The FAS/cd95 promoter single-nucleotide polymorphism -670 A/G and lupus erythematosus.

An increased level of circulating nuclear antigens caused by apoptosis is thought to be responsible for the production of autoantibodies in lupus erythematosus (LE). The presentation of these antigens to immunologically competent cells may trigger systemic autoimmunity. The influence of a functional single-nucleotide polymorphism at position -670 in the promoter of the apoptosis gene FAS on susceptibility to autoimmune diseases including systemic LE has been a controversial subject. Although it has not yet been possible to assign any particular allele or genotype to the control of FAS expression, this polymorphism has been described to be associated with several autoimmune diseases including LE. When we compared the FAS -670 A/G genotypes of 107 German patients with LE and those of 96 healthy controls, we found a trend for association between LE and the homozygous A genotype in the patient group. This finding suggests that apoptosis may contribute to development of autoimmune reactions and that FAS function might be relevant for LE.

Deletion of the late cornified envelope genes LCE3B and LCE3C may promote chronic hand eczema with allergic contact dermatitis.

BACKGROUND: Genetically determined defects in epidermal skin barrier function may contribute to the development of irritant and/or allergic contact dermatitis in chronic hand eczema (CHE). OBJECTIVES: To assess whether a deletion in the late cornified envelope genes LCE3B and LCE3C may constitute a genetic predisposition for the development of CHE or any of its subtypes. PATIENTS AND METHODS: A total of 153 German patients with clearly defined CHE subtypes and 268 healthy individuals were screened for the deletion LCE3C_LCE3B-del by allele-specific polymerase chain reaction. RESULTS: Classification of the patients by etiologic subtypes revealed an association between the LCE3C_LCE3B-del allele and CHE due to allergic contact dermatitis. In this subtype, 19/37 patients (51.4%) were homozygous deletion carriers, 11/37 (29.7%) were heterozygous carriers, and just 7/37 (18.9%) were wild-type individuals. Compared to the other CHE subgroups and the healthy control group (homozygous, 88/268 [32.83%]; heterozygous, 133/268 [49.63%]; and wild-type, 47/268 [17.54%]), the prevalence of LCE3C_LCE3B-del in these patients reached statistical significance (P = .03977), as did homozygous deletion carrier status (P = .01044 for other subtypes and P = .02695 for controls). CONCLUSIONS: A deletion of LCE genes may promote the development of allergic contact dermatitis, which is a form of CHE involving delayed-type hypersensitivity.

Filaggrin mutations may confer susceptibility to chronic hand eczema characterized by combined allergic and irritant contact dermatitis.

BACKGROUND: The pathogenesis of chronic hand eczema (CHE) is multifactorial and involves both endogenous predisposition and environmental triggers. OBJECTIVES: Filaggrin is a structural protein of the cornified envelope and important for the formation of the epidermal skin barrier. The aim of this investigation was to evaluate the role of mutations in the filaggrin gene (FLG) in the development of CHE. METHODS: In total, 122 German patients with clearly defined CHE subtypes were screened for the FLG variants R501X and 2282del4 by polymerase chain reaction and restriction enzyme digest analysis. The prevalence of these variants in CHE patients was compared with that in 95 healthy individuals. RESULTS: Overall, allele frequency and the number of mutation carriers were similar in both the CHE and control groups. When classified according to clearly defined CHE subtypes, however, the nonfunctional FLG variants showed an association with CHE involving an aetiological combination of contact allergy and irritant factors [P = 0.04; P (exact test) = 0.06; P (difference in rates) = 0.09; 95% confidence interval (CI) 0-56.8)], or with excessive daily exposure to water and irritants [P = 0.003; P (difference in rates) < 0.001; 95% CI 29.3-67.9]. CONCLUSION: Heterozygosity for nonfunctional mutations in the FLG gene may contribute to the manifestation and maintenance of a particular CHE subtype that is characterized by the combination of allergic and irritant contact dermatitis.

