Abstract
CVI is a primary immunodeficiency characterized by a failure of B cell differentiation associated with an array of T cell defects, such as enhanced T cell apoptosis. In this study we investigated the mechanisms underlying CVI enhanced T cell death. We analysed both the expression of Fas using flow cytometry techniques and the expression of FasL mRNA using RT-PCR in CVI T cells. We could not find any significant differences between CVI and normal subjects with regard to Fas expression, although there was a subgroup of CVI patients with very high Fas expression which was accompanied by an up-regulation of FasL mRNA. However, attemps to induce Fas-mediated apoptosis in these high Fas expressing cells, as evaluated by propidium iodide staining and APO2·7 staining, were unsuccessful. We also investigated intracellular levels of Bcl-2, bcl-xl and bax in CD4+ and CD8+ CVI T cells, as well as the bax/Bcl-2 ratio, using flow cytometry techniques but could not detect any differences between CVI and normal subjects. Finally we analysed TNF-RI and TNF-RII mRNA expression in CD4+ and CD8+ CVI T cells using semiquantitative RT-PCR and found a significant increase in expression of both TNF-Rs in CD4+ T cells from CVI patients. Our data suggest that the increased expression of both TNF-Rs on T cells may be one of the mechanisms responsible for the accelerated T cell apoptosis in CVI.
Keywords: apoptosis, Bcl-2 protein family, CVI, Fas/FasL, T lymphocytes, TNF-Rs
Introduction
CVI is a primary immunodeficiency disease characterized by hypogammaglobulinaemia and recurrent bacterial infections [1,2]. Some patients demonstrate a failure in B cell differentiation with impaired secretion of immunoglobulins [3], while others have an array of T cell abnormalities, such as cutaneous anergy, decreased lymphocyte proliferation [4] and reduced production and/or expression of IL2, IL4, IL5 and IL10 in response to antigens and mitogens [5–8], suggesting an impairment in early signalling events following triggering of the TCR [9].
Programmed cell death (PCD) is a mechanism of cell death with distinct morphological features which results in the phagocytosis of the dying cell without the release of its contents [10]. Recently, T cell PCD has been studied extensively because it plays an important role in the development and mantainance of the immune system. Apoptosis is especially important for the deletion of autoreactive T and B lymphocytes [11]. Apoptosis is regulated by a number of gene products which either promote cell death or extend cell survival [12]. Fas (CD95) is a 48-kDa cell surface Ag which belongs to the TNF receptor/nerve growth factor receptor family and which mediates apoptosis in a wide variety of cell types [13]. Fas ligand (FasL) is a 40-kDa type II transmembrane protein which belongs to the TNF receptor/nerve growth factor family; FasL is predominantly expressed on activated T cells and it mediates cell death by cross-linking to the Fas receptor on apoptosis sensitive Fas+ cells [14]. The susceptibility of T cells to undergo apoptosis is also controlled by the family of Bcl-2 homologues [15]. Overexpression of Bcl-2 and the long form of its alternatively spliced homologue, Bcl-xl, enhances the survival of T cells that are induced to undergo apoptosis [16]. In contrast, bax, a 21-kDa protein, homodimerizes or heterodimerizes with Bcl-2 or bcl-xl to counter their antiapoptotic effect and so promote apoptosis [17]. TNF-α is a 17-k-Da pleiotropic cytokine produced by a large number of cell types and is able to induce cytotoxicity which resembles apoptosis because it is characterized by membrane blebbing and DNA fragmentation [18].
T lymphocytes from patients with CVI show increased levels of apoptosis involving both CD4+ and CD8+ T cells, in comparison with cells from normal subjects, both spontaneously and after γ-irradiation after short-term culture in vitro [19,20]. In order to clarify the possible pathogenic mechanism of this accelerated apoptosis, we evaluated Fas and FasL expression on CVI T cells, as well as the ability of Fas to signal apoptosis, in the same group of patients in which we have previously shown increased PCD [20]. We also investigated the expression of Bcl-2, bax and bcl-xl and of TNF-RI and TNF-RII in CD4+ and CD8+ subsets of CVI T cells.
