fig. 5B). Effect of age and gender on dendritic cell numerical and functional parameters

parameter between patients older and younger than the median age, as well as between male and female patients in both group of tested subjects, patients and controls. Both older subgroups, of the patients and of the controls, had higher HLA-DR expression on immature dendritic cells, as compared to the corresponding younger subgroup, and the difference was statistically significant. A higher expression among older subjects was also found for the CD14 antigen but the difference was significant only in the patient group. Following TNF-α stimulation, younger controls exhibited an upregulation of HLA-DR, while older patients did so for CD14 surface antigen, findings not observed among the patient group. Female patients had a significantly higher CD80 antigen expression on their immature dendritic cells, and the same was true following TNFα stimulation for both CD80 and CD40 antigens, while such differences were not found in the control group. Following LPS stimulation, normal male subjects showed significantly higher expression of HLA-DR than did female. Finally, a uniform finding was the significantly higher allo-stimulatory capacity of dendritic cells in all three states of maturation of the younger controls, as compared to the corresponding MLRs of the older subjects. A similar difference was not encountered among patients with Gaucher disease. However, in both patient and

To evaluate the effect of age and gender on the numerical and functional parameters of dendritic cells, we compared each

Fig. 3. Dextran-FITC endocytosis of immature MoDCs from patients with Gaucher disease and controls.

Fig. 4. Allogeneic stimulatory activity of MoDCs. The mean percentage of BrdU positive proliferating lymphocytes for ratio 1:10 stimulator to responder cells before and after stimulation with TNF-α or LPS in MDS patients and controls.

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Fig. 5. Phenotype of DCs generated from BM CD34+ cells in the presence of SCF/GM-CSF/TNF-α/IL-4 after gating for CD1a+. Comparison of the DCs surface antigen expression on CD34-DCs in healthy donors and MDS patients. The mean percentage of expression ± standard deviation is presented on the graph (A). Allogeneic stimulatory activity of FACSorted CD1a+ CD34-DCs. The mean percentage of BrdU positive proliferating lymphocytes for ratio 1:10 stimulator to responder cells in normal donors and MDS patients is represented on the graph (B).

control group, there was a significant negative correlation between age and the percentage of MLR-induced BrdU+/CD3+ cells (Controls: unstimulated dendritic cells r = −0.626, TNF-αstimulated cells r = −0.674, LPS-stimulated dendritic cells r = −0.709, Patients: unstimulated dendritic cells r: not significant, TNF-α-stimulated cells r = −0.439, LPS-stimulated dendritic cells r = −0.333). Similar significant negative correlations were found between age and the percentage of circulating myeloid dendritic cell precursors (patients: r = −0.314, controls: r = −0.672), of circulating plasmacytoid dendritic cells (patients: r = −0.441, controls: r = −0.648) and the strength of Dextran-FITC endocytosis (patients: r = −0.634, controls: r = −0.489). A significant negative correlation was also found between age and the yield of monocyte-derived dendritic cells but only in the control group (r = −0.455). The results of all comparisons between older and younger subjects, as well between males and females, in both patient and control group are presented in Table 4. Discussion It has been recognized that patients with Gaucher disease exhibit an increased incidence mainly of lymphoid malignancies, but also of solid tumors [5–8], as well as a higher susceptibility to systemic infections. Although these observations imply that the immune surveillance of the patients might be impaired, the underlying mechanisms leading to such immunological disturbances have not yet been fully clarified. It has been hypothesized that the accumulated glycosylceramide in the organs of the reticuloendothelial system provides a chronic antigenic stimulation to the immune system [4], and this becomes evident by the polyclonal hypergammaglobulinemic pattern of the serum protein electrophoresis [17] and by the increased occurrence of monoclonal B-lymphocyte and plasma cell populations [18]. Alternatively, the increased risk of