Association of MICA-TM and MICB C1_2_A microsatellite polymorphisms with tumor progression in patients with colorectal cancer.

PURPOSE: The major histocompatibility complex class I related A (MICA) and MICB molecules are ligands of NKG2D receptors on natural killer cells, gamma/delta T cells, and CD8ass T cells that mediate host antitumor immune response. The role of MICA-TM and MICB C1_2_A alleles in patients with colorectal cancer has not yet been investigated. METHODS: We have analyzed the MICA-TM and MICB C1_2_A polymorphisms in colorectal cancer patients (n = 79) by polymerase chain reaction amplification, subsequent electrophoresis, and sequencing in comparison to a previously analyzed cohort of healthy controls (n = 306). Allele frequencies obtained for MICA-TM and MICB C1_2_A were compared to histopathological data regarding tumor invasion, disease progression, microsatellite instability, and the presence of KRAS mutations (codon 12) and analyzed for possible impact on tumor-related survival (n = 61). RESULTS: Allele frequencies of MICA-TM and MICB C1_2_A polymorphisms were not different in patients with colorectal cancer in comparison to normal controls. In colorectal cancer patients, MICA-TM A4 allele was directly and MICA-TM A5 allele was inversely associated with lymph node involvement and advanced UICC stages. Tumor-related survival in colorectal cancer patients was significantly reduced in the presence of the MICA-TM A4 allele (p = 0.015). In patients with microsatellite stable tumors, survival was reduced in association with the MICA-TM A4 allele (p = 0.006) and MICA-TM A9 allele (p = 0.034), but increased in patients showing the MICA-TM A5 allele (p = 0.042). CONCLUSIONS: Specific MICA-TM alleles seem to influence tumor progression and midterm survival of patients with colorectal cancer, indicating an important role of host innate immune predisposition involving NKG2D mediated antitumor response.

MICA*055: a new allele with eight GCT repeats in the exon 5 microsatellite.

The new allele MICA*055 contains eight GCT repeats within the exon 5 MICA-TM microsatellite polymorphism.

Soluble HLA-G promotes Th1-type cytokine production by cytokine-activated uterine and peripheral natural killer cells.

Soluble forms of HLA-G (sHLA-G) have been implicated in immune regulation. Fetal trophoblast cells are a prime source of HLA-G. Hence, an interaction between sHLA-G and uterine lymphocytes in the decidual tissues can easily be envisaged. These lymphocytes, when properly activated, are implicated in successful trophoblast invasion, placental maturation and maintenance of pregnancy. However, so far, no data are available on the effect of sHLA-G on the function and phenotype of these cells. Herein, we used a recombinant sHLA-G construct to determine the effect of sHLA-G on uterine lymphocyte cells present in endometrium at the time that it is optimally receptive to trophoblast invasion. In addition, we ascertained the effect of sHLA-G on peripheral lymphocytes. We found that upon co-culture with sHLA-G, proliferation of unfractionated IL-15-stimulated uterine mononuclear cells (UMCs) was inhibited. However, sHLA-G increased both interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha production by these cells. Vascular endothelial growth factor (VEGF) production was reduced. Notably, in contrast to membrane-bound HLA-G, sHLA-G did not affect the natural cytolytic activity of UMCs. Similarly, sHLA-G inhibited proliferation but stimulated pro-inflammatory cytokine production by cytokine-activated, unfractionated peripheral blood mononuclear cells (PBMCs). In addition, we showed that the overall inhibitory effect of sHLA-G on proliferation of the whole cell population could be ascribed to selective inhibition of CD4(+) T cells. In contrast, sHLA-G induced proliferation and IFN-gamma production by both uterine and peripheral natural killer (NK) cells. In conclusion, our data show that the sHLA-G modulates both UMC and PBMC function. sHLA-G, by promoting IFN-gamma production by uterine NK cells, may contribute to vascular remodelling of spiral arteries to allow for successful embryo implantation.

