CD4 mRNA Expression Research Articles

Reduced IL-2 and IL-4 mRNA Expression in CD4 + T Cells from Bovine Leukemia Virus-Infected Cows with Persistent Lymphocytosis

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4 + T cells to the expression of mRNAs encoding interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-γ, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4 + T cells from cows seronegative to BLV (BLV −), seropositive without persistent lymphocytosis (BLV +PL −), and seropositive with PL (BLV +PL +) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-γ, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV +PL + cows as compared to BLV − cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4 + T cells from BLV +PL + cows. In contrast, Con A stimulated PBMCs and CD4 + T cells did not differ significantly in expression of IFN-γ, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4 + T cells and semiquantitative RT-PCR the frequencies of CD4 + T cells transcribing IFN-γ, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV −, BLV +PL −, and BLV +PL + cows were determined. There were no significant differences in the frequencies of CD4 + T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4 + T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4 + T cells, thus suggesting aberrant Th regulation in subclinically infected animals.

Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

Anti-tumor mechanism of Z-100, an immunomodulatory Arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, on pulmonary metastases of B16F10 melanoma: restoration of helper T cell responses via suppression of glucocorticoid-genesis.

In the present study, the anti-tumor mechanism of Z-100 was investigated with the use of pulmonary metastasis of B16F10 melanoma. In B16F10 mice, Th1 cytokine production (IL-2, IFN-gamma) was suppressed in comparison with normal mice. On the other hand, Th2 cytokine production (IL-4, IL-10) was increased in the B16F10 mice. The administration of Z-100 to B16F10 mice restored the balance of Th1/Th2 cell responses from the Th2 dominant state to the normal state. Z-100 significantly suppressed the pulmonary metastasis of B16F10 melanoma in a dose-dependent manner. These results suggest that Z-100 restored the breakdown of Th1 cell responses, resulting in the suppression of pulmonary metastasis of B16F10 melanoma. Moreover, Z-100 decreased the corticosterone levels, which is known to suppress the Th1 cell responses, in both serum specimens and splenic tissue, and the steroidogenic CYP11A1 mRNA expression in CD4+ T cells. These results suggest that a suppression of pulmonary metastasis and restoration of Thl/Th2 cell responses by Z-100 may be due to the decrease in the corticosterone levels and the steroidogenic CYP11A1 mRNA expression of CD4+ T cells in B16F10 mice. Further, the role of Th1 cytokine, IFN-gamma, on these activities of Z-100 was examined. The suppressive effects of Z-100 on pulmonary metastasis and restoration of Th1/Th2 cell responses were eliminated by the administration of anti-IFN-gamma mAb. Moreover, the suppressive effects of Z-100 on glucocorticoid-genesis were eliminated by the administration of anti-IFN-gamma-mAb. These results suggest that Z-100 restores the balance of Th1/Th2 cell responses via the suppression of glucocorticoid-genesis by Z-100-induced IFN-gamma. IFN-gamma acts as a key cytokine in anti-tumor activities of Z-100.

Quantitative analysis of Cyclin D1 and CD23 expression in mantle cell lymphoma and B-chronic lymphocytic leukemia

We studied Cyclin D1 ( CyD1) and CD23 mRNA expression with real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) method. CyD1 expression in peripheral blood of seven mantle cell lymphoma (MCL) patients was found to be 1305.4 times higher than in 24 B-chronic lymphocytic leukemia (CLL) patients. CD23 expression in CLL was found to be 54.8 times higher than in MCL. These differences were statistically significant, and no overlap was found in CyD1 expression intensities between MCL and CLL. RQ-PCR allows rapid, simple and accurate quantification of CyD1 and CD23 expression, even from small samples, and is thus useful for the diagnosis of MCL and CLL.