malignancies may be attributed to a possible leukocyte dysfunction, related to the underlying enzyme defect [19]. In order to clarify the possible implication of dendritic cells as the most potent antigen-presenting cells, in the present study, we analyzed different quantitative and functional parameters of dendritic cells in patients with type I Gaucher's disease. We first detected the two dendritic cell subsets in the peripheral blood. The circulating dendritic cells represent distinct lineages of functionally diverse immature dendritic cells that upon appropriate signals can give rise to two types of mature dendritic cells [20,21]. Functionally, myeloid and plasmacytoid dendritic cells induce polarization of naive Tcells, driving Th1 and Th2 immune response, respectively [22]. Besides, when stimulated with viruses or CD40L in vitro, plasmacytoid dendritic cells can efficiently promote Th1 polarization, mediated by the synergistic effect of IL-12 and type-I interferon [23,24]. Thus, plasmacytoid dendritic cells constitute a critical link between innate and adaptive immunity. We found that myeloid and plasmacytoid dendritic cells were decreased in all patients with Gaucher disease, with the exception of one 8-year old child, with mild symptoms of the disease, who presented a normal dendritic cell subset profile. Potential causes for the reduction of the peripheral blood dendritic cells may be central or peripheral. In the context of chronic antigenic stimulation, dendritic cells of the peripheral blood may receive continuous stimuli, inducing their migration and homing to lymphoid organs. Alternatively, a suppressed production of dendritic cells from pluripotent hematopoietic stem cells in the bone marrow might be the cause of dendritic cell reduction in these patients. It is believed that dendritic cells originate from bone marrow stem cells via different developmental pathways related to myeloid or lymphoid cell types [12,25]. In vitro, human hematopoietic progenitors purified from cord blood or bone marrow can be induced to proliferate and differentiate into dendritic cells [26,27]. We, therefore,

Table 4 Peripheral blood dendritic cells' numerical, phenotypical and functional parameters, in relation to age and sex, among patients with Gaucher disease and healthy controls All patients

Controls

≥median

bmedian

P

Male

Female

P

≥median

bmedian

P

Male

Female

P

CD14 expression (%) Myeloid DC (%) Plasmacytoid DC (%) Dextran endocytosis a Immature DC CD1α (%) Immature DC CD14 (%) Immature DC CD40 b Immature DC CD54 b Immature DC CD80 b Immature DC CD83 (%) Immature DC HLA-DR b TNF-α stimulation–CD1α (%) TNF-α stimulation–CD14 (%) TNF-α stimulation–CD40 b TNF-α stimulation–CD54 b TNF-α stimulation–CD80 b TNF-α stimulation–CD83 (%) TNF-α stimulation–HLADR b LPS stimulation–CD1α (%) LPS stimulation–CD14 (%) LPS stimulation–CD40 b LPS stimulation–CD54 b LPS stimulation–CD80 b LPS stimulation–CD83 (%) LPS stimulation–HLA-DR b MLR immature cells c MLR TNF-α stimulation DC c MLR LPS stimulation DC c

10.7 ± 7.63 0.16 ± 0.08 0.18 ± 0.17 2.82 ± 0.78 74.1 ± 15.3 3.41 ± 1.56 3.98 ± 1.53 58.4 ± 11.2 1.38 ± 0.42 14.1 ± 2.39 39.9 ± 9.10 60.9 ± 19.3 4.94 ± 3.04 9.39 ± 2.66 103 ± 20.3 2.04 ± 0.65 42.7 ± 7.12 106 ± 27.7 51.3 ± 9.76 6.09 ± 1.69 15.0 ± 4.23 132 ± 17.7 3.86 ± 1.16 74.8 ± 10.5 243 ± 35.3 26.2 ± 4.52 34.3 ± 8.36 37.0 ± 10.2

11.7 ± 4.46 0.23 ± 0.10 0.20 ± 0.06 3.33 ± 0.64 71.7 ± 27.8 0.88 ± 0.72 2.39 ± 1.26 56.3 ± 14.2 1.45 ± 0.43 14.4 ± 8.51 23.1 ± 6.81 61.1 ± 34.7 3.47 ± 4.69 9.94 ± 3.35 89.5 ± 12.6 2.22 ± 0.39 46.6 ± 19.1 70.5 ± 30.8 43.8 ± 19.8 3.33 ± 4.26 11.1 ± 2.95 120 ± 55.0 4.83 ± 2.70 67.5 ± 14.4 165 ± 65.3 24.5 ± 7.76 40.1 ± 11.6 41.4 ± 9.13