Binding analysis of HLA-G specific antibodies to hematopoietic cells isolated from leukemia patients.

Expression of HLA-G on the surface of malignant hematopoietic cells isolated from leukemia patients was analyzed by flow cytometry using monoclonal antibodies (mAbs) recognizing both, intact HLA-G complex (87G, 01G and MEM-G9) as well as HLA-G free heavy chain (4H84, MEM-G/1 and MEM-G/2). Prerequisite of HLA-G detection by mAbs specific to free heavy chain was mild acid treatment, which dissociates intact HLA-G complex. All mAbs, with the exception of 4H84 mAb, did not indicate the presence of HLA-G antigen in leukemia cells. Positive staining with 4H84 mAb was detected in acid-treated cells isolated from 16 out of 30 patients. Intensity of staining increased after IFN-g pre-incubation in most cases. Immunoblot analyses and RT-PCR, however, failed to detect HLA-G antigen or HLA-G transcripts in cells that bind 4H84 mAb after acid-treatment. The binding of 4H84 mAb can be explained by the acid-induced cross-reactivity of this HLA-G specific mAb with classical HLA class I molecules [15]. The results described here further demonstrate that the HLA-G molecule is not expressed in freshly isolated human leukemia cells.

Th1- and Th2-like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E.

During normal early pregnancy, a particular immune environment in the decidua and the expression of non-classical HLA-G and HLA-E molecules on the invading trophoblast are assumed to be essential for the tolerance of the fetus. To assess whether HLA-G and HLA-E influence the cytokine production of their putative target cells [large granular lymphocytes (LGL)], we analysed the concentrations of tumour necrosis factor (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-10, IL-13 and granulocyte-macrophage colony stimulating factor (GM-CSF) in supernatants of isolated first trimester LGL co-cultured with HLA-G or HLA-E transfected K-562 leukaemia cells lacking the classical HLA class I and II molecules. In comparison with that observed with untransfected K-562 cells, co-culture of LGL with HLA-G-expressing cells significantly reduced the concentration of all cytokines investigated (TNF-alpha, IL-10 and GM-CSF, P < 0.01; IFN-gamma and IL-13, P < 0.05). In contrast, co-culture of LGL with HLA-E-expressing cells significantly (P < 0.01) decreased only IL-10 production, although a strong tendency towards reduced IL-13 levels was also observed. In the co-culture system presented, membrane-bound HLA-G and, to a lesser extent, HLA-E expression affected cytokine release by decidual LGL in a manner not consistent with the Th1/Th2 paradigm. In conclusion, our data are indicative of a general immune-suppressive effect of HLA-G on LGL activity.

Linkage disequilibria between HLA-B, C1_4_1, MICA and MICB.

The polymorphisms of MICA exon 5 (5 alleles), MICB intron 1 (13 alleles), C1_4_1 (6 alleles), HLA-B (29 alleles) and HLA-A (15 alleles) were investigated in a healthy German population. Sequencing was performed for the MICB alleles CA14, CA15, CA17, CA23 and CA26 isolated from different cell lines. Variation to the published sequence was observed for CA14, CA15 and for CA17. At the C1_4_1 locus a new allele (CAAA)9 was identified and confirmed by sequencing. Linkage disequilibria were investigated for two-point- and three-point-haplotypes. Although the average relative delta value correlates loosely with the physical distance from HLA-B to MICB: HLA-B-C1_4_1>HLA-B-MICA>HLA-B-MICB, there are several exceptions to this rule. Analyzing three-point-haplotypes for the segment MICB to HLA-A a wide variation of linkage disequilibria for some of the classical HLA-A, B haplotypes has been observed. While the HLA-A1, B8 haplotype displays strong relative delta values over the entire distance from HLA-A to MICB, other haplotypes have linkage disequilibria only in a limited region.

MICA, MICB and C1_4_1 polymorphism in Crohn's disease and ulcerative colitis.

