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Down-regulation of lymphocyte proliferation to mitogens by granulocyte colony-stimulating factor (Rhg-CSF)

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Down-regulation of lymphocyte proliferation to mitogens by granulocyte colony-stimulating factor (Rhg-CSF)
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  25 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA une 1997 - Poster presentations Cytokines and lymphaid differentiation maturation 419 an immune response, and during treatment an anti-angiogenic effect develops. These actions in concert lead to tumour cell killing by apoptosis. P.3.11.04 Expression of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ccRl subunits durlng the in vitro dlfferentlation of human dendritic cells A.E. Semper, J.A. Hartley, ST. Holgate. University Medicine, General Hospital, Southampton, UK cytokeratin (75%) and glial fibtillaty acidic protein (GFAP) (42%). In the present study it was determined that both ProTa and tNF-y were capable of inducing the expression of MHC ctass II and enhanced expression of CD-40 antigens on the surface of fetal human epithelial cells. Duling the cultivation of cells (96 h) in the present of IFN-y (250 U/ml) or ProTa (1O-6 M) the percentage of the surface antigens expression was equal to: HLA-DR 75% and 25%, CD-40 72% and 25%, respectively. In addiiion, it was obsenred that INF-y enhanced the CD-I 6 and CD-36 expression on the epithelial cells. Introduction:The high affinity IgE receptor (FcsRI), srcinally identified on mast cells and basophils, has also been found on antigen presenting cells, including skin Langemans cells and pulmonary dendrttic cells (DCs). Numbers of Fc&I+ DCs are elevated in the bronchial mucosa of atopic asthmatics compared to normal lungs. It is not known whether this increase reflects enhanced recfuit- ment of Fc#I+ DCs from the blood or whether FcsRl expression is induced upon DC migration into the tissue and subsequent in situ differentiation. To investigate ifthe local cytokine environment modulates FcERI expression, we have studied the effects of cytokines on the expression of this receptor during in vitro differentiation of DCs from CD34+ progenitors or from peripheral blood monocvtes. Conclusion: The result of this investigation indicates that a new normal and transformed human fetal epithelial cell lines could be used as an adequate model system to study the diierent aspects of immune response. LP.3.11.06 1 Preferential induction of expression of CD134 (0X40), a member of the TNF receptor family, in a Th2-type environment A. Roos, E.J.M. Schilder-Tol, N. Claessen, M.A. Chand. J.J. Weening, J. Aten. Department of Pathof Academic Medica/ Center, University of Amsterdam, Amsterdam, The Netherlands Matewials nd Methods To differentiate DCs from CD34+ progenitors, CD34+ cells were positively selected from human umbilical cord blood using magnetic cell sorting (MiniMACS), and were cultured in medium containing 5% human serum, recombinant human (rh) GM-CSF and rhSCF. Parallel cultures were supplemented with till-4 or rhTNFcr, or with both cytokines. After 7 or 14 days, cells were harvested and analysed: (1) by flow cytometry using mAbs against the a subunit of Fc&RI (FcsRI-cu) nd (2) by semi-quantitative RT-PCR to measure levels of transcripts for all 3 Fc&RI subunits (a, @ & y). To differ- entiate DCs from human monocytes, peripheral blood mononuclear cells were depleted of T cells, B cells and NK cells by immunomagnetic separation wkh mAbs against CD3, CD19 and CD16, respectively. Monocytes were cultured in the presence of rhlL-4 and rhGM-CSF for 5 days; further differentiation of the resulting DCs was induced by 24-46 h exposure to rhTNFa. Fc&RI expression was analysed as already described. Results: CD34+ progenitors differentiated in the presence of GM-CSF, SCF and TNFa yielded only low numbers of CD1 a+ DCs on which surface expression of FcsRla was not detected. Only (I and y subunit mRNA could be detected by RT-PCR. Cultures exposed to GM-CSF, SCF and IL-4 expressed transcripts for all 3 subunits although B subunits were expressed only at low levels. However, levels of Fc&RI-(x surface expression on CDla+ DCs in these cultures were again negligible. Only in cultures exposed to GM-CSF, SCF, TNFol and IL-4 could surface expression of FcsRla be detected. In monocyte-derived DCs, transclipts for Fc.sRI$J ould not be detected at the start of culture but appeared by day 2; after 5 days, a subpopulation of CDla+ DCs expressed cell surface FcsRla. Conclusion: In two in vitro systems, exposure to IL-4 duling DC differentia- tion is accompanied by the appearance of mRNA encoding F~ERI-b. Inclusion of TNFa further increases expression of transcripts for F-RI subunits with the appearance of surface FcERI-(Y n a subpopulation of CDla+ DCs. How this relates to DC maturation is currently under investigation. P.3.11.05 New eplthellal human cell lines of fetal srcin as target cells for prothymosin a and gamma interferon actlon G.K. Grechko’. E.L. Panchenko’, A.V. Khodvakova*, A.M. Vasiliev’, T.V. Chemovskaya *, G.D. Kozlov&aya2, N.A. Belian&aya, V.P. Zav’ialov, G.T. Sukhich I. ’ ntemationat institute of Biological Medicine, Russian Research Centre Ibr Perinatolog) Obstetdes and Gynaelog~ Moscow, Russia, 21nstitute of Immunological Engineering, Lyubuchany, Russia Introduction: CD134 (0X40), a TNF receptor family member expressed on activated T cells, is involved in T cell activation and IL-4 production, and in T cell- dependent antibody production. Until now, information on regulation of CD134 expression is limited. We examined expression of CD134 in HgClp-induced Th2-mediated systemic autoimmunity in the BN rat, and the regulation of CD134 expression in vitro. Materlals and Methods: Expression of CD134 was studied by tri-colour flowcytometry on T lymphocytes from BN rats at day 14 after the first injection of HgCI2. Furthermore, lymphnode cells from untreated BN rats were exposed to HgCI2, IL-4 and several other cytokines and T cell activating compounds in vifm; expression of CD134 on CD4+ lymphocytes was measured by flowcytometty, using propidium iodide for identification of dead cells. Results: CD134 expression was strongly increased on CD4+CD45RCb T lymphocytes in lymphnodes, spleen and peripheral blood from BN rats suffering from HgClp-induced autoimmunity. Exposure of lymphnode cells to HgCl2 in vitro induced a rapid increase of the fraction of CD134+ cells within the CD4+ cell population. Experiments using Con A or PMA and ionomycin revealed an early and parallel upregulation of CD134, CD25 and CD71. A dose-dependent increase of CD134 expression was induced by 11-4, but not by IFN-y or TNFa. The effect of IL-4, but not the effect of HgCl2, could be totally inhibited by an IL+blocking monoclonal antibody. Effects of IL-4 and PMA on CD134 expres- sion could be blocked by the protein kinase-inhibitor staurospottn. Combination of these stimuli with ionomycin resulted in a synergistic increase of CD134 ex- pression, which was blocked by cyclosportn A, an inhibitor of calcineuttn. HgC12 also synergized with PMA in increasing CD134 expression, but its effects were resistent to both cyclosporin A and staurosporin. Flow cytometric analysis of intracellular hiol levels revealed that all stimuli which increased CD134 expres- sion induced a decrease of thiol levels in CD4+ T cells, whereas CD4+CD134+ cells were relatively protected against thiol depletion induced by HgCl2 or IL-4. Conclusion: Optimal induction of CD134 expression requires protein kinase and calcineurin activity, and may involve modification of the redox balance. Furthermore, the data indicate that CD1 34 expression is favoumd in a ThP-type cytokine environment. h vivomodulation studies to investigate the relevance of the CD134-CD134L interaction for HgClp-induced systemic autoimmunity are currently underway. P.3.11.07 Down-regulation of lymphocyte prollferatlon to mitogens by granulocyte colony-stimulating factor WWSF) Introduction: Numerous observations upport he fact that cells of epithelial srcin could play an active role in the immune system by responding to inflam- m&xv cvtokines and oarticioatina in antiaen oresentation. The current studv was c d out to examine ‘the ability of-human recombinant prothymosin (I (ProTti) and INF-y to induce MHC class II and enchanced CD-40 expression on the surface of diimnt human epithelial