Granulocyte‐colony stimulating factor enhances the expression of transforming growth factor‐β mRNA in CD4‐positive peripheral blood lymphocytes in the donors for allogeneic peripheral blood stem cell transplantation

The degree of acute graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) has been observed to be, unexpectedly, of an equal level to that after bone marrow transplantation. To explain this phenomenon, we hypothesized that granulocyte-colony stimulating factor (G-CSF) administration may induce transforming growth factor (TGF)-beta producing T cells in the donors. Five donors received 10 microg/kg G-CSF subcutaneously for 4 days. The TGFbeta mRNA expression in CD4(+) cells as measured by real time reverse transcription-polymerase chain reaction increased after G-CSF administration. This elevation is considered to be one additive mechanism of repression of acute GVHD after allo-PBSCT.

Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease.

It has been proposed that CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, is preferentially up-regulated on Th2-type human T cells. In order to investigate a correlation between infection with Echinococcus multilocularis and CD30 expression, we analysed regulation of CD30 mRNA, a variant form of CD30 mRNA (CD30v) and CD30 ligand (CD30L) mRNA expression on PBMC from patients with alveolar echinococcosis (AE) using reverse transcriptase-polymerase chain reaction (RT-PCR). In PBMC of patients with AE as well as healthy donors, spontaneous expression of CD30L mRNA and the CD30v mRNA could be detected. However, the intact form of CD30 mRNA could be detected neither in freshly isolated PBMC of patients nor in PBMC of healthy individuals. Expression of CD30L mRNA and the variant form of CD30 mRNA was frequently detected at individual time points during 72 h of culture of PBMC stimulated with crude Echinococcus antigen. In contrast to CD30v or CD30L mRNA expression, induction of CD30 mRNA expression was detected only in three out of six (50%) healthy donors and in 10 out of 21 (48%) patients with alveolar echinococcosis after 72 h of incubation. As a control, mitogenic stimulation of PBMC of both healthy individuals and infected patients led to expression of intact CD30 mRNA within 24 h of culture. These data demonstrate the different expression of two different forms of CD30 mRNA in PBMC of human individuals. The specific induction of CD30 expression is correlated only in rare cases with the clinical status of patients with AE, indicating the lack of a general induction of CD30 mRNA in this Th2-type-dominated helminthic disease. The data provide further evidence that the CD30 receptor is not an exclusive marker for a Th2-type response.

Enhanced T cell apoptosis in common variable immunodeficiency: negative role of the fas/fasligand system and of the Bcl-2 family proteins and possible role of TNF-RS.

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, attempts 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.

Interleukin‐5 in growth and differentiation of blood eosinophil progenitors in asthma: effect of glucocorticoids

1. There are increased numbers of circulating CD34(+) progenitor cells for eosinophils in patients with atopic asthma, with a further increase following allergen exposure or spontaneous worsening of asthma. We investigated the expression of IL-5 and IL-5Ralpha receptor in circulating CD34(+) progenitor cells in allergic asthmatics and the effects of corticosteroids. 2. Using double-staining techniques, up to 50% of CD34(+) cells expressed intracellular IL-5, and by RT - PCR, there was significant expression of IL-5 mRNA. When cultured in a semi-liquid methylcellulose medium, there were more eosinophil colony-forming units grown from asthmatic non-adherent mononuclear cell depleted of T cells in the presence of the growth factors GM-CSF, SCF and IL-3, but not of IL-5. 3. An anti-IL-5Ralpha receptor antibody and an anti-sense IL-5 oligonucleotide reduced the number of eosinophil colony forming units. No IL-5 mRNA or protein expression on T cells was observed in asthmatics or normal subjects. In the presence of growth factors including IL-5, there were significantly greater colony numbers with eosinophilic lineage grown from either asthmatics or normal subjects. 4. Dexamethasone (10(-6) M) suppressed IL-5 mRNA and protein expression in CD34(+) cells, and reduced eosinophil colony-forming units in asthmatics, but not in normal subjects. Dexamethasone did not change the expression of IL-5Ralpha on CD34(+) cells. 5. We conclude that there is increased expression of IL-5 on blood CD34(+) cells of patients with asthma and that this expression may auto-regulate eosinophilic colony formation from these progenitor cells. Corticosteroids inhibit the expression of IL-5 in circulating CD34(+) progenitor cells.