MICA and MICB belong to a multicopy gene family located in the major histocompatibility complex (MHC) class I region near the HLA-B gene. They encode for MHC class I molecules, which are induced by stress factors like infection, heat shock or neoplastic transformation and which are mainly expressed on gastrointestinal epithelium. They are recognized by gammadelta T lymphocytes and natural killer (NK) cells. Additionally they are located within a linkage region on chromosome 6p around HLA-B and TNFalpha. Thus the polymorphic MICA and MICB genes are excellent candidate genes for providing the genetic background of inflammatory bowel disease. A strong association of allele A6 of the MICA exon 5 trinucleotide microsatellite polymorphism with ulcerative colitis has been found in Japanese patients. Therefore, we have analysed the MICA exon 5 polymorphism, the MICB intron 1 dinucleotide polymorphism and in addition the tetranucleotide polymorphism C1_4_1, which is located between the MICA gene and the HLA-B gene, in patients of Caucasoid origin with Crohn's disease (n=94) and ulcerative colitis (n=94). In this study we could not find any associations of particular alleles of the MICA, MICB and C1_4_1 polymorphisms with Crohn's disease or ulcerative colitis. We could also not discover any associations of specific two-point or three-point haplotypes with these diseases. Thus it is unlikely that the MICA and MICB genes are involved in causing susceptibility for inflammatory bowel disease, although it cannot be excluded that a weak association could be identified in a larger patient sample.

Muscle fibers in inflammatory myopathies and cultured myoblasts express the nonclassical major histocompatibility antigen HLA-G.

We demonstrate that HLA-G, a nonclassical major histocompatibility complex class I antigen, is expressed in muscle fibers in various inflammatory myopathies. Further, interferon-gamma induces surface expression and upregulation of mRNA transcripts corresponding to different isoforms of HLA-G in myoblasts cultured from control subjects and patients. HLA-G may have important immunological functions in inflammatory myopathies and other local immune reactions as they occur during vaccination, myoblast transplantation, and gene therapy.

Recombinant versus natural human 111In-beta2-microglobulin for scintigraphic detection of Abeta2m amyloid in dialysis patients.

BACKGROUND: We previously introduced scintigraphy with 131I-labeled beta2-microglobulin (beta2m), purified from uremic hemofiltrate, that is, "natural" beta2m, to specifically detect beta2m-associated amyloidosis (Abeta2m) in hemodialysis (HD) patients. METHODS: To improve the safety and resolution of the scan, we covalently bound the chelator diethylenetriaminepentaacetic acid to natural beta2m to allow radiolabeling with 111In. In a second step, we generated and evaluated the usage of recombinant human beta2m (rhbeta2m) for scintigraphy. RESULTS: Using natural 111In-labeled beta2m, eight patients on HD for 0 to 17 years, without evidence of Abeta2m, were scanned. Whole-body scintigraphy at 48 to 72 hours postinjection revealed no significant tracer accumulation over joint regions. In contrast, nine patients on HD for 10 to 21 years with clinical, radiological, or histologic (N = 4) evidence of Abeta2m showed selective tracer uptake over various joint regions. Tracer accumulation in visceral organs, which could not be related to tracer elimination or metabolism, was not detected. Compared with the previous 131I beta2m scan, scintigraphy with 111In-labeled beta2m offered highly improved image contrast, increased sensitivity, and a 50 to 70% reduction of the radiation exposure. Scanning with 111In-labeled recombinant human beta2m was performed in six patients: No significant tracer accumulation was observed over joint regions in two patients on short-term HD without evidence of Abeta2m; in contrast, local tracer accumulations similar to those observed with natural beta2m could be demonstrated in four long-term (10 to 27 years) HD patients with clinical, radiological, and histologic (N = 1) evidence of Abeta2m. CONCLUSION: Scintigraphy for Abeta2m with 111In-labeled rhbeta2m provides a homogenous and safe recombinant protein source and leads to enhanced sensitivity and lower radiation exposure.

Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis.

