Transmembrane protein tyrosine phosphatase IA-2 (ICA512) is expressed in human midgut carcinoids but is not detectable in normal enterochromaffin cells.

A potential upregulation of receptor type protein tyrosine phosphatase IA-2 (ICA512) expression was detected by differential display and investigated in midgut carcinoid tumours. Normal intestine tissue and tumour tissue from 13 midgut carcinoid patients were studied by in situ hybridisation using an IA-2 ribonucleotide probe and confocal microscopy using specific IA-2 antibodies. Previously, it had been shown that IA-2 is located in the secretory granules of virtually all neuroendocrine cells. However, we found that IA-2 was not detectable in resting normal enterochromaffin (EC) cells of the small intestine, while high expression of IA-2 mRNA and protein was confirmed in both primary and metastatic carcinoid tissue. This difference in expression was not observed with chromogranin A or serotonin, two secretory granule hormones known to be expressed in EC cells, indicating that IA-2 was seemingly not necessary for the basal production and packaging of these hormones. When comparing patients receiving biotherapy before operation with untreated patients, we found expression of IA-2 to be lower in tumours from patients that had been treated with a combination of alpha-interferon and the somatostatin analogue, octreotide. There was no correlation between IA-2 expression and proliferation rates as measured by immunohistochemistry with antibodies against the Ki 67 antigen. Furthermore, we show that IA-2 is co-localised with serotonin in carcinoid tumours as well as in the pancreatic tumour cell line, BON1, which is interesting as serotonin secretion rate is presumably higher in tumour cells than in resting EC cells. Taken together, these findings may indicate a role for IA-2 in the later stages of the regulated secretory process.

Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism.

Recently the multiple endocrine neoplasia type 1 (MEN-1) tumor suppressor gene was cloned. MEN-1 encodes a nuclear protein, called menin, of hitherto unknown function. In order to investigate the biological function of menin we employed the yeast two-hybrid system to identify menin-interacting proteins. Here we report that menin functions as a transcriptional repressor through interaction with the transcription factor JunD. The interaction is mediated via the N-terminal transcription activation domain of JunD, and the C-terminal part of menin. In transient co-transfection experiments, expression of menin leads to specific repression of JunD transcriptional activity, which is dependent on the integrity of the menin C-terminal region. C-Terminal truncations of the protein not only abolish repression, but increase JunD transcriptional activity, implying the existence of a functional domain separate from the JunD-binding region. Menin-mediated repression is relieved by the histone deacetylase inhibitor trichostatin A, indicating that deacetylation of histones is an essential component of this repression mechanism, as has recently been demonstrated for the retinoblastoma protein. Missense, in-frame deletions, frameshift and nonsense mutations lead to inactivation of menin or possibly to truncated proteins. This would result in loss of repression of menin/JunD target genes, as well as non-target genes through indirect mechanisms, deregulation of cellular growth control and endocrine tumorigenesis.

Molecular cloning and characterization of a cDNA encoding the rat interleukin-8 receptor.

In this study we present the cloning, characterization and expression analysis of a cDNA encoding a rat interleukin-8 receptor (rIL-8R). A 1324 bp cDNA containing an open reading of 359 amino acids with an 86.1% overall identity with the previously characterized mouse IL-8R was isolated. Genomic DNA analysis using several restriction enzymes revealed a single band suggesting that the rIL-8R gene exists as a single-copy, which is in contrast to humans where there are two different IL-8Rs genes. Expression of rIL-8R mRNA was found in several tissues including spleen, heart, lung, liver, skeletal muscle and kidney. In brain and testis rIL-8R mRNA was not detectable. Rat IL-8R mRNA expression at the cellular level was studied in the spleen using RNA-RNA in situ hybridization and immunohistochemistry. IL-8R mRNA containing cells were predominately found in the mantle zone of the germinal center. These cells were identified as B lymphocytes using the OX-33 monoclonal antibody.

Assignment of the mouse homologue of a human MEN1 candidate gene, phospholipase C-beta 3 (Plcb3), to chromosome region 19B by FISH.