CD20‐positive adult T‐cell leukemia

A 67-year-old woman was admitted to our hospital because of lymphadenopathy and lymphocytosis. Monoclonal integration of HTLV-I provirus DNA was detected, and a diagnosis of adult T-cell leukemia (ATL) was made. Flow cytometry revealed that the ATL cells expressed CD20 as well as T-cell-associated antigens, and expression of CD20 mRNA was also demonstrated. A novel T-cell subpopulation expressing CD20 molecules has recently been identified. This is the first report of CD20-positive ATL, suggesting that HTLV-I can infect and transform CD20-positive T cells. Am. J. Hematol. 66:39–41, 2001. © 2001 Wiley-Liss, Inc.

Co-transduction of cDNAs for c-kit and steel factor into single CD34+ cord blood cells further enhances the growth of erythroid and multipotential progenitors.

Previous studies have demonstrated that the c-kit encoded tyrosine kinase receptor and its ligand, steel factor (SLF), are critical for normal blood cell development. We have reported that transduction of the c-kit gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blood (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-transducing c-kit and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels of CD34 and different levels of c-kit. Cells were then prestimulated with granulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL)-3, IL-6, erythropoietin (Epo) in the presence and absence of various concentrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, and then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kit and SLF genes significantly enhanced colony formation compared with individual gene transduction, especially by erythroid and multipotential progenitors that responded to stimulation by added cytokines. Little or no growth was seen with the c-kit- and/or SLF-transduced cells without addition of cytokines. The degree of enhancement effected by co-transduction inversely correlated with the degree of expression of c-kit protein before transduction. Optimal enhancing effects were noted in CD34(+++) kit(Lo/-) cells co-transduced with both c-kit and SLF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF mRNA expression in CD34(+++) cells and enzyme-linked immunoadsorbent assay (ELISA) measurement of secreted SLF protein demonstrated that the transduced SLF cDNA was expressed and soluble SLF was released in medium cultured with SLF gene transduced MACS-separated CD34(+) cells in the presence, but not in the absence, of IL-3, GM-CSF, IL-6, and Epo. These results demonstrate the enhancement of the proliferation of growth factor responsive HPC that express transduced c-kit and SLF genes.

Sequence and functional properties of African green monkey CD4 silencer

We reported previously that in African green monkey (AGM) CD4 lymphocytes, CD4 mRNA expression undergoes a decrease following in vitro activation, and CD4 cells are therefore subject to loss of CD4 expression on the cell surface. To examine the transcriptional regulation of the CD4 gene in this species, we analyzed the CD4 silencer, which has been identified as a regulatory element responsible for the down regulation of CD4 transcription in CD8 cell lineage cells. Sequence analysis indicated that the CD4 silencer of the AGM was highly homologous to that of humans. However, two nucleotide substitutions were present in one of the nuclear protein binding sites, which was characterized as the FP II site having a strong enhancing effect on transgene expression in CD4 cells. By performing transient transfection assays, we found that the enhancing activities of the CD4 silencer or FP II-containing fragment of the AGM were greatly reduced in a human CD4 cell line as compared to those of human materials. The CD4 mRNA level was significantly decreased in the human CD4 cell line when synthetic oligonucleotide corresponding to the human FP II sequence was added to the culture. These observations imply that FP II-protein interaction might be required for the maintenance of sufficient expression of the CD4 gene, and the enhancing activity mediated by the above interaction might be decreased in the AGM CD4 silencer, due probably to the nucleotide changes occurring at the FP II site.