Materials and methods
Patients
Forty CVI patients, age range 7–67 years, with well-documented CVI according to the diagnostic criteria of the WHO expert group for primary immunodeficiency diseases [1], were included in the study after informed consent was obtained. Specifically, these subjects had significantly reduced serum IgG and IgA and/or IgM levels. T and B cell numbers and the number of CD4+ and CD8+ T cells were determined for each subject. The range for CD4+ cells was 174–2490/mm3; the ratio of CD4/CD8 ranged from 0·33 to 4·64. All patients were free from acute or exacerbated chronic bacterial infections as well as from viral infections when studied. All patients were receiving monthly IG i.v. infusions but none were receiving corticosteroids. Blood samples were always collected before the monthly IG i.v. infusion was given. Healthy blood donors matched for age and and sex were studied in parallel with the patients.
Cell isolation and culture
Peripheral blood mononuclear cells (PBMC) were isolated from the patients' heparinized whole blood and from the buffy coats of the healthy donors by density gradient centrifugation over Ficoll Hypaque (Pharmacia, Piscataway, NJ, USA). T cells were obtained by rosetting with neuraminidase-treated (EN) sheep red blood cells (SRBC), lysis with NH4Cl and depletion of monocytes and macrophages by adherence to plastic. Fluorescence staining of the T cell population showed 93–98% CD3+ staining cells. T cells were cultured in RPMI 1640 medium supplemented with fetal calf serum (10%), l-glutamine (2 mm) and penicillin–streptomycin (100 U/ml, 100 µg/ml, Gibco, Grand Island, NY, USA). In some experiments CD4+ and CD8+ lymphocytes were isolated with conjugated aCD4 and aCD8 immunomagnetic beads (Dynal, Great Neck, NY, USA). Following the manufacturer's protocol, PBMCs were incubated with the specified beads for 30 min at 4°C on a rotating shaker. Cells bound to the beads were placed directly into Trizol Reagent (Gibco BRL) for RNA extraction. The positively selected subsets had a purity greater than 90% as analysed by flow cytometry.
Fas expression
PBMC were washed twice with PBS and stained with PeCy5-labelled anti-CD3 antibody (Coulter Corporation, Miami, FL, USA) and FITC-labelled anti-Fas (Pharmingen, San Diego, CA, USA) for 30 min at 4°C. FITC-labelled and PeCy5-labelled mouse IgG were used as isotype-matched background controls. Following incubation, the cells were washed twice with PBS with 1% BSA and 0·01% sodium azide and 5000 cells were acquired using flow cytometry (Epics Profile II, Coulter Corporation). Lymphocytes were gated and the percentage of double-positive cells (CD3+ Fas+) was determined.
Fas-induced apoptosis
PBMC freshly isolated were stimulated for 16 h with either 2 µg/ml CH-11, an anti-Fas IgM antibody (Upstate, Lake Placid, NY, USA) or a control IgM (Upstate). T cell death was then evaluated by propidium iodide inclusion. In some experiments, PBMC were stimulated with anti-CD3 (Ortho Diagnostics Ltd, Raritan, NJ, USA) at a final concentration of 25 ng/ml for 48 h and kept in IL2-containing (10 ng/ml) complete medium for 6 days. Then PBMC were washed and stimulated for 16 h with 2 µg/ml CH-11 (Upstate) or with a control IgM (Upstate) and T cell death evaluated by staining with APO 2·7.
Propidium iodide inclusion
Cells were washed in PBS and stained for 30 min with a FITC-conjugated anti-human CD3 monoclonal antibody (Coulter Corporation) or a non-binding, matched isotype control (Coulter Corporation). Cells were washed twice and re-suspended in 0·5 ml of PBS with 1% BSA, 0·01% sodium azide and PI (1 µg/ml, Sigma, St Louis, MO, USA). Samples were analysed using flow cytometry (Epics Profile II, Coulter Corporation). The percentage of cell death was determined by dividing the number of PI+CD3+ cells by the total number (PI+ and PI−) CD3+ cells. Forward light scatter characteristics of living cells were used to delete debris from the analyses.
