© Birkhäuser Verlag, Basel, 2007
Inflamm. res. 56 (2007) 527–532 1023-3830/07/120527-6 DOI 10.1007/s00011-007-7081-7
Inflammation Research
Altered dendritic cell function in response to sera of common variable immunodeficiency patients M. Nourizadeh1, A. Aghamohammadi1, S. M. Moazzeni2, M. Mahdavi2, A. Jalili3, N. Rezaei1, J. Hadjati4 1
Immunology, Asthma and Allergy Research Institute, Medical Sciences University of Tehran, Tehran, Iran Department of Immunology, Faculty of Medicine, Tarbiat Modarres University, Tehran, Iran 3 Division of Immunology, Allergy and Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, Allgemeines Krankenhaus, Vienna, Austria 4 Department of Immunology, Faculty of Medicine, Medical Sciences University of Tehran, Tehran, Iran, Fax: ++98 21 6641 9536, e-mail:
[email protected] or
[email protected] 2
Received 22 April 2007; returned for revision 20 June 2007; accepted by G. Wallace 20 July 2007
Abstract. Objective: CVID is characterized by hypogammaglobulinemia and T cell disorder in most cases. Dendritic cells might be severely perturbed in differentiation and maturation but it is not clear whether this perturbation is intrinsic or because of alterations in microenvironmental factors. We evaluated the effects of CVID patient’s sera as a source of microenvironmental factors on monocyte-derived DCs (MDCs). Methods: Monocyte derived DCs (MDCs) were generated in the presence of GM-CSF, IL-4 and 10 % concentration of CVID (n = 10) and healthy control (n = 8) serum samples. MDCs were matured with monocyte conditioned medium and TNF-a. Mature MDCs were used for: (i) immunophenotyping, (ii) MLR, (iii) co-culture with allogeneic lymphocytes for cytokine production assays and (iv) DC-cytokine production assays after stimulation with CD40L. Results: Treatment of MDCs with sera derived from CVID patients as compared to control sera: (i) causes lower surface expression of HLA-DR after maturation, (ii) leads to production of higher amounts IL-18 by activated MDCs and, (iii) results in lower allostimulatory capacity of MDCs in MLR assays. Conclusions: Our findings argue for constitutive presence/ absence of soluble factors e. g. cytokines in CVID patients’ sera steering the immune response toward the cellular rather than the humoral arm. Our observations deserve further studies to identify these factors. Key words: Common variable immunodeficiency – Dendritic cells – Cytokines – Antibody
Correspondence to: Jamshid Hadjati PhD
Introduction Common variable immunodeficiency (CVID) is a heterogeneous group of disorders, characterized by defective antibody production and increased susceptibility to recurrent pyogenic infections as well as autoimmune and neoplastic diseases [1– 5]. In CVID patients serum levels of at least 2 immunoglobulin isotypes are lower than normal [1, 3–6]. The number of circulating B cells is reduced or normal, but these cells can proliferate and produce immunoglobulins in vitro when stimulated with anti-CD40 and cytokines such as IL-4 and IL-10 [7]. Although CVID is a largely unknown disorder, several causes leading to alteration of immunoglobulin concentrations in the blood have already been identified. These include: T-cell abnormalities [8], accelerated T cell apoptosis [9], impaired cytokine production [10–12] and reduced generation of antigen-specific memory T cells [13]. Initially it had been believed CVID to be a defect in B cell maturation and function, but recently it has been shown that CVID-derived B cells are able to secrete IgM and IgG upon in vitro stimulation with B cell polyclonal activators despite these patients being hypogammaglobulinemic [7, 14]. Therefore, it is speculated that B cells may not receive appropriate signals from T helper lymphocytes or dendritic cells (DCs) and perturbed interaction in secondary lymphoid organs may be involved in the pathogenesis of the disease [15]. In addition to T cell stimulation, DCs regulate B-cell growth and immunoglobulin secretion/class switching and differentiation toward plasma cells [16, 17]. Although malfunctioning of DCs appears to be one of the prominent features of CVID patients [17], it is unclear whether the malfunction is associated to the effect of CVID patient’s microenvironmental factors such as blood cytokines or the DCs are inherently impaired. The aim of the preset study was to study the effects of CVID patients’ sera on development, maturation, cytokine production and induction of adaptive immune response by monocyte-derived dendritic cells (MDCs) in vitro.