Human CMV has evolved multiple strategies to interfere with immune recognition of the host. A variety of mechanisms target Ag presentation by MHC class I molecules resulting in a reduced class I cell-surface expression. This down-regulation of class I molecules is expected to trigger NK cytotoxicity, which would have to be counteracted by the virus to establish long-term infection. Here we describe that the human CMV open reading frame UL40 encodes a canonical ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide. Expression of UL40 in HLA-E-positive target cells conferred resistance to NK cell lysis via the CD94/NKG2A receptor. Generation of the UL40-derived HLA-E ligand was also observed in TAP-deficient cells. The presence of a functional TAP-independent HLA-E ligand in the UL40 signal sequence implicates this viral gene as an important negative regulator of NK activity.

LST1: a gene with extensive alternative splicing and immunomodulatory function.

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3' end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.

Association of beta(2)-adrenoreceptor variants with bronchial hyperresponsiveness.

Because of its involvement in the regulation of airway tone, the beta(2)-adrenoreceptor is considered a candidate for bronchial hyperresponsiveness (BHR) associated with asthma. This notion is supported by several reports that have implicated the chromosomal region 5q31-q33 harboring the gene for the beta(2)-adrenoreceptor in the genetics of asthma and related phenotypes. We performed a population-based association study focusing on BHR as a qualitative trait and omitting other asthma-related phenotypes. From a German population sample of 1,150 individuals we extracted all 152 bronchohyperreactive probands, who were compared with 295 bronchonormoreactive control subjects. All individuals were genotyped for three single nucleotide polymorphisms of the beta(2)-adrenoreceptor gene resulting in variants at the amino acid positions 16, 27, and 164. The genotyping protocol used allowed the determination of haplotypes of these polymorphisms. Whereas no individual polymorphism was associated with BHR, the Gly16/Gln27/Th164 haplotype was significantly underrepresented in the case group indicating a protective effect of this haplotype with regard to BHR. Upon reanalysis by sex a significant association persisted only for female probands.

Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis.

Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.

Implications of HLA-E allele expression and different HLA-E ligand diversity for the regulation of NK cells.

The interaction of HLA-E with CD94/NKG2A is dependant on the binding of HLA class I signal sequence derived peptides to HLA-E. In the caucasoid population two HLA-E alleles are observed at equal frequencies. Here we study the functional differences between the two HLA-E molecules with regard to cell surface expression, peptide binding, and potential to inhibit lytic activity of a CD94/NKG2A(+) NK cell line. In contrast to the HLA-E(R) allele, the HLA-E(G) allele shows considerable cell surface expression even in the absence of endogenous HLA class I signal sequence derived HLA-E ligands. Eighteen HLA-E allele/HLA-E ligand combinations were analyzed. No correlation between cell surface expression of HLA-E and NK cell inhibition was observed. The peptides present in the signal sequences of HLA-B15, -Cw0402, and -Cw7 bound to both HLA-E alleles but did not lead to an inhibition of NK cell lysis. In our experimental system the peptides A2 and G were not effective with regard to NK cell inhibition when bound to the HLA-E(R) allele. These results may be of functional significance particularly in the placenta where the only HLA-E ligands are derived from HLA-G and -C.

Cell surface detection of HLA-E gene products with a specific monoclonal antibody.

An HLA-E-specific monoclonal antibody (mAb) was obtained by immunization of human beta2-microglobulin transgenic mice (M-TGM) with spleen cells from double transgenic mice expressing HLA-E molecules (EM-TGM). This mAb, designated V16, specifically recognizes in flow cytometry analysis the HLA-E expressing mouse cells, whereas it does not bind to mouse cells expressing various HLA class I molecules (HLA-A2, -A3, -A11, -A26, A29, -B7, -B27, -Cw3, -Cw7, and HLA-G). V16 mAb binds efficiently to human EBV-infected B lymphocytes, PHA blasts and PBL, thus establishing the surface expression of HLA-E in vivo on these cells.