Apo 2·7 staining
In some experiments the appearance of a mitochondrial membrane protein, APO2·7, was used as a marker to detect apoptotic cells [21]. Cells were washed twice in PBS and stained for 30 min with APO2·7-PECy5-conjugated antibody (Coulter) and either FITC-labelled anti-human CD4 or anti-human CD8 Ab (Coulter). FITC-labelled and PECy5-labelled mouse IgG served as isotype-matched background controls. Following incubation, cells were washed twice, resuspended in 0·5 ml of PBS with 1% BSA and 0·01% sodium azide, and analysed using flow cytometry (Facscalibur, Becton Dickinson).
Reverse transcriptase-PCR analysis of FasL, TNF-RI and TNF-RII
Total RNA was extracted from freshly isolated T cells using Trizol (Gibco BRL). RNA, 1 µg, was reverse transcribed at 42°C for 45 min in 20 µl buffer containing 10 mm Tris pH 8·3, 50 mm KCL, 5 mm MgCl2, 10 mm DTT, 1 mm each of dATP, dCTP, dGTP and dTTP, 20 U Rnasin (Promega, Madison, WI, USA), 0·1 mg oligo(dT)15 (Genelink, Thornwood, NY, USA) and 200 U M-MLV Reverse Transcriptase (Gibco). Reactions were stopped by heat inactivation at 99°C for 5 min and chilled on ice. Subsequently 1 µl of the cDNA product was amplified by PCR in 25 µl of 10 mm Tris pH 8·3, 50 mm KCl, 2 mm MgCl2, 200 mm each of dATP, dCTP, dGTP and dTTP in the presence of 6·5 pmol each of the sequence-specific primers and 1·25 U of Taq polymerase (AmpliTaq Gold-DNA polymerase, Perkin Elmer, Branchburg, NJ, USA). Amplification was performed for 32 cycles after 10 min initial denaturation at 95°C. Each cycle of PCR included 1 min of denaturation at 95°C, 1 min of annealing at 58°C (FasL) or 60°C (TNF-RI and TNF-RII) and 1 min of extension/synthesis at 72°C in a DNA thermal cycler (Programmable Thermal Controller PTC 100, MJ Research, INC Watertown, MA, USA). All PCR cycles were terminated with a final extension step at 72°C for 10 min. As a control β-actin amplification was performed under the same conditions. The sequence of the primers (Integrated DNA Technologies INC, Coralville, IA, USA) were (in a 5′−3′ orientation) as follows: β-actin: 5′-TGACGGGGTCACCCACACTGTGCCCATCTA and 3′-CTAGAAGCATTGCGGTGGACGATGGAGGG; FasL: 5′-ATTCTTTGTTACAGGCACCG and 3′-GAGTTGATTGTCA GGAAGCA; TNF-RI: 5′-ATTTGCTGTACCAAGTGCCACAA AGGAACC and 3′-GTCGATTTCCCACAAACAATGGAGTAG AGC; TNF-RII: 5′-GAATACTATGACCAGACAGCTCAGAT GTGC and 3′-TATCCGTGGATGAAGTCGTGTTGGAGAACG. Amplified products were analysed by 1·5% agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. The FasL product was 696 bp, the β-actin product was 661 bp, the TNF-RI product was 587 bp, the TNF-RII product was 403 bp. The bands were quantified using the Genequant program and the ratios β-actin/TNF-RI and β-actin/TNF-RII were calculated.
Analysis of intracellular Bcl-2, bax and bcl-xl expression
PBMC were washed twice with PBS and stained with PeCy5-labelled anti-human CD4 or anti-human CD8 antibody (Coulter Corporation) for 30 min at 4°C. They were then permeabilized using Permeafix (Ortho Diagnostics Ltd), washed with PBS containing 1% BSA and 0·01% sodium azide and incubated with FITC-labelled anti-Bcl-2 (Dako, Carpenteria, CA, USA) or with anti-bax rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with anti-bcl-xl rabbit polyclonal antibodies (Santa Cruz Biotechnology). FITC-labelled goat anti-rabbit immunoglobulins (Dako) were used as the secondary antibody for bax and bcl-xl staining. FITC-labelled and PeCy5-labelled mouse IgG were used as isotype-matched background controls. Following incubation the cells were washed once with PBS containing 1% BSA and 0·01% sodium azide and 5000 cells were acquired using flow cytometry (Becton and Dickinson, Lincoln Park, NJ, USA). Because Bcl-2, bax and bcl-xl are intracellular antigens that are present in all lymphocytes, changes in their expression were measured by changes in fluorescence intensity. Therefore lymphocytes were gated and the fluorescence intensity of double-positive cells as measured by mean fluorescence channel (MFC) number was determined.