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Materials and Methods
Flow cytometry
Patients and volunteers
The following monoclonal antibodies were used in flow cytometry: FITC–conjugated mouse anti-human CD14 (Clone TUK4), CD86 (Clone BU63), CD11c (Clone KB90) and PE–conjugated mouse antihuman HLA-DR (Clone AB3). These and the corresponding isotype control mAbs were purchased from DakoCytomation (Hamburg, Germany). FITC-conjugated monoclonal anti-human CD83 (Clone HB15e) and corresponding isotype control were obtained from Ancell (Bayport, MN). Stainings were performed according to manufacturer’s protocols. Briefly, 105 MDCs were washed twice with PBS/1 % FCS (staining buffer) and incubated for 30 min with anti-CD14, CD1a (Clone NA1/34), CD86, CD11c, and HLA-DR and for 45 min with anti-CD83 at 4 °C. Cells were subsequently washed with staining buffer and analyzed by FACS using a FACS Caliber and CELLQuest software (Becton & Dickenson, San Joes, CA).
Following informed consent and approval of the local Ethics Committee, 10 CVID patients, who had been referred to the Children Medical Center of Tehran University of Medical Sciences, were investigated. The diagnosis of CVID was based on standard criteria, which has been introduced by the Expert Committee of International Union of Immunological Societies (IUIS) on Primary Immunodeficiency [4]. To exclude other causes of hypogammaglobulinemia, patients with B cells number of less than 1 % were subjected to BTK (Bruton´s Tyrosin kinase) [18] mutation analysis and the patients with B cells number of more than 1 % were analyzed for AID (activation-induced cytidine deaminase) [19] (males and females), SH2D1A [20] and CD40L [21] (just in males) defects. Eight age and sex matched healthy volunteers served as control group.
Allogeneic Mixed Lymphocyte Reaction Assay (MLR) Serum preparation Serum samples were collected from patients before immunoglobulin replacement therapy (or four weeks after the last immunoglobulin replacement therapy) and controls. Briefly, 5 ml of whole blood obtained in order to get 2 ml of serum. Blood was allowed to clot for 30 minutes at room temperature, subsequently rimmed with a Pasteur pipette and after centrifugation at 3000 rpm for 10 min/37 °C, serum was separated and heat-inactivated, (56 °C for 30 min) then stored at –70 °C.
Generation of monocyte-derived dendritic cells (MDCs) MDCs were generated as described elsewhere [22]. Briefly, heparinized peripheral blood was obtained from healthy volunteers (O+ blood group) different from control serum donors, mixed with an equal volume of PBS. PBMCs were collected over half volume of lymphoprep 1.077 ± 0.001 g/ml (Axis-Shield, Oslo, Norway) after 20 min centrifugation at 2000 rpm/37 °C and washed twice with RPMI-1640 (Invitrogen, Gibco, United Kingdom) to reduce platelets. MDCs were generated from PBMCs adherent to plastic flasks (Nunc, Roskilde, Denmark) in RPMI1640 + 10 % heat-inactivated human AB serum (Central Blood Bank, Tehran, Iran) after 2 h incubation at 37 °C. and after 5 day culture of adherent monocytes in 500 IU/ml rhIL-4, 1000 IU/ml rhGM-CSF (both from Bender MedSystems, Vienna, Austria) and 10 % of CVID patients or controls sera. Finally non-adherent and loosely adherent cells were harvested, washed and used for immunophenotyping and considered as MDCs. Mature DCs were generated by culturing of MDCs with 40 ng/ ml TNF-a (Bender MedSystems, Vienna, Austria) and 30 % MCM (see Monocyte Conditioned Medium (MCM) section under Materials and Methods) [23] for 48 hrs. Yields of DCs averaged around 8 × 105 per 107 PBMCs.
Monocyte Conditioned Medium (MCM) MCM was prepared as previously described with some modifications [24]. Briefly, Ig coated bacteriologic plates were prepared immediately before use by the addition of 10 ml of 5 mg/ml human gamma globulin (Baxter Healthcare, Deerfield, IL) for 5 min. The plates were washed three times with sterile PBS before use. PBMCs (108) isolated as above were layered onto the Ig-coated bacteriologic plates in 10 ml complete medium with 20 % human AB serum. After 2 hours nonadherent cells were washed away and discarded. Ig-adherent cells were incubated in fresh complete medium with 10 % human AB serum at 37 °C for 24 hrs. The medium was collected, centrifuged at 3000 rpm for 10 min and the cell-free supernatant was passed through a 0.22 µm filter and frozen at –20 °C until use.
