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.

Endogenous FLT-3 ligand serum levels are associated with disease stage in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients' sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system.

Expression and regulation of the thrombopoietin receptor variants MPLP and MPLK in PBMC.

The human c-mpl proto-oncogene encodes the receptor for thrombopoietin (TPO), which plays a central role in magakaryopoiesis and platelet production. Recent results showed that c-mpl mRNA expression is restricted to CD34(+) cells, megakaryocytes, and platelets. The authors analysed mRNA expression by reverse transcription and PCR (RT-PCR) of the transmembrane Mpl variants, MPLK and MPLP, in magakaryocytic cell lines, peripheral blood mononuclear cells (PBMC), lymphocytes, and monocytes. Transcription of both Mpl variants was detected in freshly isolated PBMC and the megakaryocytic cell line, DAMI, whereas none could be detected in the megakaryocytic cell line Meg-01. The cytokines TPO, interleukin 11 (IL-11), erythropoietin (EPO), and stem cell factor (SCF), which are known stimulators of megakaryopoiesis, but not interleukin3 (IL-3), increased MPLP mRNA detection in PBMC during 96 h of incubation. However, concomitantly the detection of MPLK transcripts decreased in these cells. MPLP was also detected in monocytes, B-lympocyte and T cell populations. In contrast, MPLK mRNA expression was low in monocytes, B and T cells, and no clear increase in transcription detection was observed during stimulation of these cells.

Endogenous serum thrombopoietin concentrations in patients with myelodysplastic syndromes.

Prognosis in patients with myelodysplastic syndromes (MDS) is closely correlated with cytopenia. To date, no factor is available which is able to reliably stimulate megakaryopoiesis in vivo. Recently, the ligand of the mpl receptor was cloned and found to be thrombopoietin (TPO). We measured endogenous TPO serum levels in 69 patients suffering from MDS using an TPO receptor-based ELISA. Twenty-six of the patients suffered from refractory anemia (RA), 12 from RA with excess of blasts (RAEB), 25 from RAEB in transition (RAEBt), and six from chronic myelomonocytic leukemia (CMML). This assay uses a chimeric molecule consisting of the human TPO receptor fused to the Fc portion of human IgG as the capture reagent. Biotinylated antibody to TPO IgG was used for detection and the lower detection limit in serum was found to be 160 pg/ml. Samples obtained from healthy individuals with a normal platelet number were shown to be below detectable levels. In patients with RA, high endogenous serum TPO concentrations correlated with low platelet count and significantly elevated TPO concentrations were only found when megakaryopoiesis was absent or decreased in the bone marrow. This correlation was not detected in RAEB and RAEBt patients and no detectable TPO serum concentrations were found in six CMML patients whether they showed decreased or normal platelet count. Our data show that endogenous TPO production is upregulated by decreased circulating platelet count only in patients with refractory anemia. In the more advanced stages of MDS where the leukemic clone has further progressed, an inadequate TPO response occurs, possibly due to overexpression of the mpl receptor by the malignant clone.

Spontaneous or interferon-gamma-induced T-cell infiltration, HLA-DR and ICAM-1 expression in genitoanal warts are associated with TH1 or mixed TH1/TH2 cytokine mRNA expression profiles.

The purpose of the study was to investigate the cytokine gene expression patterns and immunohistochemical characteristics of genitoanal warts in order to obtain a clue as to the immunological mechanisms possibly relevant for wart regression or persistence. We analysed surgically removed warts from 11 patients, 2 of whom were immunosuppressed. Lesions of five of the nine otherwise healthy individuals were additionally treated with intralesional interferon-gamma (IFN gamma) prior to surgery. Invasion of CD4+ T cells into the papillomas and HLA-DR and ICAM-1 expression on keratinocytes were found in two otherwise healthy patients and were intensified by intralesional IFN gamma in four of five patients. The mRNA expression patterns in seven of eight non-recurrent warts were compatible with a predominant TH1 or mixed TH1/TH2 cytokine profile. In contrast, in recalcitrant warts of three patients (one healthy, two immunocompromised) histological signs of immunore-activity and TH1-like cytokine mRNA expression were not detected. In recurrent warts of a renal transplant patient, IL-4 and IL-5 mRNA expression was repeatedly found suggesting a predominant TH2 response. In conclusion, immunoreactivity to genitoanal warts such as T-cell infiltration, HLA-DR and ICAM-1 expression was associated with a predominant TH1 or mixed TH1/ TH2 cytokine mRNA expression profile.

