Biomedical subjects

C Röpke

Publications and source records attributed to C Röpke.

At least 19 recordsLinked to original sources

PubMed2004

Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1.

To study cell motility in different phases of the cell cycle, time-lapse recording by computer-assisted microscopy of unsynchronised cells from three mammalian cell lines (L929, BT4Cn, HeLa) was used for the determination of the displacements of individual cells. The displacements were used for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner, and that an enhancement of Rac1 activity is sufficient for a delay of the reduced cell displacement otherwise seen in G2.

Animals↗

PubMed2002

IL-15 induces unspecific effector functions in human peptide-specific CD8+ T-cell cultures.

Antigen (Ag)-specific CD8+ T cells are a major host defence against viral infections. In the present study, we generated human CD8+ T-cell lines specific towards influenza matrix peptide (IMP)-pulsed Ag-presenting cells. We compared the effect of interleukin-2 (IL-2) and IL-15 on the proliferation and cytotoxic activity of primary and secondary IMP-specific cytotoxic T lymphocyte (CTL) culture. In primary CTL cultures, IL-15-induced cell expansion was considerably reduced as compared with IL-2-induced cell expansion, and IL-15 favoured the outgrowth of CTLs without peptide specificity in these cultures. Secondary IMP-specific CD8+ T cells were generated by the addition of IL-2 during two cycles of restimulation. From the third restimulation, identical CTL cultures were expanded with either IL-2 or IL-15 in parallel. Cell expansion as well as Ag specificity was considerably reduced after a 5 day culture period in the presence of IL-15. No or low CD69 expression was observed in IL-15-cultured CTLs, whereas IL-2-cultured CTLs contained high fractions of CD69+ cells. Furthermore, a high fraction of these latter cells coexpressed the cytotoxic marker CD56. However, IL-15-cultured CTLs exhibited cytotoxic activity without detectable expression of CD56, suggesting that CD56 is not essential for cytotoxic activity. Thus, the results presented suggest that IL-15 favours the outgrowth of unspecific cytotoxic effector T cells.

Antigens, CD↗

PubMed2001

Superantigen presentation by human retinal pigment epithelial cells to T cells is dependent on CD2-CD58 and CD18-CD54 molecule interactions.

Human retinal pigment epithelial (RPE) cells are capable of presenting bacterial superantigens (SAg) to T cells in vitro by ligation of MHC class II molecules on RPE cells with the T cell receptor. The purpose of this study was to evaluate the involvement of adhesion molecules in presentation of SAg. Cultured human fetal and adult RPE cells were treated with interferon-gamma (IFN-gamma, 500 U ml(-1) for 72 hr) and afterwards pulsed with the SAg staphylococcal enterotoxin A (SEA, 500 ng ml(-1) for 2 hr) followed by coculture with freshly obtained T cells isolated from peripheral blood. Proliferation was measured by (3)H-thymidine incorporation assay. In selected experiments, either RPE or T cells were pre-treated with blocking antibodies specific for cell surface molecules. For comparison, dendritic cells were used as superantigen presenting cells for T cells. This study showed that presentation of SEA by RPE cells to resting T cells was dependent on the presence of the molecules CD2, CD58 and CD18, CD54. The cycling status of T cells was decisive, thus resting T cells but not activated T cells were capable to proliferate in response to SEA presentation. Proliferation of T cells induced by adult RPE cells was comparable to the proliferation induced by dendritic cells at concentrations of SAg above 100 ng ml(-1), but at concentrations of SAg below 10 ng ml(-1) the response was significantly lower for SAg presented by RPE cells compared to dendritic cells. The results demonstrate that CD2-CD58 and CD18-CD54 interactions are critical for SAg presentation by RPE cells to T cells. The findings thus suggest that also presentation of peptides to resting T cells by RPE cells may be dependent upon these interactions.

Antibodies, Monoclonal↗

PubMed2001

Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells.

Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Ralphabetagamma) triggers mitogenesis in T cells. IL-2Ralpha expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Ralpha expression, survival, and growth of malignant SS cells.

Antineoplastic Agents↗

PubMed2001

Induction of cytotoxic CD8+CD56+ T cells from human thymocytes by interleukin-15.