cells lines. S. Rutella’, C. Rumi , S. Sica’, G. Leone 2. ’ Center for the Flow Cytometric Study of Blood Cells, Catholic Universiw Rome, Itatx *Chair of Hernato/ow, Catholic University; Rome, ltaly The effects of rhG-CSF administration (16 &q$day S.C. or 5 days) on lympho- cyte phenotype and blastogenesis were evaluated in 14 HLA-matched healthy subjects. Lymphocyte count increased in all donors 1.5-fold (range 1.2-2.6) from baseline values @ < 0.0001.) Major changes of lymphocyte subsets cc- curred on day +4 of rhG-CSF: CD3+ and CD19+ cells increased 1.6 (range 1 l-2.6) and 3-fold (range 1.6-5.5) compared to baseline (p = 0.0062 and p < 0.0001 respectively) and NK cells (CD3-/CD16+CD56+) increased P-fold (range 1.2-2.6, p = 0.0011.) T helper (CD4+, Th) and T suppressor (CD6+, Ts) subsets increased 1.6-fold (range 1 Z-3.5; p c O.OfXJ5) and 1 &fold (range 1.2-2.2, p = 0.0021) respectively: consequently, CD4+/CD6+ ratio remained unaffected (1.0 vs 1.2 on day +4, p = MS.) No changes of type-2 cytokine producing T lympho- cytes (CD4+CD30+) occurred in any subject compared to pm-treatment levels. Monocyte count raised 5.5 fold (range 2.5-12.4) in all subjects @ -Z O.oooo3 on day +4. Lymphocyte blastogenic responses were evaluated by propidium iodide staining and subsequent flow cytometric analysis (BIastest@, Ylem, Italy). S-phase percentages of phytohemoagglutinin stimulated cultures markedly de- Materials and Methods: Epithelial cells isolated rom various organs by collagenase digestion of human embryos (16-20 wk age gestation) have been established both in primary cell cultures and as SV40 virus transformed cell lines too. Resufte~ Ten cell lines from retina, ids, lens, conjunctiva, pancreas, thymus, spleen, kidney and skin were developed. All lines demonstrated a typical epithe- lial motphology as judged by ligth microscopic studies after 2wo passages. They were keeping the differentiated phenotype, for example, their ability to secrete insulin for , J-cells of pancreas, and crystallins for lens epithelial cells, and extracellular matrix for keratinocytes of skin, and so on. By means of flow cytometry it was evaluated that both normal and transformed epithelial cells displayed the following cell surface and intracellular phenotype: Thy1 (62%), HlA-A, B, C (65%), HLA-DR (O%), CD-40 (159/o), CD-16 (IO%), CD-36 (5%),  420 Cytokines and lymphoid differentiatiorhatration 25 Jane 1997 - Poster presentations creased from 25.4% (range X-35.5) prior to rhG-CSF to 7% (range l&11.9. p = 0.0026,) 8 (range 4-12, p = 0.0091 nd 15% (range 9-22, p = 0.00.W) on days +2. +4 and +6 of rhG-CSF treatment. Similar changes were observed in concanavalin-A stimulated cultures; conversely, no significant modifications of lymphocyte reactivity to pokeweed mitogen were evident in rhG-CSF-primed lymphocytes compared to untreated samples. S-phase values of lectin-stimu- lated lymphocytes inversely correlated with neutrophil (Rs = 0.69; p = 0.0075~ and monocyie count (Rs = 0.61; p = 0.005.) RhG-CSF in healthy donors tran- siently downmodulates lymphocyte proliferative responses to lectin mitogens; rhG-CSF primed neutrophils and monocytes could release immuno-modulating cytokines. critically involved in down-regulation of lymphocyte proliferation. P.3.11.08 Identlflcatlon of genes controlllng the Tcell prollferatlve response to IL-2 uslng recombinant congenlc stralns Conclusion: CD4+CD45RA+ Tcells isolated rom adult and cord populations are capable of producing ThO-type cytokines following ptimary stimulation in a CD3 system. However, the data also suggests that newborn T cells are more readily directed, by 11-4, owards a ThP-type response. P.3.11 lO Studying of immunomodulating and antltumor properties of recombinant prothymosln a L.M. Khromykh’, A.V. Khodyakova*, I.P. Brisgalov’, G.D. Kozlovskaya2, A.M. Vasiliev2, T.V. Chemovskaya*, A.B. Vartapityan3, V.M. Abramov*, V.P. Zav’yalov2. 