Statistical analysis
The Mann–Whitney U-test was used to compare data obtained in CVI subjects with that obtained from normal controls.
Results
Fas/FasL expression in CVI
To clarify whether increased Fas expression might contribute to the accelerated T cell death that we found in a group of CVI patients [20], we analysed and compared freshly isolated MNC from the same 40 CVI patients and 32 normal subjects for Fas expression on T cells by flow cytometry. As shown in Fig. 1, T cells from both CVI and normal subjects had a variable expression of Fas. Due to this variability, we did not find any significant difference between CVI and controls, although there was a subgroup of CVI patients in whom the percentage of Fas+ T cells was clearly higher than in the normal subjects. In order to evaluate whether increased Fas expression was accompanied by increased FasL mRNA expression, we investigated FasL mRNA expression in normal and CVI T cells by RT-PCR. Figure 2 shows that in normal subjects FasL mRNA was undetectable, whereas it was expressed in those CVI patients with high Fas expression but was undetectable in the others. When we analysed the immunological and clinical characteristics of the subgroup of CVI patients, we were unable to correlate the increased Fas and FasL mRNA expression with either a reduced T cell count or any clinical manifestation.
Fig. 1.
Percentage of Fas expression on T cells from 40 CVI patients and 32 normal subjects. n.s. not significant. Horizontal lines represent the median values.
Fig. 2.
FasL mRNA expression in lymphocytes from six normal subjects (Nl) and eight CVI patients (P1-P8). + + positive control (PBMC cultured in the presence of PHA and IL2 for 4 days); C = negative control; MW = molecular weight marker.
Fas-induced cell death in CVI
To determine whether enhanced expression of Fas and FasL mRNA correlates with enhanced susceptibility of T cells to undergo Fas-induced apoptosis in CVI, we treated the PBMC of 5 ‘high Fas expression’ CVI patients with anti-Fas (2 µg/ml) MoAb or its isotype-matched control for 16 h. In the same experiment we used cultured Jurkat T cells as a positive control. The treatment with anti-Fas failed to induce cell death in CVI T cells (median CD3+ PI+ without treatment = 2·5% range 2–2·6; median CD3+ PI+ after anti-Fas = 2·6% range 2·4–4·7; median CD3+ PI+ after isotype-matched control = 2·5% range 2–3·4; in the control group median CD3+ PI+ without treatment = 1·2% range 0·8–1·6; median CD3+ PI+ after anti-Fas = 1·3% range 1–1·5%; median CD3+ PI+ after isotype-matched control = 1·2% range 0·8–1·6%). We also cultured CVI PBMC for 6 days in IL-2 containing medium after an initial stimulation with anti-CD3 MoAb for 48 h and then stimulated the cells with anti-Fas for 16 h. As shown in Table 1, after Fas ligation both CD4+ and CD8+ T cells from CVI and normals underwent apoptosis as evaluated by examining the appearance of a 38-kDa mitochondrial membrane protein (APO2·7), an early marker of apoptosis (21); however, the percentage of CD4+ APO2·7+ and CD8+ APO2·7+ cells in CVI was not higher than in the control group.
Table 1.
Fas-induced cell death in activated T cells from CVI and controls†
| CH-11 | IgM | |||
|---|---|---|---|---|
| CD4+ APO2·7+ | CD8+ APO2·7+ | CD4+ APO2·7+ | CD8+ APO2·7+ | |
| CVI(4) | 33(25–41) | 36(31–39) | 15(13–19) | 16(14–19) |
| Controls(3) | 35(31–43) | 39(37–43) | 18(17–22) | 17(13–17) |
Data expressed as median and range. T lymphocytes were activated with anti-CD3 and IL2 for 6 days and then stimulated with either CH-11 or with an isotype control for 16 h. CD4+ and CD8+ percentage death was evaluated through APO2·7 staining. No difference was found between CVI and controls.