A recent study using comparative mapping analysis suggests that the proximal segment of mouse chromosome 19 contains the mouse homologs of the human multiple endocrine neoplasia type 1 (MEN1) flanking markers proximal to the locus. We have recently shown that phospholipase C-beta 3 (PLCB3) is a candidate gene for the MEN1 syndrome. In the present investigation we used fluorescence in situ hybridization with a genomic DNA clone for mouse Plcb3, and mapped the locus to chromosome region 19B. This is in agreement with the comparative mapping of the MEN1 flanking markers in mouse.

Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage.

Three human monocytic cell lines, U-937, THP-1 and Mono Mac 6 have, because of their morphology and staining properties, been classed as cell lines frozen in a window of the monocyte differentiation lineage corresponding to monoblasts and/or immature monocytes. These cell lines were analyzed for expression of a panel of hematopoietic differentiation markers by Northern blot analysis. They were all found to express one or several biochemical markers characteristic of immature cells in monocytic development, including myeloperoxidase, N-elastase, cathepsin G, myeloblastin, and azurocidin. Normal peripheral blood monocytes did not express these markers. Moreover, several markers expressed at high levels in mature monocytes, such as lysozyme, CD14, MHC class II and alpha-1 antitrypsin were either not expressed or were expressed only at low levels in the three cell lines analyzed. These results show that arrested differentiation at a relatively early stage of monoblast development is a common denominator for these human monocytic cell lines. Thus, transforming mutations acting at such an immature differentiation stage may frequently lead to neoplastic transformation, whereas similar mutations occurring at a more mature differentiation stage never give rise to any leukemias due to the loss of proliferative potential in committed cells.

Expression of a mast cell tryptase in the human monocytic cell lines U-937 and Mono Mac 6.

Expression of a mast cell tryptase mRNA was detected in two human monocytic cell lines, the U-937 and the Mono Mac 6, and in normal human peripheral blood (PB) monocytes. In the U-937 cell line but not in normal PB monocytes, the tryptase expression was upregulated 3-50 fold following phorbol ester (PMA)-induced differentiation, but no such induction was seen after retinoic acid, interferon-gamma or vitamin D3 exposure. The tryptases expressed in PMA-induced and non-induced U-937 and in Mono Mac 6 were characterized by PCR amplification and nucleotide sequence analysis. The U-937 cell line was found to express a tryptase identical to one of the previously cloned mast-cell beta tryptases (Tryptase I), and the tryptase expressed in Mono Mac 6 was found to be nearly identical to the previously cloned alpha tryptase. By northern blot analysis with oligonucleotide probes specific for the alpha and beta tryptases both cell lines were found to express only one type of tryptase. Densitometric quantifications of tryptase mRNA levels, in the two cell lines, showed approximately 80 times higher mRNA levels in Mono Mac 6 compared to non-induced U-937. Immunohistochemical staining for tryptase showed a marked heterogeneity in the Mono Mac 6 cell line. Only one out of 10 cells were positive for the protein but the levels in these cells were very high, equivalent, or even higher than the levels seen in the human mast cell line HMC-1. This shows that the expression of a single tryptase, in this case the alpha tryptase, is sufficient for the production of a stable protein and probably also a stable proteolytically active tetramer. The family of human mast-cell tryptases has been considered to represent a class of proteases specifically expressed in mast cells and basophilic leucocytes. The expression of tryptases in two monocytic cell lines and in normal PB monocytes indicate that in humans, the lineage specificity of these serine proteases is less restricted than earlier expected. The cloning of a full length cDNA for the murine counterpart to the human mast cell tryptases, the MMCP-6, is presented. No expression of the MMCP-6 was detected in a panel of mouse monocyte or macrophage cell lines indicating a species difference in the lineage specificity of the 'mast cell tryptases'.

Interleukin-4 down-regulates Sendai virus-induced production of interferon-alpha and -beta in human peripheral blood monocytes in vitro.