0.791 0.258 0.771 0.279 0.877 0.017 0.146 0.822 0.809 0.958 0.018 0.993 0.647 0.821 0.322 0.639 0.743 0.153 0.558 0.318 0.231 0.764 0.574 0.482 0.150 0.681 0.442 0.543

11.8 ± 3.82 0.19 ± 0.13 0.22 ± 0.16 2.65 ± 0.68 66.9 ± 24.9 2.69 ± 2.13 2.40 ± 1.05 50.2 ± 8.70 1.07 ± 0.25 11.8 ± 4.09 33.1 ± 15.4 77.5 ± 19.4 3.51 ± 3.59 7.84 ± 2.82 104 ± 18.3 1.78 ± 0.48 36.2 ± 9.67 81.6 ± 42.6 53.6 ± 16.5 4.19 ± 3.16 14.5 ± 2.47 151 ± 29.6 5.32 ± 2.38 67.9 ± 11.3 223 ± 71.4 26.8 ± 6.77 38.0 ± 12.4 38.9 ± 11.3

10.6 ± 7.96 0.19 ± 0.06 0.16 ± 0.07 3.50 ± 0.57 79.9 ± 18.0 1.44 ± 1.23 3.97 ± 1.69 64.4 ± 12.4 1.77 ± 0.24 16.6 ± 7.08 29.9 ± 4.99 40.5 ± 25.4 4.89 ± 4.55 11.9 ± 1.26 84.7 ± 9.04 2.48 ± 0.27 55.5 ± 13.8 92.4 ± 18.1 39.0 ± 12.5 5.01 ± 4.11 14.5 ± 4.68 97.7 ± 42.1 3.26 ± 1.25 74.2 ± 14.8 165 ± 48.2 24.0 ± 5.68 35.9 ± 6.72 39.5 ± 7.33

0.778 0.995 0.442 0.058 0.400 0.336 0.153 0.098 0.004 0.274 0.698 0.065 0.665 0.048 0.130 0.037 0.066 0.689 0.239 0.773 0.993 0.121 0.208 0.546 0.286 0.500 0.785 0.933

10.7 ± 4.01 0.30 ± 0.11 0.36 ± 0.13 3.18 ± 0.80 61.2 ± 17.3 3.51 ± 1.34 4.16 ± 1.58 58.5 ± 13.6 1.54 ± 0.57 5.32 ± 1.51 57.0 ± 14.9 70.3 ± 10.1 8.27 ± 2.21 10.0 ± 3.03 97.0 ± 26.9 2.47 ± 1.06 48.3 ± 14.8 64.0 ± 15.6 84.0 ± 9.27 8.93 ± 2.86 13.6 ± 3.59 92.3 ± 7.13 4.86 ± 2.21 70.0 ± 21.4 181 ± 52.2 25.7 ± 4.12 40.6 ± 5.18 45.0 ± 4.06

8.6 ± 2.64 0.39 ± 0.05 0.43 ± 0.12 3.88 ± 0.90 72.1 ± 13.5 2.17 ± 0.70 4.70 ± 0.93 52.3 ± 12.5 2.31 ± 0.73 14.0 ± 7.11 24.8 ± 3.94 81.3 ± 4.92 3.45 ± 1.05 14.4 ± 4.39 99.5 ± 15.2 3.76 ± 1.49 67.3 ± 16.9 154 ± 33.5 81.3 ± 9.18 5.80 ± 1.95 17.7 ± 3.89 89.0 ± 9.80 6.95 ± 3.94 79.7 ± 15.1 199 ± 50.3 33.9 ± 5.27 53.1 ± 6.32 55.3 ± 5.61

0.401 0.190 0.506 0.277 0.658 0.281 0.697 0.661 0.307 0.164 0.042 0.238 0.049 0.310 0.914 0.374 0.298 0.025 0.767 0.269 0.331 0.717 0.548 0.630 0.746 0.039 0.016 0.023