1 nstitute f Carcinogenesis, Cancer Research Centce, Moscow, Russia, 9Wtute of Enginekng Immunology state Stock Company. Moscow State University, Russia, 3nBiopreparationn, hcow State University; Russia Introduction: he attention of many scientists s rivet to prothymosin (I (ProTa), its role as a nucleus protein, its participation in regulator m&haniti of Cell proliferation and other. In the same lime it is known that (ProTa) is a mediator of immune system, factor of differentiation of T-cell chain of immunity. In our previous research we have shown data abilities of recombinant analogue of (ProTa) in vitro. It was established, that (ProTa) induced the differentiation of eadv precursors of bone marrow cells to mature T-cell forms. The previous treaiment of man or mouse thymocytes lead to the ability of this cells to’respond on suboptimal dose of mitogen and alloantigens. Present work is devoted to studying the recombinant (ProTa) in vivo, its abilw in restoration of immune status of immunodeficient (nude) mice. M. Krulov& I, H. Havelkov& ‘, M. Kosaiovti ‘, V. HOI&Y , A.A.M. Hart*, P. Demand , M. Lipoldov& I. ’ Institute of Molecular Genetics, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB cademy of Sciences of the Czech Republic, Prague, Czech Republic, *The Netherlands Cancer /nstitute, Amsterdam, The Netherlands Intmductlon: The induction of the expression of IL-2 and its homologous receptor is one of the major events in ? lymphocyte activation. Lympho&tes of mouse strains BALB/cHeA (BALB/c) and STS/A (STS) differ in IL-2 induced proliferative response, STS being a high and BALB/c a low responder in the range of concentrations 125-2000 IE/ml. We analyzed the genetic basis of this strain difference using the Recombinant Congenic Strains (RCS) of the BALB/c-c-STS/Dem (CcS/Dem) series. Materials nd Methods: n the work nude mice (H-2d) were used. (ProTa) injected subcutaneously in dose 10, 50, 100 fig/ mouse. The first group of animals was injected for 5 and IO days, and then lymph node and spleen cells were tested on their ability to react on the presence of mitogens -ConA (2.5 &ml) and PHA (IO &ml). B-cell immunity was studied for the ability to genemte the plaque forming cells on T-dependent antigen (SRBC). Second group of animals after injection of (ProTa) for IO days was inoculated allogeneic (H-2b) tumour cells EL-4 (50 103 cells per mouse) and continued the injection of preparation for 15 days. MaterMs and Methods: cS/Dem series comprises 20 homozygous strains all derived from parental background strain BALB/c and parental donor strain STS. Each CcSIDem strain contains different, random set of approximately 12.5% genes of the donor strain STS and approximately 67.5% genes of the background strain BALB/c. Proliferative response was tested by lymphocyte proliferation assay, genolyping of simple sequence length polymorphism (SSLP) was done by PCR. The role of genetic factors in the proliferative response was examined by analysis of valiance (ANOVA, NCSS). Results: We analysed 540 F2 hybrids between one of the high responder RC strains, CcS-4, and the low responder parental strain BALB/c. We found that the response to high IL-2 concentrations is controlled by a loci Cindal (Cytokine induced activation I), on chromosome 11 near the marker DllMil4. The response to a lower dose of IL-2 tested on lymphocytes of the same mice was found to be controlled by another locus, Cinda2, in the centrometic part of chromosome 12 near the marker Dl2Mit37. Con&don: Identification of genetic factors like Cindal and Cinda2, that control T-cells unction and understanding their action Is expected to contribute to the efficient analysis of the genetic control of susceptibility to infections and autoimmune diseases as well as testing the possible role of their human homologues in responsiveness to cytokine immunotherapy. Reeults: The experiments carrying out have shown that (ProTa) induced the ability of lymph node cells of nude mice o react on the presence of mitogens after first 5 injections of the protein. The proliferate level of the lymphocytes increase in 6-10 times compared to control. The analogue testing after IO injections of (PmTa) defined the ability of splenocytes (group 100 &mouse) to respond on the mitogens use. The level of proliferation increase in 6-8 times compared to control. Studying of the humoral immune response on SRBC have shown practically its full restoration compared with normal (Balblc) mice. Studying the process of tumour development have shown, that injections of (ProTa) lead to significance slowing of tumour growth. The volume of tumour knot decrease in 2-3 times compared to control. Conclusion: The recombinant analogue of (PmTa) induced relatively restoration of cellular and humoral immunity of T-immunodeficieni mice and also enhanced the tumour growth resistance of an organism. P.3.11.09 Enhanced sensltlvlty of newborn naive T cells to IL-4 in Th cell differentlatlon E. Rainsford, D.J. Reen. Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin. Dublin 12. Ireland Introduction: he factors controlling the transition from naive CD4+CD45RA+ P.3.11.11 Cytoklne expression by allergen-activated CD4+ and CD8+ cells R.J. Dearman. I. Kimber. Zeneca Central T~icotoov Laboratom Aldedev Park. Macclesfield, UK “. <. , tive selection using Dynabeads and Lymphokwik. The purified cells (<i% T cells to effector or memory cell phenotype, secreting various cytokines, CD45RO+) were stimulated using anti-CD3 plus anti-CD28 in the presence of IL-2 and IL-1 and in the presence or absence of IL-4. The cells were re- is still poorly understood in humans. Using anti-CD3 and highly purified peatedly stimulated at various timepoints from day 6 onwards and supematants were harvested for measurement of IFN-y, IL-4 and IL-5 by ELISA. CDCCD45RA+ T cells from umbilical cord blood and adult peripheral blood, the nature and kinetics of the cytokine response was examined under the influence of various cc-stimulatory molecules, in an APC independent system. Materiale nd Methods: CD4+CD45RA+ T cells were isolated bv neoa- iymphocytes, wher& the ixclusive source of the IFN-y is CD8+ cells’. We have now examined the influence of accessory cells on cytokine expression by Introduction: Prolonged topical exposure of mice to the respiratory allergen CD4+ and CD8+ cells. trimellitic anhydride (TMA) elicits the production by draining lymph node cells (LNC) of substantial amounts of interieukin 10 (IL-lo) and mitogen-inducible intetfeukin 4 (IL-4). but little of the Type 1 cytokine interferon y (IFN-y). Us- ing complement depletion techniques, we have demonstrated previously that production of the Tvoe 2 cvtokines IL-4 and IL-IO is associated with CD4+ T Materials nd Methods: ALB/c strain mice received 50 ~1 of 10% TMA dissolved in 4:1 acetone:olive oil vehicle on both shaved flanks on days 0 and 5. Five days later animals received a further 25 ~1 of chemical on the dorsum of both ears daily for three consecutive days. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised and a single cell suspension oreoared bv mechanical disaaareoation. CD4+ and CDB+ ractions (=-65% purej a;d CW-depleted (<I%) c& C D8 depleted (t5%) populations vmre prepared using magnetic mmunobeads. Cells (IO’ cells/ml) were cultured in the presence of 2 &ml concanavalin A (con A) for 24 hr or in the absence of con A for 120 hr. Cytokine content of culture supematants was analyzed using enzyme-linked immunosotbent assays. Rwultez Culture of unfractionated LNC and CD4+ fractions with con A for 24 hr resulted in vigorous IL-4 pmduction. Furthermore, the CD4 putifed population retained the capacily to express spontaneoudy comparable levels of IL-10 to those observed for unfractionated LNC. CD4 depleted and CD8+ Results: Primary stimulation of CD4+CD45RA+ T cells, in the absence of 11-4, resulted in IFN-y and IL-5 production in all cultures and IL-4 production in 3 out of 7 adult (AD) and 4 out of 6 cord blood (CB)samples tested at day 6. All cultures produced IL4 on day 8 following a second stimulation (Mean f SD: AD, 446 i 606 pglml; CB, 2i5 f 251 p&ml). There were no significant differences n evels of individual cytoklnes between adult and cord T-cell cultures following repeated stimulation over 8 to 12 days of culture. Culture of cells in the presence of IL4 resulted in a significant reduction of IFN-y produced by all samples following ptimaty stimulation (Mean f SD: AD, from 447 f 429 pg/ml to 68 + 39 pg/ml, p -z 0.05, and CB, fmm 523 f 262 p@ml o 95 f 33 pglml, p < 0.05). IL-5 production by cord T cells was significantly increased n the presence of IL-4 (Mean f SD: 278 f 90 p@ml o 933 f 381 pg/ml, p < 0.05) but adult T-cell IL-5 levels were unaffected.
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