Published online before print June 14, 2004
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* Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, and
Ematologia, Dipartimento di Medicina Clinica e Sperimentale, Università Federico II, Naples, Italy
1 Correspondence: Cattedra di Immunologia-Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II", Via Pansini 5, 80131 Naples, Italy. E-mail: giruggie{at}unina.it
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and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.
Key Words: GPI-linked molecules • cytokine secretion • accessory signals
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Hemolytic anemia, thrombophylia, and cytopenia characterize this disease [1 7 11 ]. The lack of GPI+ molecules CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis), critically involved in the protection of red cells from the lytic attack of activated, complement fractions [12 ], accounts for chronic hemolysis with capricious exacerbation. Platelet surface activation by small amounts of complement and urokinase plasminogen receptor deficiency [13 14 ] might explain thrombophylia, and the origin of the underlying BM failure is still unknown [15 16 17 18 ].
Others and our data [19 20 ] have shown that a majority of granulocytes and monocytes in peripheral blood of PNH patients is GPI-defective. The functional analysis of the GPI-defective leukocytes from PNH patients is still controversial [21 22 23 24 ].
Monocytes play a key role in achieving an effective immune response. Indeed, they mediate essential effector functions leading to the direct control of pathogen [25 ] and are involved in the activation of the adaptive immune response [26 ]. In this regard, a role for monocyte-derived cytokines in driving the activation of specialized T cell-dependent effector pathways, as represented by the T helper cell type 1 (Th1)/Th2 cytokine secretion pattern, has been demonstrated already [27 ]. Neutropenia, frequently present, is responsible for the increased susceptibility to infections of PNH patients [2 21 ]. Whether a functional impairment of neutrophils and monocytes may have a role in the reduction of host immune response in PNH has yet to be defined [9 14 16 ].
Pathogen recognition as well as the indirect sensing of infections through inflammatory cytokines are involved in entering monocytes into an integrated developmental program, dendritic cell (DC) differentiation [28 29 ]. Monocyte-derived DCs represent a key cell population for antigen-dependent activation of naive T lymphocytes [27 30 31 ]. Functional properties of GPI-defective monocytes and their ability to differentiate into DCs are largely undefined in PNH patients.
This study investigated the ability of GPI-defective monocytes from PNH to differentiate in vitro into functional DCs.
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Table 1. Patient Characteristics
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(TNF-), interleukin (IL)-12, and isotype-matched controls were purchased from BD PharMingen (San Jose, CA).
To analyze the intracellular production of TNF- and IL-12, staining with the specific mAb was performed using a fixing/permeabilization kit purchased from Caltag (Burlingame, CA), following the manufacturer’s instructions. To avoid extracellular cytokine export, the cultures were performed in the presence of 5 µg/ml Brefeldin-A (Sigma-Aldrich, St. Louis, MO), as described [32
].
All DC phenotypes referred to the flow cytometry analysis of the DC population gated by using forward- and side-scatter parameters. Flow cytometry and data analysis were performed using a two-laser-equipped FACSCalibur flow cytometer and the CellQuest analysis software (BD PharMingen), as described [33 ].
DC and resting T cell populations
Peripheral blood mononuclear cells (PBMC) were isolated from patients and controls by centrifugation of peripheral blood over Lymphoprep (Nycomed, Oslo, Norway) gradient. To obtain a purified monocyte population, the cells collected from the plasma/Lymphoprep interface were washed in phosphate-buffer saline (PBS) and incubated at 37°C in the presence of RPMI-1640 medium (Gibco-BRL, Grand Island, NY) in six-well plates (Falcon, Seattle, WA). After a 2-h incubation, the nonadherent cells were removed, and each well was washed three times with PBS. Adherent cells were cultured for a 7-day period in the presence of 80 ng/ml human recombinant granulocyte macrophage-colony stimulating factor (hrGM-CSF) and 1000 IU/ml hrIL-4 (Sigma-Aldrich), as described [34
]. When indicated, lipopolysaccharide (LPS; 100 ng/ml, Sigma-Aldrich) or human trimeric CD40 ligand (CD40L) molecule (100 ng/ml, BenderMedSystems, Austria) was added to the culture for an additional 16–24 h to induce terminal DC differentiation [34
]. The purity of the DC population was defined by immune fluorescence phenotype analysis. Only the populations showing less than 5% of lymphocyte contaminants were used in the study. The ability of these DC populations to elicit antigen-dependent T cell proliferation was assessed by incubating DC with 10 µg/ml purified protein derivative (PPD; Staten Serum Institute, Copenhagen, Denmark) for 2 h at 37°C. After extensive washing, PPD-treated DCs were 30 Gy-irradiated and cultured for 5 days in the presence of autologous PBMC.