Bcl-2, bax and bcl-xl expression
To determine whether an imbalance between pro-apoptotic and anti-apoptotic proteins might contribute to the accelerated T cell apoptosis in CVI, we evaluated Bcl-2, bax and bcl-xl expression in both CD4+ and CD8+ T cells as well as the bax/Bcl-2 ratio. As shown in Table 2, we could not find any difference between CVI T cells and those from normals.
Table 2.
Bcl-2, bax and bcl-xl expression and bax/Bcl-2 ratio in T lymphocytes from CVI and controls†
| Controls (8) | CVI (6) | |||
|---|---|---|---|---|
| CD4+ | CD8+ | CD4+ | CD8+ | |
| Bcl-2 | 423 | 407 | 471 | 444 |
| (394–527) | (382–466) | (347–534) | (341–519) | |
| Bax | 914 | 937 | 605 | 626 |
| (555–1366) | (578–1156) | (269–981) | (252–950) | |
| Bcl-xl | 814 | 738 | 771 | 671 |
| (370–1295) | (336–1080) | (507–1070) | (443–927) | |
| Ratio Bax/Bcl-2 | 2·2 | 2·2 | 1·5 | 1·5 |
| (1·5–3·5) | (1·4–2·8) | (0·8–2·7) | (0·5–2·8) | |
Data expressed as median and range. In freshly isolated PBMC from CVI and controls mean fluorescence intensity (MFI) for Bcl-2, bax and bcl-xl expression has been evaluated. No difference was found between CVI and normals.
TNF-RI and TNF-RII mRNA in CVI
To clarify whether increased TNF-RI and/or TNF-RII expression might play a role in the accelerated T cell death in CVI, we analysed TNF-RI and TNF-RII mRNA in 11 CVI patients and in 10 normal subjects using semiquantitative RT-PCR. Figures 3 and 4 show the data from six CVI patients and six normal controls with regard to TNF-RI and TNF-RII mRNA, respectively, in CD4+ T cells. Table 3 summarizes the results showing that CD4+ T cells from CVI patients had significantly higher levels of both TNF-RI and TNF-RII mRNA than the control group, whereas there was no significant difference between patients and normals regarding TNF-RI/II expression in CD8+ T cells. We were unable to correlate the increased TNF-RI and TNF-RII mRNA expression in CD4+ T cells in CVI with either a reduced CD4+ cell count or any clinical manifestation.
Fig. 3.
TNF-RI and β-actin mRNA expression in CD4+ T cells from six CVI patients and six normal controls. N = negative control, M = molecular weight marker.
Fig. 4.
TNF-RII and β-actin mRNA expression in CD4+ T cells from six CVI patients and six normal controls. N = negative control, M = molecular weight marker.
Table 3.
TNF-RI and TNF-RII mRNA expression in T lymphocytes from CVI and controls†
| Controls (10) | CVI (11) | |||
|---|---|---|---|---|
| CD4+ | CD8+ | CD4+ | CD8+ | |
| TNF-RI | 10 | 100 | 3·1* | 2·8 |
| (2·7–100) | (1·4–100) | (1·9–100) | (0·9–100) | |
| TNF-RII | 100 | 100 | 1·15* | 1·9 |
| (1–100) | (0·6–100) | (0·1–100) | (0·5–100) | |
Data expressed as median and range. TNF-RI and TNF-RII RT-PCR was performed in CD4+ and CD8+ T cells, isolated using magnetic beads. β-actin/TNF-RI and β-actin/TNF-RII ratios were calculated. CVI CD4+ T cells express higher levels of TNF-RI and TNF-RII than controls (P < 0·05). A ratio of 100 represents no TNF-R expression; the lower the ratio, the greater the level of TNF-R expression.
