1 Departments of Molecular Immunogenetics and
2 Rheumatology, 5th Floor, Thomas Guy House, Guy's, King's and St Thomas' School of Medicine, Guy's Hospital, London SE1 9RT, UK,
3 Department of Rheumatology, Westmead Hospital, NSW 2145, Australia and
4 H. Lee Moffitt Cancer Center and Veteran's Administration Hospital, Departments of Medicine and Microbiology/Immunology, University of South Florida, 12902 Magnolia Drive, Suite 3157, Tampa, FL, USA
Correspondence to:
J. S. Lanchbury, Molecular Immunogenetics, 5th Floor, Thomas Guy House, Guy's, King's and St Thomas' School of Medicine, Guy's Hospital, London SE1 9RT, UK.
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Abstract |
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Methods. Thirty-five patients with Felty's were studied, along with 18 patients with LGL syndrome and arthritis, 17 patients with LGL syndrome but no arthritis, and 128 controls. The polymorphism was typed by polymerase chain reaction followed by digestion with the restriction enzyme MvaI.
Results. No significant difference was found in genotype or allele frequencies between the groups.
Conclusion. This promoter polymorphism is not a significant risk factor responsible for the LGL expansions seen in Felty's and LGL syndromes. Abnormal, constitutive expression of Fas ligand may be more relevant to the aetiology of these diseases.
KEY WORDS: Felty's syndrome, Large granular lymphocyte, Fas, Polymorphism, Rheumatoid arthritis, Fas ligand.
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Introduction |
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One explanation for the occurrence of T-cell expansions and autoimmune disease in these patients is a failure of apoptosis of self-reactive T cells [4]. There are several pathways by which a lymphocyte might undergo apoptosis. One of these involves Fas/Fas ligand interactions. Fas is a type I transmembrane protein that belongs to the tumour necrosis factor receptor superfamily, and maps to the long arm of chromosome 10q23 in humans [5]. It mediates apoptosis induced by Fas ligand. One important animal model supports a role for the Fas gene in these diseases. The MRL-lpr/lpr mouse model of autoimmune disease, like FS and LGL syndrome, is characterized by massive T-cell proliferation, arthritis and splenomegaly [6]. It is caused by a 5.3 kb insertion of DNA within the second intron of the Fas gene, which results in abnormal Fas expression [7]. Children with defective Fas genes have also been identified, with various mutations leading to a failure of surface expression of Fas, or to normal expression of a non-functional molecule [8, 9]. These patients have phenotypes very similar to those of the lpr mice, with autoimmune disease and lymphoproliferation.
Two polymorphisms have been described recently within the Fas gene promoter [10]. One of these is an A to G substitution at nucleotide position -670 in the enhancer region. This creates an MvaI restriction fragment length polymorphism (RFLP), and abolishes the binding site of the nuclear transcription element GAS. Although the functional significance of this polymorphism is as yet unknown, its location implies that it might be associated with altered Fas gene transcription. The resemblance between the human and murine diseases caused by mutations in the Fas gene and FS/LGL syndromes stimulated us to study the role of Fas promoter polymorphism in our patients.
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Methods |
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The LGL syndrome was defined as the presence of >1x109 LGL/l in the peripheral blood, persistent for at least 6 months. LGL were defined either by morphology or by immunophenotyping by flow cytometry (FACS) using fluorescent antibodies to CD3 and CD57, as described previously [12]. Since lymphopenia can be present in the LGL syndrome, an alternative criterion was the presence of LGL comprising >25% of lymphocytes. Eighteen patients with LGL and arthritis were studied (seven female, 11 male, mean age 72 yr, range 6385), eight of whom have been described previously [3]. A further two patients were recruited in the UK. Eight patients from the USA were studied, and these have been described previously [13]. All fulfilled the ACR criteria for RA.
Seventeen patients with LGL, but no arthritis, were studied (nine female, eight male, mean age 69 yr, range 5886). Four of these have been described previously [3], and a further two patients were recruited in the UK. Eleven USA patients were also studied, and these have been described previously [13]. All patients and controls were of North European Caucasoid origin.