CD8(+) CD56(+) cells isolated from human peripheral blood lymphocytes have been shown recently to represent a population of cytotoxic active T cells. However, it is not known if these cells are intrathymically or extrathymically developed or how these cells are influenced by growth factors. In the present study, we investigated the effects of interleukin-2 (IL-2) and IL-15 on human thymocytes with respect to development of CD8(+) CD56(+) T cells. Freshly isolated thymocytes contain few CD8(+) CD56(+) cells, but the number of these cells increases significantly when thymocytes are grown in the presence of IL-15 or IL-2. However, IL-15 induced a significantly higher fraction of CD8(+) CD56(+) cells compared with IL-2. Thus, although IL-2 and IL-15 are known to have a number of redundant functions, we here demonstrate that IL-15 is superior to IL-2 in inducing CD8(+) CD56(+) T cells from cultures of thymocytes. The majority of the IL-15-grown CD8(+) CD56(+) cells were CD45R0(+), representing a memory phenotype, and showed high expression of the IL-15R-complex and high numbers of CD69(+) cells. Moreover, cytotoxic activity was confined to this cell population.

Blood↗

PubMed2001

Lack of FasL expression in cultured human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.

Adult↗

PubMed2001

Interferon-alpha induces transient suppressors of cytokine signalling expression in human T cells.

The suppressors of cytokine signalling (SOCS) proteins comprise a newly identified family of negative feedback regulators of cytokine signalling. SOCS expression is differentially induced upon cytokine stimulation in different cell types. Here we show that interferon-alpha (IFNalpha) is a potent inducer of SOCS expression in human T cells, as high expression of CIS, SOCS-1, SOCS-2, and SOCS-3 was detectable after IFNalpha stimulation. After 4 h of stimulation, CIS, SOCS-1, and SOCS-3 expression had returned to baseline levels, whereas SOCS-2 expression had not declined. In contrast, after IL-2 induction neither CIS, SOCS-1, nor SOCS-2 expression levels declined after 6 h. In conclusion, we provide the first evidence that IFNalpha induces SOCS expression in human T cells. Moreover, we show that IFNalpha and IL-2 induce distinct patterns of expression kinetics, suggesting that dynamic changes in cytokine sensitivity might be mediated via induction of SOCS expression with different kinetics in T cells.

CD4-Positive T-Lymphocytes↗

PubMed2001

Gab2 is phosphorylated on tyrosine upon interleukin-2/interleukin-15 stimulation in mycosis-fungoides-derived tumor T cells and associates inducibly with SHP-2 and Stat5a.

Cutaneous T cell lymphomas (CTCLs) often show abnormal interleukin-2 (IL-2) receptor signaling. In this study, we investigated the role of Gab2, a recently identified adaptor molecule involved in IL-2 receptor signaling in CTCLs. We show that Gab2 was transiently phosphorylated by tyrosine in human mycosis fungoides (MF) tumor T cells upon IL-2 stimulation and that SHP2 as well as Stat5a associated inducibly with Gab2. IL-15, but not IL-4, also induced tyrosine phosphorylation of Gab2, suggesting that the IL-2 receptor beta-chain is important for IL-2-induced Gab2 phosphorylation. Preincubation of cells with the Src family kinase inhibitor, PP1, surprisingly increased the IL-2- and IL-15-induced tyrosine phosphorylation of Gab2, indicating that an Src family kinase member negatively regulates IL-2 receptor signaling in MF T cells. Thus, although Gab2 seems to function normally in MF T cells compared to normal T cells, Gab2 itself might be abnormally regulated by an Src family kinase.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Oligonucleotide fishing for STAT6: cross-talk between IL-4 and chemokines.

Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline epsilon transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1beta and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.

Animals↗

PubMed2000

Interleukin 15 is a growth factor for human thymocytes with preferential effect on CD8(+)cells.

The effects of IL-15, as compared to IL-2, on growth of human thymocytes has been evaluated. Expression of comparable amounts of receptor chains was found on IL-2 and IL-15 cultured thymocytes, as well as comparable receptor signalling. However, IL-15 was superior to IL-2 in promoting CD8(+)thymocyte growth, whereas CD4(+)cells proliferated to a higher extent in IL-2 cultures. CD4(+)8(+)and CD4(-)8(-)thymocytes expanded equally well in both culture types.