9.30 ± 3.96 0.36 ± 0.11 0.39 ± 0.14 4.02 ± 0.86 64.5 ± 20.5 2.32 ± 1.27 4.73 ± 1.57 62.5 ± 12.9 2.01 ± 0.70 8.60 ± 4.41 51.0 ± 21.9 75.3 ± 10.9 5.98 ± 3.52 13.1 ± 5.60 111 ± 17.6 3.17 ± 1.34 53.7 ± 14.0 142 ± 46.9 79.7 ± 9.01 6.43 ± 3.44 16.5 ± 5.55 96.0 ± 7.79 5.12 ± 2.34 68.0 ± 20.6 233 ± 30.1 29.3 ± 7.61 46.4 ± 10.5 50.2 ± 9.83

10.0 ± 3.05 0.33 ± 0.08 0.39 ± 0.12 3.04 ± 0.68 68.5 ± 9.50 3.36 ± 1.02 4.13 ± 0.91 48.4 ± 9.73 1.84 ± 0.80 10.7 ± 8.32 30.8 ± 8.79 76.3 ± 8.22 5.73 ± 2.27 11.2 ± 2.23 85.8 ± 17.6 3.13 ± 1.51 66.3 ± 16.8 76.3 ± 33.1 85.7 ± 8.65 8.37 ± 1.91 15.1 ± 2.26 85.3 ± 5.91 6.70 ± 3.98 81.7 ± 14.7 147 ± 29.4 30.3 ± 4.47 47.3 ± 5.88 50.1 ± 3.32

0.779 0.637 0.989 0.113 0.876 0.419 0.664 0.284 0.845 0.763 0.294 0.922 0.937 0.681 0.242 0.978 0.459 0.179 0.534 0.525 0.802 0.197 0.654 0.488 0.045 0.823 0.889 0.973

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Significant differences are stressed in bold letters. a Arbitrary units. b Mean intense of fluorescence. c % BrdU+ cells.

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studied the potential of CD34+ bone marrow hematopoietic progenitor stem cells to produce dendritic cells in a cytokine supplemented liquid culture system [16]. In this system, bone marrow progenitors of patients with Gaucher disease appear not to be directly affected by the enzyme defect, since, at least in vitro, they successfully differentiated into phenotypically and functionally potent dendritic cells. However, in the “in vivo” setting, the presence of abundant bone marrow macrophages, replete with glycosylceramide, and/or additional microenvironmental factors, such as suppressive cytokines and various active immune competent cells, may negatively influence “dendritopoiesis”. This is highly suggested by the obtained low peripheral blood dendritic cell precursor subsets, as demonstrated in the present study. We also studied dendritic cells generation from another potential dendritic cell precursor population—the circulating monocytes that in vivo provide a boost to the antigenpresenting cell population in sites of significant infection or inflammation [28]. We observed that, although no difference in the percentage of peripheral blood monocytes initiating the culture of both patients and controls was found, the dendritic cells yield of patients was significantly low. It is well established that the differentiation and maturation of dendritic cells are dependent on a number of cytokines. The induction of dendritic cells from patients with neoplastic disorders for example is assumed to be difficult as a result of the negative effect of several cytokines, such as IL-10, transforming growth factor-β, monocyte colony-stimulating factor (MCSF), IL-6 or vascular-endothelial growth factor (VEGF), which are often produced by the malignant cells and can prevent dendritic cell differentiation, and reduce their allostimulatory capacity [29–31]. The low yield of monocytederived dendritic cells, which we found in patients with Gaucher disease, may be ascribed to activities of macrophagederived cytokines. The role of the cytokines in Gaucher disease has been previously suggested by several studies [32– 34]. Increased M-CSF and IL-8 have been correlated with the severity of the disease [35]. Interleukin-6 and IL-10 have also been found elevated in the serum of the patients with this lysosomal storage disorder [36]. In this regard, the maturation pathway of the dendritic cell precursors could be influenced by different cytokines that compete in their action. On the other hand, a primary defect associated with the enzyme deficiency of monocytes probably affects the potential of peripheral blood monocytes in Gaucher disease to differentiate into dendritic cells. However, the fact that monocyte-derived dendritic cells obtained in the culture did not exhibit phenotypical and functional defects shows the prevalent role of the microenvironment on dendritic cell differentiation that in vitro seems to be partially overcome. There was a negative effect of age upon some immunophenotype markers, as well as upon the allo-stimulatory capacity of dendritic cells among patients, but basically among normal controls. A decline of many immunologic functions is a well-known consequence of the growing age. The difference in some activation markers of dendritic cells, between male