Allostimulatory capacity of CVID and control sera treated MDCs was evaluated by co-culturing of different numbers (5000–40000) of irradiated (30 Gy) mature MDCs (effector) with 1 × 105 allogeneic PBMCs/ well/200 µl (responder) in RPMI 1640 medium supplemented with 10 % human AB serum in 96-well plates (Greiner Bio-one, CELL STAR. Frickenhausen, Germany) [10, 25]. After 5 days of culture, proliferation of effector cells was measured by incorporation of 1 µCi/well [3H]-thymidine (Amersham, Aylesbury, UK) which was added during the final 18 h of the culture. Cells were then harvested onto glass-fiber filter papers (Titertek, Lier, Norway) by a mini-cell harvester (NUNC, Roskilde, Denmark) and incorporation of [3H]-thymidine was determined by liquid scintillation beta counter (LKB, NY). Thymidine incorporation was measured by standard liquid scintillation counting, and results were expressed as counts per minute (mean ± SD of triplicate values) [26]. Control wells contained only T cells, DCs, or T cells stimulated with 1 % phytohemoagglutinin (Invitrogen, Gibco, UK).
Cytokine production assays A major trigger for cytokine secretion especially IL-12 in vivo is based on the T cell CD40L interaction with CD40 on DCs. The soluble CD40L molecule closely mimics the signals delivered to DC by CD40L expressed on the CD4+ T cells [26, 38]. To investigate the effect of CVID and controls sera on cytokine production by MDCs, these cells were stimulated with 1 µg/ml CD40L (Alexis, Lausen, Switzerland) in 24-well plates (Greiner Bio-one, CELL STAR, Frickenhausen, Germany) for 24 hrs and cell free supernatant were collected and used for cytokine assays (IL-10, IL-12 and IL-18) using Sandwich ELISA kits obtained from BMS (Bender MedSystems GmbH, Vienna, Austria). Cytokine production by MDC-stimulated T cells was measured in supernatants collected 48 hr after co-culturing of MDCs and allogeneic PBMCs. Cytokine cocentration (IFN-g and IL-4) measured in the supernatants using Sandwich ELISA kits obtained from BMS (Bender MedSystems GmbH, Vienna, Austria).
Statistical analysis One-Sample Kolmogorov-Smirnov Test was performed in order to test normal distribution of quantitative variables in subgroups of qualitative variables. Parametric test were used for variables with normal distribution. Statistical analysis was performed by unpaired t or one-way ANOVA tests with Welch`s or Tukey corrections, respectively using GraphPad-InstatTM software. p values less than 0.05 were considered significant. Results were expressed as mean ± standard deviation (SD).
Vol. 56, 2007
Altered dendritic cell function in response to sera of common variable immunodeficiency patients
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Table 1. General characteristics and immunological parameters of CVID patients and general features of controls enrolled in this study. CVID patients had more than two fold decreases in the mean values of at least two immunoglobulin super families but normal frequency of CD4+, CD8+ T– and CD19+ B-lymphocytes in peripheral blood. Normal controls Patients and Controls
Sex
Age (years)
Serum IgG mg/dl
Serum IgA mg/dl
Serum IgM mg/dl
CD4%
CD8%
CD19%
CD4/CD8 ratio
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 C1 C2 C3 C4 C5 C6 C7 C8 Reference Values
Male Male Male Female Male Male Female Male Male Female Male Female Male Male Female Male Male Male
26 27 12 55 8 10 11 47 14 27 27 30 24 27 25 40 14 30
80 233 225 170 140 469 114 76 103 160 ND* ND* ND* ND* ND* ND* ND* ND*
17 4 70 0 13 36 52 0 28 50 ND* ND* ND* ND* ND* ND* ND* ND*
8 4 50 0 14 0 30 11 30 68 ND* ND* ND* ND* ND* ND* ND* ND*
38 41.56 29 40 31 29.1 35.23 44.14 31.7 44.14 ND* ND* ND* ND* ND* ND* ND* ND*
31 45.7 23 45 25 38.99 31.29 31.9 34.2 31.98 ND* ND* ND* ND* ND* ND* ND* ND*
9.1 6.37 10 10.7 12 7.08 24.9 9.78 31.4 9.7 ND* ND* ND* ND* ND* ND* ND* ND*
1.22 0.9 1.26 0.89 1.24 0.74 1.12 1.38 0.93 1.38 ND* ND* ND* ND* ND* ND* ND* ND*
639–1349
70–312
56–352
30–50
20–35
6–19
0.7–1.9
* ND: Not Defined
Table 2. Immunophenotyping of monocytes and MDCs. MDCs are from 8 normal donors, and the serum from different subjects (10 CVID and 8 controls). MDCs cultured in the presence of CVID patient’s sera expressed lower levels of HLA-DR on their surface as compared to those cultured in the presence of control sera in the maturation phase (two-tailed p value of 0.0136, one sample t test). Maturation of MDCs leads to significant up regulation of CD86 surface expression independent on the type of serum source (CVID or control) in the culture (p value