CD8-Positive T-Lymphocytes↗

PubMed2000

Inhibition of Huntington synthesis by antisense oligodeoxynucleotides.

The Huntington disease gone encodes the protein huntington, which is widely expressed during embryonic development and in mature tissues. In order to elucidate the physiological function of huntington, which so far is unknown, we intend to study the effect of antisense down-regulated huntington expression. We have found an inhibiting effect of a phosphorothioated oligodeoxynucleotide (PS-ODN) added to the culture medium of embryonic teratocarcinoma cells (NT2) and postmitotic neurons (NT2N neurons) differentiated from the NT2 cells. Specific inhibition of expression of endogenous huntington was achieved in NT2N neurons in the concentration range of 1-5 microM PS-ODN, whereas no inhibition was obtained in NT2 cells. We describe in detail the selection of the target sequence for the antisense oligo and the uptake, intracellular distribution, and stability of the antisense PS-ODN in the two cell types. Antisense down-regulation of huntington in this model of human neurons represents a suitable approach to study its normal function.

Animals↗

PubMed2000

Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway.

The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.

Base Sequence↗

PubMed1999

Compromised virus control and augmented perforin-mediated immunopathology in IFN-gamma-deficient mice infected with lymphocytic choriomeningitis virus.

To define the role of IFN-gamma in the control of acute infection with a noncytopathogenic virus, mice with targeted defects of the genes encoding IFN-gamma, perforin, or both were infected i.v. with two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in wild-type mice. Our results reveal that IFN-gamma is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus-specific TCR are adoptively transferred before virus challenge, indicating that the disease is the result of an unfortunate balance between virus replication in internal organs, e.g., liver and spleen, and the host response; resetting this balance by increasing host responsiveness will again lead to a rapidly controlled infection and limited tissue damage. Thus, the presence or absence of IFN-gamma determines whether CTLs will clear infection with this noncytopathogenic virus or induce severe immunopathology.

Adoptive Transfer↗

PubMed1999

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.

Antibodies, Monoclonal↗

PubMed1999

Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

Animals↗

PubMed1999

Superantigens are presented by and activate thymocytes from infants.

A high percentage of human fetal and postnatal thymocytes express MHC class II molecules. This raises the possibility that human thymocytes in early life are able to present peptides to other immature T cells and thereby initiate thymic selection of these cells. Here we address this question by exposing newly harvested infant thymocytes to superantigen (Sag) which binds to the T-cell receptor and to MHC class II chains outside the peptide binding groove. The results show that the thymocytes are able to present Sag and to be activated to proliferation as well as apoptosis by Sag presented by other thymocytes. The absence of responses to Sag with mutations in class II binding sites showed that class II molecules were necessary for the responses, and very low expression of class II molecules on CD4-8- cells indicates that the demonstrated T-cell/T-cell interactions are confined to T-cell receptor-positive CD4+8+, CD4+8-, and CD4-8+ cells. These latter subsets were shown to be able to present Sag to each other. These findings suggest that class II+ thymocytes may participate in the selection of self-restricted T cells during a critical period in the shaping of the human immune system.

Antigen Presentation↗

PubMed1998

Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells.

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Role of interferon-gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-gamma knock-out mice.

Generally, interferon-gamma (IFN-gamma) is considered a critical regulator of T cell mediated inflammation. For this reason, we investigated the pathogenesis of lymphocytic choriomeningitis in mice with a targeted defect of the gene encoding this cytokine. Our results revealed that IFN-gamma is redundant in the afferent phase of the antiviral T cell response as well as a local mediator of this T cell mediated inflammatory disease. However, IFN-gamma may play an indirect role as it is involved in reducing extraneural infection that may compete with CNS for available effector cells. Analysis of the inflammatory exudate disclosed that leucocyte recruitment was unimpaired in the absence of IFN-gamma as was the upregulation of ICAM-1 and VCAM-1 on endothelium at the inflammatory site. However, local macrophage activation (production of tumor necrosis-alpha and NO) was significantly impaired. Notably, a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN-gamma knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may be induced in the absence of IFN-gamma and local macrophage activation.

Animals↗