and female patients, which was observed in this study may be attributed to the younger age of the female patients (median age 27.5 years vs. 37 years for the males) and/or to the relatively more severe disease status of the male patients (Zimran's Severity Score Index: males 9 ± 2.2, females 6.2 ± 1.3, P = 0.055), than reflecting true difference between the two genders. In conclusion, we demonstrate that dendritic cells of patients with type I Gaucher disease exhibit mainly quantitative but not qualitative defects. It may be hypothesized that, in the “in vivo” setting, the chronic antigenic stimulation, the bone marrow infiltration or the elevated levels of macrophagederived cytokines (IL-10, M-CSF) may negatively influence “dendritopoiesis”. The decreased numbers of dendritic cell precursors in Gaucher disease may also lead to a decreased IL12 secretion, which, together with the low number of natural killer cells, may contribute to the poor immune response against various infectious agents and also to impaired immunologic surveillance, allowing neoplastic cells to emerge and perpetuate, and thus proceed to malignancy. It is unknown whether these numerical dendritic cell defects are reversible with the administration of the appropriate enzyme replacement therapy. In our group of patients, only two young female (one adult and one pediatric patient) have been treated with imiglucerase for 18 and 12 months, but both still exhibited similar levels of peripheral blood dendritic cell subsets to those observed among untreated patients. Despite the clinical improvement of both these patients, we may assume that the treatment period was probably short for the dendritic cell populations to be corrected, and therefore, the study of additional patients before and after effective treatment, may clarify whether dendritic cell defects may correlate with disease severity and could be reversed after successful enzyme replacement therapy. References [1] T.M. Cox, Gaucher disease: understanding the molecular pathogenesis of sphingolipidoses, J. Inherited Metab. Dis. 24 (2001) 106–121. [2] E. Beutler, G.A. Grabowski, Gaucher disease, in: C.R. Scriver, D. Valle, A. Beudet, W.S. Sly (Eds.), The Metabolic and Molecular Bases of Inherited Diseases, vol. III, McGraw-Hill, New York, 2001, pp. 3635–3668. [3] L. Marodi, R. Kaposzta, J. Toth, A. Laszlo, Impaired microbicidal capacity of mononuclear phagocytes from patients with type I Gaucher disease: partial correction by enzyme replacement therapy, Blood 86 (1995) 4645–4649. [4] Y. Shoenfeld, L.A. Gallant, M. Shaklai, E. Livni, M. Djaldetti, J. Pinkhas, Gaucher's disease: a disease with chronic stimulation of the immune system, Arch. Pathol. Lab. Med. 106 (1982) 388–391. [5] J.A. Paulson, G.E. Marti, J.K. Fink, N. Sato, M. Schoen, D.S. Karcher, Richter's transformation of lymphoma complicating Gaucher's disease, Hematol. Pathol. 3 (1989) 91–96. [6] Y. Burstein, G. Rechavi, A.R. Rausen, B. Frisch, Z. Spirer, Association of Gaucher's disease and lymphoid malignancy in 2 children, Scand. J. Haematol. 35 (1985) 445–447. [7] D. Garfinkel, Y. Sidi, M. Ben-Bassat, F. Salomon, B. Hazas, J. Pinkhas, Coexistence of Gaucher's disease and multiple myeloma, Arch. Intern. Med. 142 (1982) 2229–2230. [8] A. Zimran, I. Liphshitz, M. Barchana, A. Abrahamov, D. Elstein, Incidence of malignancies among patients with type I Gaucher disease from a single referral clinic, Blood Cells Mol. Dis. 34 (2005) 197–200.

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