Recombinant interleukin-4 (rIL-4) caused a 65-70% reduction of the interferon-alpha (IFN-alpha) and -beta production induced by Sendai virus (SV) in human peripheral blood monocytes in vitro. Significant inhibition was seen at concentrations as low as 0.1 ng/ml. The rIL-4 also reduced levels of IFN-alpha and -beta mRNA by 60% and 67%, respectively, as well as the frequency of IFN-alpha and -beta mRNA-containing cells by 65% and 54%, respectively. The frequency of IFN-alpha/beta mRNA-containing cells was inhibited by rIL-4 throughout the whole course of induction by SV. IL-4 caused a shift of the grain count distribution towards less heavily labelled cells, suggesting an inhibitory effect of rIL-4 on most IFN-alpha/beta mRNA-containing cells. Antibodies to rIL-4 did not influence the normal IFN-alpha/beta response induced by SV, but abolished the inhibitory effect of the rIL-4. When rIL-4 was added to cells 4 h after start of stimulation by SV, at which time much mRNA has accumulated but little IFN-alpha/beta has been secreted, no inhibition of the IFN-alpha/beta production by rIL-4 was seen. IFN-gamma had only a minor reversing effect on the rIL-4 inhibition, but if cells were precultivated in medium with or without IFN-gamma for 6 h before SV induction, rIL-4 paradoxically enhanced the IFN-alpha/beta response. Our results suggest that rIL-4 inhibits an early step of IFN-alpha/beta induction in monocytes, at the level either of transcription of IFN-alpha/beta genes or of the processing or stability of mRNA. The IL-4 effects may however depend on the state of activation of the monocytes.

Interferon-alpha but not -beta genes require de novo protein synthesis for efficient expression in human monocytes.

Monocytes produce interferon-alpha (IFN)-alpha) and -beta when human peripheral blood mononuclear cells (PBMCs) are stimulated in vitro by Sendai virus (SV). We found that about 70% of the IFN-producing cells (IPCs) expressed both IFN-alpha and -beta mRNA; the rest expressed only IFN-beta mRNA. In the presence of the protein synthesis inhibitor cycloheximide (CHX), the frequency of IFN-alpha mRNA-containing cells, measured after 6h, was decreased by 85-90%. Results of nuclear run-on transcription assays showed that CHX inhibited IFN-alpha gene expression. The frequency of IFN-beta mRNA-containing cells was not reduced by CHX. Actually, a threefold increase was observed at the lower CHX concentrations. Studies on the kinetics of IFN-alpha/beta mRNA induction showed that CHX accelerated the appearance of IFN-beta mRNA-containing cells, increased IFN-beta mRNA levels, and delayed the normally occurring post-inductional decrease of IFN-beta mRNA. Unexpectedly, an initially normal or even accelerated IFN-alpha mRNA response was seen in the presence of CHX during the first 3-4 h after SV stimulation. This occurred in a small proportion of the potential IPCs. However, CHX prevented the subsequent marked increase of IFN-alpha mRNA levels. Preincubation of PBMCs for 6 h in conditioned medium (CM) containing IFN and other cytokines prevented the CHX-mediated inhibition of IFN-alpha mRNA. Without preincubation this was not seen. The preincubation in CM caused an accelerated appearance of IFN-alpha mRNA, resembling that of IFN-beta mRNA. The results suggest that IFN-alpha and -beta genes are differentially regulated in the same monocytes, the former requiring de novo synthesis of intracellular protein(s) for efficient expression.

The induction of interferon-alpha and interferon-beta mRNA in human natural interferon-producing blood leukocytes requires de novo protein synthesis.

The induction of interferon-alpha (IFN-alpha) and IFN-beta mRNA in natural IFN producing (NIP) cells in cultures of human peripheral blood mononuclear cells (PBMCs), stimulated by glutaraldehyde-fixed Herpes simplex virus type 1 (HSV)-infected WISH cells, was studied. The protein synthesis inhibitor cycloheximide (CHX) totally prevented the appearance of both IFN-alpha and IFN-beta mRNA, also in cultures supplemented with a conditioned medium (CM) assumed to contain soluble factors necessary for the IFN induction. However, when PBMCs were preincubated for 4 h in medium supplemented with fetal bovine serum (FBS) with or without addition of CM, the subsequent induction of IFN-alpha/beta mRNA became partially resistant to CHX. In serum-free medium containing interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF), the early induction of IFN-alpha mRNA became resistant to CHX, and, in contrast to FBS and CM supplemented medium, this was observed also without a preincubation of the PBMCs. In contrast, IL-1, IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, or IFN-gamma had no such effects. Our results suggests that de novo synthesis of proteins normally is required for the induction of IFN-alpha/beta mRNA. Such proteins might be cytokines, possibly CSFs, which in turn also may require protein synthesis for their actions. In contrast, the actual triggering signal provided by the HSV-inducer is independent of protein synthesis.