Breakdown of Th Cell Immune Responses and Steroidogenic CYP11A1 Expression in CD4+ T Cells in a Murine Model Implanted with B16 Melanoma

The association between the balance of Th1/Th2 cell responses and CYP11A1 expression in CD4+ T cells was investigated in a murine model implanted with highly metastatic B16F10 melanoma cells (B16F10 mice). When 2 × 105 cells/mouse of B16F10 cells were inoculated into C57BL/6 mice, Th2 cell responses and pulmonary metastasis were increased. In addition, corticosterone levels in splenic tissue or serum and CYP11A1 mRNA expression (mRNA encoding cholesterol side-chain cleavage p450 enzyme) in CD4+ T cells were increased in these mice. When the anti-corticosterone drug aminoglutethimide (CYP11A1 inhibitor) was administered to B16F10 mice, corticosterone levels in splenic tissue or serum and CYP11A1 mRNA expression were decreased at 14 days after tumor inoculation. In addition, Th1 cell responses were restored and pulmonary metastasis was reduced by aminoglutethimide. These results indicated that the breakdown of Th cell responses and increase of pulmonary metastasis were due to an increase in steroidogenic CYP11A1 mRNA expression in CD4+ T cells. Moreover, it was suggested that promotion of CYP11A1 mRNA expression in Th2 cells was partially involved due to an increase in level of corticosterone in splenic tissue and the breakdown of Th cell responses locally in the splenic tissue, which then affected the maintenance of Th2 cell functions in the microenvironment of tumor-bearing mice.

CXCR4 and CCR5 expression by H9 T-cells is downregulated by a peptide-nucleic acid immunomodulator

Product R (Reticulose TM) is a peptide-nucleic acid immunomodulator with broad-spectrum antiviral activity that was recently shown to increase expression of mRNAs encoding the proinflammatory cytokines, IFN- γ, IL-1 β, IL-6 and TNF- α. Since these cytokines induce expression of the chemokines, MIP-1 α, MIP-1 β, RANTES, and SDF-1, all of which inhibit viral infectivity, we were interested to determine if Product R also alters chemokine expression. In addition, the finding, that Product R decreases HIV-1 RNA and extracellular p24 antigen in H9 T-lymphoma cells, suggested to us that this drug may block viral infection by reducing the expression of chemokine receptors on target cells. We have therefore utilized H9 cells to test the effects of Product R on expression of mRNAs encoding the chemokine receptors, CD4, CXCR4 and CCR5, as well as their ligands, IL-16, SDF-1, MIP-1 α, MIP-1 β, and RANTES, by RT-PCR. We also assayed the effect of Product R on surface receptor expression by flow cytometry, and on the chemotactic activity of these cells towards the CXCR4 ligand, SDF-1, and the CCR5 ligands, MIP-1 α and RANTES. H9 cells were cultured for 3–21 days in medium containing 5% or 10% Product R, or 5% or 10% PBS. We found that, compared to control cultures, cells cultured in media containing Product R expressed lower amounts of CXCR4 and CCR5 mRNA and surface antigen at all time points. Culture for 3 days in media containing Product R also reduced the ability of cells to migrate towards 10–20 ng/ml SDF-1 and 100–250 ng/ml RANTES. In contrast, Product R had no effect on the expression of CD4 mRNA and receptor protein, or on expression of IL-16 mRNA. These findings suggest that Product R may have clinical efficacy in HIV-1-infected patients by downregulating viral coreceptors on target T-cells.

Infection of mesangial cells with HIV and SIV: Identification of GPR1 as a coreceptor