A control panel of 128 individuals (female 61, male 67, mean age 38 yr, range 2460) was donated by the departments of tissue typing at Guy's and King's College Hospitals, London. All were healthy Caucasoid volunteers, most of whom worked in or near the respective laboratories, as described previously [14].
Molecular typing
DNA was obtained from peripheral venous blood by proteinase K digestion and phenol/chloroform extraction. The MvaI RFLP was studied by polymerase chain reaction (PCR) amplification followed by MvaI (Boehringer Mannheim) restriction enzyme digestion as described previously [10]. Briefly, PCR reactions were carried out in a final volume of 20 µl. Reaction mixtures consisted of 200 µM dNTP, 1 mM MgCl2 , 50 mM KCl2 , 10 mM TrisHCl (pH 8.5), 0.5 U Taq polymerase (Gibco), 0.25 µM primer and 60 ng DNA. Primers had the following sequences. Forward primer: 5'-CTACCT AAGAGCTATCTACCGTTC-3'; reverse primer: 5'-GG CTGTCCATGTTGTGGCTGC-3'. The reaction mixtures were amplified in a Perkin Elmer 9700 thermal cycler for 34 cycles, with each cycle consisting of 30 s at 94°C, 30 s at 58°C and 1 min at 72°C. The cycling was preceded by a single 6 min denaturation at 94°C, and followed by a single cycle of 10 min at 72°C for extension. After the reaction, 8 µl of each sample were digested overnight at 37°C with 1 µl of MvaI restriction enzyme and 1 µl of buffer supplied by the manufacturer. The product was run on a 3% low-melting-point agarose gel for 30 min at 150 V, stained with ethidium bromide and visualized with UV transillumination (Eagle Eye, Stratagene). Two polymorphic alleles, allele 1 (189 bp) and allele 2 (233 bp), were produced depending on the presence of a G or A allele at position -670 (see Fig. 1).
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Results |
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Conclusions |
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There has been some controversy regarding the applicability of data from mouse models and in vitro experiments on cell lines to apoptosis in normal circulating human T cells. `Resting' CD45RO- T cells do not express Fas, but Fas is upregulated on stimulation in vitro. However, freshly isolated Fas+ human T cells do not undergo apoptosis after exposure to Fas stimulating antibodies that are effective on Fas+ cell lines unless subject to prior stimulation [15]. This led to the view that Fas on human T cells was non-functional. Recent data have shed new light on this area [16]. It is now clear that membrane-bound FasL does induce apoptosis in freshly isolated human T cells, whereas soluble FasL or stimulating antibodies do not. Indeed, soluble FasL appears protective in this regard [16]. The stimulus has to be given in a physiologically appropriate form to have an effect. Hence, genetic polymorphisms that give rise to greater or lesser Fas expression on human T cells could indeed predispose to or protect from autoimmune disease.
Despite the resemblance between FS/LGL syndrome and the lpr mouse model [7] and Fas-deficient humans [8, 9], Fas expression on LGL is not decreased. On the contrary, LGL from FS (manuscript in preparation) and LGL syndrome [17] patients express Fas to a higher degree than LGL from age-matched controls. Recent evidence suggests that the Fas receptor in leukaemic LGL transduces the apoptotic signal less effectively than normal LGL, rendering them less susceptible to apoptosis [17]. However, human equivalents of the mutations in murine Fas were not found in that study.
The simple model in which lack of Fas expression by LGL leads to failure of peripheral clonal deletion is clearly inadequate. Indeed, one explanation is the converse of this model: abnormal, constitutive expression of FasL. Two groups have noted this in leukaemic LGL [18, 19]. Constitutive FasL expression has also been noted in melanoma cells [20] and is thought to mediate immune privilege. In this model, about which there has been some controversy [21, 22], Fas+ cytotoxic T cells in the vicinity of FasL-expressing cells are killed by apoptosis. Hence, polymorphisms in the regulatory region of the FasL gene might reveal more about the aetiopathogenesis of Felty's and LGL syndromes. Various other proteins have apoptosis-inducing (granzymes and tumour necrosis factor alpha) and inhibiting (Bcl-2) activities [23], and abnormalities in their expression will also merit study in these diseases.
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Acknowledgments |
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References |
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