Phorbol ester-mediated inhibition of IFN-alpha/beta gene transcription in blood mononuclear leukocytes.

The phorbol ester PMA efficiently inhibits the in vitro IFN-alpha and -beta responses in human blood monocytes induced by Sendai virus and in natural IFN-producing cells induced by glutaraldehyde-fixed herpes simplex virus-infected human amnion (WISH) cells. This PMA-mediated inhibition of IFN-alpha/beta secretion is correlated with considerably reduced levels of IFN-alpha/beta mRNA, as determined by Northern blot analysis. Nuclear run-on assays further show that, at least in monocytes, PMA inhibits transcription of the IFN-alpha and -beta genes. The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol has the same effects as PMA, and the protein kinase inhibitor staurosporine prevents the PMA-mediated inhibition of IFN-alpha/beta expression. These results suggest a role for protein kinase C in the inhibition of IFN-alpha/beta responses. The PMA does not inhibit the accumulation of IFN-beta mRNA in monocytes stimulated by Sendai virus in the presence of cycloheximide, indicating that the inhibitory action of the phorbol ester requires de novo protein synthesis.

Characterization of the blood mononuclear leucocytes producing alpha interferon after stimulation with herpes simplex virus in vitro, by means of combined immunohistochemical staining and in situ RNA-RNA hybridization.

A procedure is described for combined immunohistochemical staining and in situ RNA-RNA hybridization of human peripheral blood mononuclear leucocytes (PBMC). These cells were first stimulated in vitro by herpes simplex virus (HSV)-infected fibroblasts and after 6 h fixed and stained with a panel of antibodies against differentiation antigens. Alpha interferon (IFN-alpha)-producing cells (IPC) were identified by in situ hybridization by means of a 35S-labelled IFN-alpha 2 cRNA probe. The IPC were infrequent, one in 200-5000 PBMC, but heavily labelled with the cRNA probe. They lacked antigens typical of T and B lymphocytes, and were also essentially negative for the Leu-M5 antigen, present on a majority of monocytes. However, 50% of IPC expressed OKM5 antigens, corresponding to the thrombospondin receptor. The IPC lacked the antigens present on null lymphocytes detected by OKT16, but most of them expressed HLA-DR, -DP and -DQ antigens. The IPC may represent a small subpopulation in the monocyte/macrophage lineage, resembling cells described as antigen presenting and stimulators of autologous mixed lymphocyte reactions. Alternatively, they constitute a subpopulation among the null lymphocytes.

Different induction patterns of mRNA for IFN-alpha and -beta in human mononuclear leukocytes after in vitro stimulation with herpes simplex virus-infected fibroblasts and Sendai virus.

Human peripheral blood mononuclear leukocytes (PBL) were stimulated in vitro by HSV type 1-infected glutaraldehyde-fixed fibroblasts, or Sendai virus (SV). The PBL containing mRNA for IFN-alpha 2 or -beta 1 were clearly identified by RNA-RNA in situ hybridization by using 35S-labeled alpha 2- and beta 1-probes. Although the two inducers gave similar levels of IFN in the culture medium (about 20 U/10(4) PBL), the patterns of expression of mRNA at the cellular level differed. The HSV induced only IFN-alpha mRNA in the PBL, with a lag of 1 to 2 h, and with a peak frequency of about 10 labeled cells/10(4) PBL at 6 h. Grain counts were high, the majority of cells having more than 50 grains. They were morphologically medium to large lymphocytes. The HSV-infected glutaraldehyde-fixed fibroblasts therefore induce IFN-alpha 2 mRNA in infrequent but highly efficient PBL, each cell capable of producing as much as 2 antiviral units of IFN-alpha. In contrast, SV induced both IFN-alpha 2 and -beta 1 mRNA in PBL, and without clear lags. IFN-beta 1 mRNA-positive PBL peaked somewhat earlier (4 h) than cells containing IFN-alpha 2 mRNA (6 h), and their mean frequencies were approximately 80 and 60/10(4) PBL, respectively, in a panel of PBL from six blood donors. Grain counts were lower than with the HSV inducer, the majority of cells having less than 50 grains, and most labeled cells were morphologically monocytes. The frequency of labeled PBL rapidly decreased with increasing culture time with both the SV and HSV inducers, was low at 12 h and almost absent at 24 h.