Mesangial cells are an important component of the glomerulus. Dysfunction of mesangial cells is thought to be involved in the development of human immunodeficiency virus type 1 (HIV-1)-associated nephropathy (HIVAN). HIVAN is a structural renal failure frequently observed in patients with acquired immune deficiency syndrome. However, the susceptibility of mesangial cells to HIV-1 is disputable. More than ten G protein-coupled receptors, including chemokine receptors, have been shown to act as HIV-1 coreceptors that determine the susceptibilities of cells to HIV-1 strains with specific cell tropisms. We examined the susceptibility of mesangial cells to various HIV-1, HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) strains. Expression of CD4 and HIV/SIV coreceptors was examined by Western blotting and polymerase chain reaction. Mesangial cells were found to be susceptible to HIV-1 variant and mutants that infect brain-derived cells, but highly resistant to T-tropic (X4), M-tropic (R5) or dual-tropic (X4R5) HIV-1 strains. In addition, mesangial cells were also susceptible to HIV-2 and SIV strains that infect the brain-derived cells. Among HIV/SIV coreceptors we tested, the expression of GPR1 mRNA was detected in mesangial cells. Expression of CD4 mRNA and protein was also detected in them. Mesangial cells and GPR1-transduced CD4-positive cells showed similar susceptibilities to the HIV-1 variant and mutants and HIV-2 and SIV strains. CD4 and GPR1 mRNAs were detected in mesangial cells. Mesangial cells were susceptible to HIV/SIV strains that use GPR1 as a coreceptor. Our findings suggest that an orphan G protein-coupled receptor, GPR1, is a coreceptor expressed in mesangial cells. It remains to be investigated whether the interaction of mesangial cells with specific HIV-1 strains through GPR1 plays a role in the development of HIVAN.

Detection of IL-3 and g-csf mRNA and protein in CD34 progenitor cells and serum from cml patients

Abstract Our previous studies demonstrated that primitive subsets of leukaemic cells isolated from patients with chronic phase CML exhibit growth factor independent proliferation, in both single cell and bulk cultures, which is at least partially dependent on autocrine production of bioactive IL-3 and to a lesser degree G-CSF (PNAS 96; 12804, 1999). In those studies ∼ 85% of the cases investigated were CML patients in whom the stem cell compartment (LTC-IC) was already predominantly Ph + . Since such cases represent less than 10% of newly diagnosed patients, it was of interest to examine IL-3 and G-CSF mRNA expression in CD34 + progenitor cells and any corresponding protein production in serum in a larger series of CML patients independent of the Ph status of their LTC-IC compartment. CD34-enriched lineage-negative cells were isolated using STEMSep TM from leucapheresis samples obtained at diagnosis from chronic phase patients and viable, propidium iodide (PI)-negative, annexin V-negative, CD34 + cells then isolated by FACS. Total RNA was extracted from ≥ 5 × 10 4 such cells and c-ABL, BCR-ABL, IL-3 and G-CSF expression examined using a 2-step RT-PCR procedure. 8 of the 10 CML samples tested to date are positive for IL-3 expression. Using sensitive ELISA kits, G-CSF was detected at significantly lower levels in the sera of CML patients (7.2 ± 2.9 pg/ml, n=13) than in normal controls (24.5 ± 3.2 pg/ml, n=9, p=0.001), with levels correlating inversely to the WBC. In untreated patients with elevated WBC (45–166 × 10 9 /L) G-CSF levels proved to be undetectable. In patients on hydroxyurea, in whom the WBC was controlled ( 9 /L), levels were low but above the threshold for detection (2–11 pg/ml). Of interest, in patients who had achieved a complete cytogenetic remission following either allogeneic BMT or interferon therapy, levels were within the normal range. These findings support the proposed negative feedback mechanism between peripheral neutrophil count and serum G-CSF levels. IL-3 was detected in sera from 1 of 9 normal controls and 5 of 29 CMLs (range 1–35 pg/ml), 3 of whom were in accelerated phase. These results confirm that IL-3 mRNA expression in the progenitor compartment is typical of the majority of cases of CML. However, this does not necessarily lead to detectable levels of this growth factor in the circulation suggesting its effects in vivo may be largely confined to extravascular regions of the marrow and other tissues that are infiltrated by CD34 + leukaemic cells.