T lymphocytes were isolated from venous peripheral blood samples obtained from healthy controls as described previously [35 ]. Briefly, small, resting T cells were purified from the PBMC population using a Percoll density gradient, after removing B cells and monocytes by plastic and nylon wool adherence. The recovered high buoyant density population was always >97% CD3+CD56–. Purity of T cell preparations was assessed using immunofluorescence and phytohemagglutinin stimulation.
Proliferation assay
The DCs, obtained after a 7-day culture in the presence of GM-CSF and rIL-4, were used as costimulators after complete maturation induced by LPS treatment. Resting peripheral T cells (1x105) were activated with anti-CD3 mAb CLB-CD3/E in 96-well, flat-bottomed microtiter plates (Falcon) in the presence of different percentages of 30 Gy-irradiated DCs, as described in Results. For the antigen-specific proliferation assay, a concentration of 20% PPD-treated DCs was used.
Anti-CD3-triggered T cell proliferation was assessed by culturing 2 x 105 PBMC obtained from normal controls and PNH patients in the presence of anti-CD3 mAb (CLB-CD3/E), as indicated.
Cultures were incubated for 3–5 days at 37°C in a humidified atmosphere containing 5% CO2 and pulsed with 0.5 µCi/well [3H] thymidine for the last 16 h. The incorporation of the labeled nucleotide was determined by scintillation counting after automatic cell harvesting. All tests were performed in the presence of RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (Gibco-BRL). Autologous serum was used for antigen-specific assays.
Statistical analysis
The statistical analysis was performed with Student’s t-test. Results were considered significant when a P value <0.05 was obtained.
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Figure 1 shows the phenotype of GM-CSF- and rIL-4 treated monocytes from one representative healthy donor and the three PNH patients. As indicated, comparable surface expression of CD40 and HLA-DR molecules has been observed. In addition, no significant differences in the expression of other surface markers (i.e., CD4, CD11c, HLA class I) were observed (data not shown). At variance, a significant impairment of CD1a up-regulation as well as a high expression of CD86 molecule was detected. These data indicate that the DC derived in vitro from PNH monocytes showed significant phenotype alterations. Moreover, we analyzed the ability of PNH monocytes treated with GM-CSF and rIL-4 to up-regulate the mannose receptor molecule associated with an immature DC status [36 37 ]. As shown in Figure 2 , control and GPI-defective PNH monocytes were able to acquire significant surface expression of the mannose receptor molecule as part of the in vitro DC differentiation pathway.
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Figure 1. Phenotype analysis of DCs derived in vitro from PNH GPI-defective monocytes. Peripheral blood monocytes derived from normal donors and PNH patients were cultured for 7 days with GM-CSF and rIL-4 to obtain DC differentiation (see Materials and Methods). At the end of the culture, the cells were analyzed for the surface expression of CD40, HLA-DR, CD1a, and CD86 molecules (as indicated). Dotted line peaks refer to the staining in the presence of the isotype control mAb. The results were obtained from peripheral blood monocytes derived from one representative healthy donor and three PNH patients. Results were confirmed in three independent experiments. Impaired CD1a up-regulation and CD86 surface expression seem to characterize the PNH patient DC phenotype.