Discussion
CVI is a disease characterized by low levels of serum Igs due to a failure of B cells to produce antibodies. There is good evidence that in most patients the B cells are intrinsically normal and that the defect lies in the inability of T cells to provide the appropriate signals. We have shown recently that CVI T cells undergo enhanced apoptosis both spontaneously and after γ-irradiation [20]. The aim of this study was to clarify the pathogenic mechanisms underlying T cell apoptosis in the same group of CVI patients as those in whom we described accelerated T cell death. First we investigated the Fas/FasL pathway, because signalling through Fas plays a central role in the induction of apoptosis, especially in chronically activated T cells, helping to terminate an ongoing immune response [14]. This is confirmed by murine models where genetic defects in either Fas or FasL result in the abnormal accumulation of double negative T cells because of an impairment in physiological T cell deletion [22]. Due to the fact that several reports have shown persistent immune activation in CVI [23], it was reasonable to suppose an involvement of the Fas/FasL pathway in CVI T cell apoptosis. However, we could not find any differences between Fas expression on CVI and normal T cells, although a subgroup of CVI patients had T cells with high levels of Fas accompanied by an up-regulation of FasL mRNA. This differs from that reported by Iglesias et al., who found high Fas expression on T cells in CVI patients with a reduced count of CD4+ lymphocytes [19]; we were unable to correlate Fas and FasL expression with either T cell count or any clinical manifestation.
To examine whether increased expression of Fas and FasL in the subgroup of CVI patients correlated with an increased susceptibility to apoptosis, we tried to induce Fas-mediated apoptosis of CVI ‘high Fas expression’ T cells, comparing their behaviour with normal T cells. In line with normal subjects where freshly isolated T cells are resistant to apoptosis [24], CVI T cells were insensitive to the cross-linking of the Fas molecule despite the high level of Fas expression. After activation with anti-CD3 and IL2, both normal and CVI T cells underwent Fas-mediated apoptosis but we could not show any difference between the two groups, although we cannot exclude that the addition of IL2 to the culture medium after CD3-activation promotes T cell survival, abrogating any possible difference between CVI and normals. Therefore the Fas/FasL system does not seem to contribute to the enhanced apoptosis of CVI T cells and the increase in Fas+ cell numbers within the CD3+ T cell population together with FasL mRNA up-regulation observed in some CVI patients may be due to a shift from a naive to memory T cell phenotype which results in increased Fas expression and resistance to Fas-mediated apoptosis [25].
Other molecules besides Fas are involved in the apoptotic pathway, especially Bcl-2 gene family members [15]. Most CVI patients show reduced in vivo IL2 production [5–7] and IL2 plays an important role in preventing cell death up-regulating anti-apoptotic Bcl-2 family proteins [26]. Therefore we hypothesized that the chronic cytokine deprivation together with the deficient co-stimulation which occurs in CVI [20] might contribute to the accelerated T cell death by down-regulating Bcl-2 and bcl-xl and up-regulating bax. However, we could not find any differences in Bcl-2, bax and bcl-xl expression or in the bax/Bcl-2 ratio in CD4+ and CD8+ subsets between CVI and normal subjects, nor Bcl-2 down-modulation during CVI T cell culture in vitro (data not shown). The relationship between Bcl-2 family members and the Fas/FasL pathway is controversial because it depends on the cell type. However, at least in human T cells, the bax/Bcl-2 ratio correlates with susceptibilty to anti-Fas induced apoptosis [27] and the normal bax/Bcl-2 ratio in CVI T cells may contribute to the resistance to anti-Fas induced apoptosis in spite of the high Fas expression detected in some patients.
Finally, several reports have shown an activation of the TNF-α system in CVI where increased serum levels of TNF-α accompanied by glutathione depletion in CD4+ lymphocytes have been reported [28]. TNF-α is a well-known inducer of apoptosis which exerts its effects by binding to two cell surface receptors: TNF-RI and TNF-RII [18]. Controversial data exist with regard to the role of TNF-Rs in the induction of apoptosis: according to some authors TNF-RI triggers cell death and TNF-RII has an anti-apoptotic effect, whereas according to others TNF-RII plays the main role in the induction of apoptosis, especially in the murine model [18,29]. More recently, it has been reported that T lymphocytes from aged humans have an increased susceptibility to apoptosis accompanied by an increased constitutive expression of TNF-RI and a decreased expression of TNF-RII [30].
Our data show that both TNF-RI and TNF-RII mRNA expression is significantly increased on CVI CD4+ T cells, without any correlation with CD4+ T cell count or any clinical manifestation. This overexpression may contribute to the accelerated T cell death enhancing T cell susceptibility to increased and/or normal levels of circulating TNF-α. However, the functional role of both TNF-Rs in CVI remains to be established.
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