Gastrointestinal Stromal Tumors May Originate from a Subset of CD34-Positive Interstitial Cells of Cajal

Most gastrointestinal stromal tumors (GISTs), a subgroup of mesenchymal neoplasms of the gut wall, express both Kit (CD117) and CD34 proteins. It has been suggested that GISTs originate from or differentiate into interstitial cells of Cajal (ICC), after several reports indicated that ICC are likely the only cells in the gut which express both Kit and CD34. ICC are among the few cell types resident in the gut which express Kit, together with mast cells. However, the question whether or not ICC express CD34 is currently disputed. Using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) on cultured murine intestinal cells, single ICC were selected by morphology and tested for the expression of c-kit and CD34 mRNA. Most ICC were only c-kit-positive, however a subset (7 out of 43) were double positive for both c-kit and CD34. In the human small intestine, sequential immunohistochemical staining for Kit and CD34 proteins on the same 3-microm sections showed that some of the ICC surrounding Auerbach's plexus and ICC within the circular muscle layer of the small intestine were positive for both Kit and CD34. In addition, CD34(+)Kit(-) cells were seen adjacent to ICC. These data from two different techniques indicate that ICC can be double positive for Kit and CD34. Thus, GISTs with the Kit(+)CD34(+) phenotype may arise from a subpopulation of CD34(+) Kit(+) ICC.

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10.

We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1alpha (SDF-1alpha)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70,000 SDF-1alpha-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 approximately 4.4 nM and Kd2 approximately 14.6 nM), and a total of approximately 130,000 SDF-1alpha-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4-SDF-1 receptor-ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.

Cloning, sequence and mRNA expression of bovine interleukin-16.

A lambda gt11 cDNA library was constructed using mRNA isolated from Theileria parva-infected bovine lymphocytes. Sequencing of random clones of this library resulted in the identification of a cDNA encoding bovine interleukin-16 (IL-16). The cDNA has an open reading frame of 1134 bp, and a 3' untranslated region of 275 nucleotides with a polyadenylation signal 16 bases upstream from the poly (A) tail. The protein predicted by the cDNA sequence contains 378 amino acids and the level of amino acid homology with the corresponding part of human precursor IL-16 is 79 %. No information is available about the tissue distribution of IL-16 in cattle, therefore we investigated the expression of IL-16 mRNA in bovine lymphoid tissues by reverse-transcription polymerase chain reaction assays. To investigate the potential of IL-16 as an immunoregulatory molecule we also analysed IL-16 mRNA expression in CD4+ and CD8+T-cell clones derived from T. parva-immunised cattle.

An immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, Z-100, restores the balance of Th1/Th2 cell responses in tumor bearing mice

The regulatory effect of Z-100 on the balance of Th1/Th2 cell responses in BALB/c mice bearing Meth-A fibrosarcoma was investigated. In tumor bearing mice, Th1 cytokine production (IL-2, IFN-γ) are suppressed and Th2 cytokine production (IL-4, IL-10) are increased, as compared with those of normal mice. The administration of Z-100 (10 mg/kg) to tumor bearing mice restored the balance of Th1/Th2 cell responses from Th2 dominant state to the normal state. This regulatory effect of Z-100 was eliminated by depletion of adherent cells from splenocytes derived from tumor bearing mice, and by the treatment with 2-ClAdo (a macrophage inhibitor). Similarly, this regulatory effect was diminished by the treatment with anti-IL-12 mAb and anti-IFN-γ mAb. In addition, the IL-12 p40 mRNA expression in splenic adherent cells and IFN-γ mRNA expression in CD4 + T cells were increased by the administration of Z-100 to tumor bearing mice. These results suggested that Z-100 restored the balance of Th1/Th2 cell responses to the normal one in tumor bearing mice through the activation of macrophages and up-regulation of IL-12 production from macrophages and IFN-γ production from CD4 + T cells.

Retroviral-Mediated Gene Transduction of c-kit Into Single Hematopoietic Progenitor Cells From Cord Blood Enhances Erythroid Colony Formation and Decreases Sensitivity to Inhibition by Tumor Necrosis Factor-∝ and Transforming Growth Factor-β1

The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.