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Figure 2. Analysis of mannose receptor expression on DCs derived in vitro from PNH GPI-defective monocytes. Bold line peaks indicate the staining with PE-labeled antimannose receptor mAb of DCs from a healthy control (A) and a PNH patient (B). Dotted line peaks refer to the staining in the presence of the isotype control mAb. Control and PNH cells show significant mannose receptor expression. Data represent one of two independent experiments and were confirmed in all the PNH patients enrolled in the study.
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after exposure to LPS, as well as after CD40L triggering (see Materials and Methods).
Figure 3
refers to one representative intracellular staining for TNF- in one healthy control (A and B) and one PNH patient (C and D) DC population. As expected, medium incubation (dotted line peaks in panels A–D) was unable to induce TNF- production in the control as well as in the PNH DC populations. At variance, LPS (bold line peak in C) and CD40L treatment (bold line peak in D) induced a lesser TNF- production in PNH DCs if compared with the normal DC counterpart (A and B). It is interesting that deficiency in CD40-dependent cytokine production was observed in the presence of normal surface expression of CD40 on PNH monocytes (Fig. 1)
. The statistical significance of such impaired cytokine production was assessed in all tested patients for LPS and CD40-dependent stimulation (P<0.05). In our experimental conditions, a similar impairment was revealed also in IL-12 production, and no difference was observed in IL-4 and IL-10 production in PNH-derived DC (data not shown).
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Figure 3. Flow cytometry analysis of TNF- production by PNH GPI-defective DCs. Intracellular staining for TNF- of monocyte-derived DCs from one representative healthy control (A and B) and one PNH patient (C and D). Bold line peaks refer to TNF- production after LPS (A and C) and CD40L treatment (B and D). Dotted line and plain line peaks indicate the cell incubation in medium alone and the staining in the presence of the isotype control, respectively (A–D). Numbers indicate percentage and mean florescence intensity (MFI) of the TNF--labeled cells in the different conditions. A significant decrease in TNF- production was observed in the PNH DC population after LPS and CD40 stimulation (P<0.05 for both stimuli). Data are representative of one of three independent experiments and were confirmed in all the PNH patients enrolled in this study.
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Figure 4A shows that DCs obtained in vitro from normal controls provided an optimal costimulation of TCR-triggered T cell proliferation at cell percentages as low as 2.5%, with a significant costimulatory activity maintained even at 0.5% DC. As shown, a strong impairment of PNH DCs in providing accessory signals for TCR-triggered T cell proliferation was observed. Similar data were observed with the purified CD45RA+ naive T cell preparations (not shown). To analyze the effectiveness of PNH DC in an antigen-dependent model, we also performed experiments by enrolling one patient and two healthy controls, referring recent bacillus Calmette-Guerin vaccination. Therefore, to assess the ability of PNH DC to take up, process, and present antigens to autologous T lymphocytes in comparison with the normal counterpart, we analyzed their ability to elicit T cell proliferation against a mixture of micobacteral antigens, as represented by PPD preparation. PPD-treated PNH and control DCs were thus cultured with autologous T cells at saturating concentration (2x104/well). As shown (Fig. 4B) , PNH-DC exhibited a significantly decreased ability to trigger antigen-dependent T cell proliferation compared with the control DC populations.
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Figure 4. In vitro DC differentiation of PNH monocytes generates cells with impaired ability to elicit TCR-dependent T cell proliferation. (A) Proliferation levels of resting T cells triggered with anti-CD3 mAb in the presence of an increasing number of irradiated LPS-treated DCs (see Materials and Methods) from normal donors (•) and from two PNH patients ( ,  ). The proliferation was measured by [3H] thymidine incorporation after 3 days of incubation. Results are presented as mean cpm of triplicate cultures without background subtraction; no proliferation was observed after incubation of the T lymphocytes with or without anti-CD3 in the absence of the irradiated DCs. Data are representative of the results obtained in four independent experiments. (B) DCs from two normal controls and a PNH patient were incubated with medium or PPD, as indicated. After extensive washing, treated DCs were irradiated and cultured for 5 days in the presence of the autologous PBMC at a concentration of 20%. Results are presented as mean cpm of triplicate cultures without background subtraction. Data are representative of the results obtained in two independent experiments.