In vitro stimulation of erythropoiesis by stem cell factor alone in myelodysplastic syndrome patients with elevated endogenous erythropoietin serum levels.

Anemia remains a therapeutic problem in patients with myelodysplastic syndrome (MDS). In view of the recently reported potential of stem cell factor (SCF) in restoring erythropoiesis in combination with erythropoietin (Epo), we first aimed to define a correlation between SCF serum levels and anemia in MDS. Endogenous SCF levels in 50 MDS patients were determined by using a quantitative sandwich enzyme immunoassay. Broad interindividual variations were observed, but SCF serum levels were in the normal range with no correlation to peripheral blood count. A soft agar culture system was used to further define the role of SCF for stimulation of erythroid growth. Bone marrow mononucleated cells of 20 MDS patients (4 refractory anemia, 5 refractory anemia with excess of blasts, 7 refractory anemia with excess of blasts in transition, and 4 chronic myelomonocytic leukemia) were investigated, and SCF plus Epo was able to stimulate burst-forming unit-erythroid significantly more than SCF or Epo alone independent of French-American-British group. When mononucleated cells from six MDS patients (two refractory anemia, two refractory anemia with excess of blasts, and 2 refractory anemia with excess of blasts in transition) with elevated serum Epo levels were incubated in the presence of SCF and autologous serum, a significant dose-dependent stimulation of burst-forming unit-erythroid number and cells per colony was detected. Erythroid differentiation was further enhanced by adding serum with high colony-stimulating activity obtained from patients with severe aplastic anemia. Our data suggest that in MDS patients with high endogenous Epo serum levels SCF alone might be effective in stimulating erythropoiesis in vivo.

Cloning and genomic characterization of LST1: a new gene in the human TNF region.

The leucocyte specific transcript - 1 (LST1) represents the human homolog of the mouse B144 transcript, encoded within the tumor necrosis factor (TNF) region of the human major histocompatibility complex class III interval. The gene is localized about 4 kilobases upstream of the lymphotoxin beta gene. It spans a polymorphic genomic region encompassing the microsatellites TNFd and TNFe in intron 3 and a polymorphic Pvu II restriction site 260 base pairs downstream of the polyadenylation signal. Isolation of a full-length cDNA clone revealed that LST1 codes for IFN-gamma-inducible 800 nt transcripts, which are present in lymphoid tissues, T cells, macrophages, and histiocyte cell lines. The cDNA contains three long open reading frames (ORF) with the most likely ORF encoding a transmembrane protein. Its close linkage to the TNF genes and pattern of expression point toward a possible role for LST1 in the immune response.

Pvu II polymorphism in the primate homologue of the mouse B144 (LST-1). A novel marker gene within the tumor necrosis factor region.

A Pvu II RFLP was mapped within the LST-1 gene, the human homologue of the mouse B144 sequence, establishing LST-1 as a new marker gene within the TNF region. We investigated the distribution of this Pvu II RFLP in 274 unrelated individuals, 132 additional HLA-DR7-positive individuals, 86 homozygous lymphoblastoid cell lines, and in four families. Seventeen of 274 individuals (6.2%) were heterozygous for the Pvu II restriction site (ADB1 = lack and ADB2 = presence of the Pvu II restriction site). In our study population the polymorphism has a much wider distribution than that previously reported in an analysis of selected haplotypes. Besides a strong association of ADB1 with HLA-B14, -DR7, we found a further association with HLA-B35. These results were also validated by family segregation studies and analyses of homozygous cell lines. In addition, five of 17 individuals carrying the ADB1 allele had HLA types other than B14 or B35, emphasizing that the presence of ADB1 is not limited to the HLA-B14, DR7 haplotype. LST-1 and its polymorphism may be used as an additional marker of the TNF region, where genes responsible for autoimmune diseases are suspected to be localized.

