Carsten Röpke

Suppressors of cytokine signalling: SOCS

The mechanism of positive regulation of cytokine signalling pathways has been well investigated, whereas our knowledge of negative regulation is relatively sparse. Here we review recent literature on important negative regulators: the family of suppressors of cytokine signalling, SOCS, consisting of eight members (SOCS‐1 to SOCS‐7 and CIS) all sharing a central SH2 domain and a C‐terminal SOCS box. Expression of CIS, SOCS‐1, SOCS‐2 and SOCS‐3 is induced by various cytokines, and overexpression studies in various cell lines have demonstrated their inhibitory roles. These family members have been implicated in the negative regulation of several pathways, particularly the JAK/STAT pathway, and since this signalling pathway is responsible for their induction, they form part of a classical negative feedback circuit. To date, at least three different modulating mechanisms have been demonstrated: through the SH2 domain they bind to phosphotyrosines on the target protein, leading to inhibition of signal transduction by N‐terminal inactivation of JAK, by blocking access of STAT to the receptor sites, or by SOCS box‐targeting bound proteins to proteasomal degradation. In gene modification studies in mice, it has been demonstrated that SOCS‐1 plays an important role in IFNγ‐regulation and T‐cell differentiation, while SOCS‐2 seems necessary for normal growth regulation. SOCS‐3−/− mice die during embryogenesis for a reason still not fully understood, but insufficient control of fetal erythropoiesis or defects in placental development may be involved. The physiological role for the other family members, as well as their molecular regulation mechanisms, remain to be revealed.

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-2Rαβγ) triggers mitogenesis in T cells. IL-2Rα expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Rα negative. An aberrant expression of IL-2Rα has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Rα 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-2Rα 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-2Rα gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Rα 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-2Rα expression, survival, and growth of malignant SS cells.

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3

A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

Immunotoxicity of the pyrethroid insecticides deltametrin and α-cypermetrin

Abstract The synthetic pyrethroids deltametrin and α-cypermetrin were studied for effects on the immune system in 28-day studies in F344 male rats. Sixteen rats per group were dosed with either deltametrin 0, 1, 5, or 10 mg/kg body wt./day or α-cypermetrin 0, 4, 8, or 12 mg/kg body wt./day in soy bean oil by gavage. Haematology, bone marrow cell counts, tests for natural killer (NK) cell activity and mitogen response (Con A and STM) as well as quantitation of lymphocyte subpopulations were performed. Spleen cells from immunized animals (six animals/group) were tested for antibody production (SRBC-PFC). Volumes of lymphoid compartments of mesenteric lymph nodes and thymus were estimated using stereological methods. In the deltametrin study an effect was found in the groups receiving 5 or 10 mg/kg body wt. The effects were: increased weight of mesenterial lymph nodes, decreased thymus weight in immunized animals and an increase in numbers of SRBC-PFC and splenic NK cell activity. An effect on relative adrenal weight was seen in the 10 mg/kg body wt. group. No severe effects on the immune system was found. The lowest effect level of α-cypermetrin was 12 mg/kg body wt./day based on increased relative adrenal weight.

Antigen expression of the pancreatic beta‐cells is dependent on their functional state, as shown by a specific, BB rat monoclonal autoantibody IC2

Antigen expression is studied corresponding to a monoclonal autoantibody (IC2) derived from a hybridoma of rat myeloma Y3 cells and splenocytes of the diabetic BB rat. The selective reactivity of IC2 with islet cells has earlier been proven. We studied the possible specificity for beta islet cells, and the possible variation in autoantigen expression. Islet cells were isolated by cautious collagenase and dispase treatment. The cells were labelled with IC2 alone or together with anti‐insulin immunoglobulin in double‐labelling experiments. Extensive series of cells were examined by immunofluorescence microscopy, and some samples also by flow cytometry. In double‐labelling examinations we found that only anti‐insulin positive cells could bind the IC2 antibody, thus showing beta‐cell selectivity. On the other hand, not all anti‐insulin positive cells were IC2‐positive. Since insulin treatment has been shown to decrease the incidence of diabetes in the BB rat, islet cells were examined after reduced beta‐cell strain. Islet cells from Lewis and Wistar Furth rats display 21.4 ± 1.4% IC2‐positive cells, while islet cells from 24‐hour fasting animals showed 7.0 ± 1.4% (p < 0.0001). Similar results were seen for BALB/c mice (25.0 ± 1.8% vs. 13.7 ± 2.3%, p < 0.002). Also, after a week of insulin treatment, autoantigen expression was significantly decreased. Thus, the IC2 antibody is beta‐cell‐specific, and expression of the corresponding cell surface antigen depends on the functional state of the beta‐cells.

Light and electron microscopic studies of the paracortical post-capillary high-endothelial venules.

SummaryThe light and electron microscopic structure of the high-endothelial postcapillary venules of lymph nodes were studied in the mouse, rat, guinea pig, and rabbit. The venules were most frequently found in the mouse and rat. In all species, they reached their highest degree of differentiation in the paracortical area. The morphology in the light microscope was identical in all four species. The venules in the rat and mouse were surrounded by a cuff of concentrically-arranged lymphocytes, which was rarely seen in the guinea pig and rabbit.The ultrastructure of the high-endothelial cells in all four species was very complex; a prominent Golgi apparatus was present which often occupied large parts of the cytoplasm. Coated and uncoated vesicles originating in the Golgi apparatus often permeated the cytoplasm. These vesicles emptied their contents into the extracellular space after fusion with the plasma membranes.Numerous lymphocytes traversed vessel walls. During their passage, they were always located between, not inside the high-endothelial cells.

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-α (IFNα) 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 IFNα 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 IFNα induces SOCS expression in human T cells. Moreover, we show that IFNα 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.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin.

PURPOSE The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. METHODS Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. CONCLUSION A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruchs Membrane (BM) for RPE survival.

Inhibition of Huntingtin 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.

T lymphocyte subsets in patients with newly diagnosed type 1 (insulin-dependent) diabetes: a prospective study

SummaryT lymphocyte subsets in peripheral blood from 11 newly diagnosed Type 1 (insulin-dependent) diabetic patients were studied prospectively at three time intervals: as soon as possible after diagnosis, 3 weeks and 5 months later. Lymphocytes were marked with monoclonal OKT antibodies and examined in a fluorescence-activated cell sorter. The percentage of T lymphocytes (OKT 3) did not change significantly at the three study times. The percentage of helper/inducer T cells (OKT 4) was high the first week after diagnosis, but decreased at the 5-month examination (p<0.05). The percentage of suppressor/cytotoxic T cells (OKT 8) was low at diagnosis but increased at 3 weeks (p<0.02) and 5 months (p<0.01). The ratio OKT4/OKT8 lymphocytes was 2.28 at diagnosis, decreasing to 1.77 at 3 weeks and 1.87 at 5 months, compared with 1.46 for 16 age-matched control subjects. There was no significant change in the absolute number of lymphocytes. It is concluded that the distribution of T cell subsets was abnormal at the time of diagnosis, but changed towards normal within a few weeks, after which there was no significant change at 5 months. It is as yet unknown whether the high proportion of helper/inducer T cells and/or the low percentage of suppressor/cytotoxic T cells at diagnosis favour immune reactions involved in the pathogenesis of Type 1 diabetes.