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Table 2. Comparative Analysis of PNH Patients and Healthy Control T Lymphocyte Proliferation
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DCs are key regulators of the immune response, and their differentiation reflects an ordered series of events involving specific gene expression patterns, intracellular protein targeting, and organelle biogenesis leading to potent immunomodulatory functions [27 30 37 40 ]. The up-regulation of GPI-linked molecules, as represented by the CD48 antigen, has already been described as part of the DC differentiation program [41 ]. Therefore, it is possible that cell-to-cell functional interactions involving GPI-linked structures affect monocyte differentiation into DCs. In addition, an involvement of GPI-linked structures in the molecular machinery underlying in vitro monocyte–DC differentiation could be hypothesized.
Our data show that the treatment of PNH monocytes with GM-CSF and rIL-4 was unable to generate in vitro functional DCs. In this respect, GM-CSF and IL-4 receptors are expected to be functional in PNH patients, as neither molecule needs a GPI anchor to be expressed on the cell membrane [42 43 ]. Indeed, GM-CSF receptor-dependent, medullar mobilization of GPI-defective precursors has been described in PNH patients [44 ].
The involvement of lipid rafts for growth factor signaling in DCs could be of some relevance. Lipid rafts are specialized plasma membrane regions rich in adapters, kinases, and GPI-linked molecules [45 46 ]. All these structures are likely involved in the assembly of functional, multicomponent transduction complexes [47 48 ]. In this context, the observation that raft microdomains can operate as a general signaling system involved in cytokine-dependent signal transducer and activator of transcription [49 ] might indicate the requirement of GPI-linked molecules for optimal, cytokine-dependent monocyte–DC differentiation. Therefore, a role for the GPI-molecule defect in the cytokine signaling and its correlation with a correct raft assembly should be investigated in PNH patients.
Our data show a severe impairment of the DCs obtained from GPI-defective monocytes to deliver optimal accessory signals to TCR-triggered T lymphocytes, even in the presence of high levels of the CD86 costimulatory molecule. In this respect, the lesser cytokine production observed and the availability of putative inhibitory molecules by DCs could account for the impairment in PNH. Notably, our data refer to an impaired DC-dependent antigen presentation to T lymphocytes. This observation confirms and extends previous results from murine models [50 ].
The level of costimulation depends on the expression of costimulatory molecules as well as on the stability of the immunological synapse [51 52 ]. In this context, the relevance of a correct recruitment of the lipid rafts, membrane domains rich in signaling molecules as well as in GPI-linked structures [45 46 ], for the achievement of an optimal, primary T cell response has been established [53 ].
In our model, the impaired expression of the CD1a molecule and the presence of CD86 and mannose receptor expression seem to be the hallmarks of DC population obtained in vitro from the GPI-defective monocytes in PNH patients. CD1a represents a third class of major histocompatibility complex molecules able to associate with a wide range of lipids and glycolipidic antigens to be presented to CD8+, CD8–CD4–, and 
T lymphocytes [54
]. In addition, the critical involvement of CD1 antigens in the DC-dependent activation of regulatory T and natural killer T lymphocytes in mice and human models has been suggested [55
]. Several observations indicate that CD1 down-modulation, combined with a variable CD86 surface expression, is a common feature of the DC functional impairment occurring in cancer [56
57
58
59
] as well as in immunodeficiency models [60
].
The observation that GPI-defective monocytes generate impaired in vitro DC differentiation suggests the existence of a defect in the physiological cross-talk between innate and adaptive immunity in PNH patients, at least when monocyte compartment is completely GPI-defective.
Received December 2, 2003; revised April 8, 2004; accepted May 20, 2004.
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