Restricted expression of Mov13 mutant alpha 1(I) collagen gene in osteoblasts and its consequences for bone development.

Cell type-specific differences in the transcriptional control of the mouse gene coding for the alpha 1 chain of collagen type I (Col1a1) have been revealed previously with the help of the Mov13 mouse strain which carries a retroviral insert in the first intron of the gene. Transcription of this mutant Col1a1 allele is completely blocked in all mesodermal cell types tested so far, with the exception of the odontoblast where it is expressed at an apparently normal rate (Kratochwil et al. [1989] Cell 57:807-816). To define the tissue specificity of the mutant allele more precisely, we have now studied its expression in osteoblasts, another skeletogenic cell type which, like odontoblasts, produces high amounts of collagen I. Evidence for transcription of the Mov13 allele was obtained by in situ hybridization in homozygous (M/M) and heterozygous (M/+) bone tissue, in grafts as well as in vivo. The presence of mouse collagen I and the development of bone tissue were demonstrated in M/M skeletal elements grown on the chick chorioallantoic membrane (CAM). Further support for expression of the mutant gene was obtained from two 16 day M/M fetuses in vivo. Bone tissue of diverse embryological origin (vertebrae and ribs of somitic origin, long bones derived from lateral plate, calvariae from head paraxial mesoderm, and mandibulae from head neural crest) expresses the mutant allele. However, in situ hybridization experiments indicate that only a subpopulation of osteoblasts is capable of transcribing it at a high rate, resulting in severe impairment of bone development in grafts and in vivo. Therefore, osteoblasts, in comparison to odontoblasts and fibroblast-like cells, assume an intermediate position with respect to transcription of the Mov13 allele. We suggest that this diversity in the utilization of the mutant collagen gene reflects cell type-specific differences in the transcriptional regulation of the wild type (wt) Col1a1 gene.

Recombinant human interleukin-8 restores function in neutrophils from patients with myelodysplastic syndromes without stimulating myeloid progenitor cells.

Prognosis in myelodysplastic syndrome (MDS) is not only correlated closely with blast cell count in bone marrow and chromosomal abnormalities but also correlated with decreased leucocyte count and function leading to acquisition of lethal infections. Recently, clinical trials in MDS have focused on the application of haemopoietic growth factors such as G-CSF or GM-CSF, which have proven to increase neutrophil count and function. However, these cytokines carry the risk of stimulating the malignant clone, particularly in patients with increased blast cell count. Therefore, investigation of cytokines which are able to stimulate neutrophil function without the potential risk of stimulating haemopoietic progenitor cells may be relevant for MDS. As the stimulatory effect of interleukin-8 on neutrophil function is well known, we investigated whether recombinant human IL-8 is also able to improve the function of neutrophils gained from patients with MDS. Using three different techniques--the E. coli killing assay (8 patients), the production of reactive oxygen as determined by cytochrome c reduction (7 patients) and chemiluminescence (8 patients)--a significant stimulation of neutrophil function at a concentration of 10 nm IL-8 was found in all test systems. No correlation with FAB classification was evident. On the other hand, IL-8 only mildly stimulated growth of myeloid progenitor cells in bone marrow culture of healthy individuals and MDS patients. This minimal stimulation was blocked by a neutralizing antibody directed against GM-CSF, suggesting an indirect effect of IL-8 via secondary GM-CSF release. Thus, IL-8 is able in vitro to repair the functional abnormalities of neutrophils from patients with MDS but has only a marginal influence on myeloid progenitor cells.