Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr ...phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-kappa B, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced.
Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor ...antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor crosslinking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.
Cross-functional coopetition (the joint occurrence of cooperation and competition between departments) has received increasing interest from academia and practice. However, there is still little ...evidence on how cross-functional coopetition can be fostered. We investigate in how far leadership styles (consideration and participation) and organizational structures (centralization and formalization) can be employed to enable a firm's management favoring cross-functional coopetition between departments. Analyzing survey data from 234 German companies, we demonstrate that both consideration and participation have a positive effect on cross-functional coopetition. Additionally, we find that formalization has positive effect on cross-functional coopetition, whereas the effect of centralization is negative. We show that our findings are valid for a multitude of organizational cultures. Finally, we derive implications for research and practice as well as avenues for future research.
In untransformed T lymphocytes, pp19/cofilin, a cytoplasmic actin-binding protein, undergoes dephosphorylation and nuclear translocation in response to costimulation through accessory receptors ...(e.g., CD2), but not following TCR/CD3 triggering. In malignant T lymphoma cells, dephosphorylation and nuclear translocation of pp19/cofilin occur spontaneously through constitutive activation of a serine phosphatase. Blockade of these processes by the serine phosphatase inhibitor okadaic acid leads to apoptosis. Moreover, lowering the intracellular pp19/cofilin concentrations by antisense-cofilin transfection results in reduced cloning efficiencies. These findings provide support for the view that pp19/cofilin plays a critical role in the growth and survival of both untransformed and malignant T lymphocytes.
Alloreactive human T lymphocytes were cloned in soft agar or by limiting dilution and subsequently propagated with interleukin 2 and alloantigen for 8 months or more. By indirect immunofluorescence ...every clone was reactive with anti-Ia antibodies as well as the T cell-specific antibodies anti-T3 and anti-T11 and expressed either T4 or T8 antigens. All 15 T8+clones were highly cytotoxic for the sensitizing alloantigen. In contrast, only two of the seven T4+clones mediated cytotoxic effector function. The specificity of T4+and T8+clones and subclones was analyzed on a panel of typing cells and by antibody blocking studies of major histocompatibility complex (MHC) determinants on the stimulating alloantigen. It was found that T8+clones killed targets that shared class I MHC antigens (HLA-A,B) with the original stimulator cells whereas cytotoxic T4+clones were directed at class II MHC antigens (Ia-related). Preincubation of the allogeneic target cell with a monoclonal antibody to a nonpolymorphic HLA α -chain determinant inhibited killing by the T8+clones but did not affect T4+cytotoxic function. In a reciprocal fashion, anti-Ia antibodies to common framework structures on the same target cell blocked killing by T4+but not by T8+clones. These results indicate that T4+and T8+T lymphocytes have receptors for different classes of MHC antigens and suggest that cytotoxic T4+subpopulations might be important in human transplantation and autoimmune disorders.
Hintergrund/Einleitung:
Der M. Crohn wird als eine Th1-dominierte Immunerkrankung betrachtet, die durch eine erhöhte Produktion proinflammatorischer Zytokine, insbesondere von Interleukin-12 (IL-12) ...gekennzeichnet ist. IL-12 besteht als heterodimeres Protein aus den zwei Untereinheiten p35 und p40. Kürzlich wurden zwei IL-12 verwandte Zytokine beschrieben, IL-23 und IL-27. Biologisch aktives IL-23 ist ein Heterodimer aus der p40 Untereinheit und einer p19 Untereinheit, die eine entfernte Verwandtschaft zu IL-12p35 aufweist. IL-27 besteht aus EBI3, einem IL-12p40 verwandten Protein sowie p28, einem neu entdeckten IL-12p35 verwandten Polypeptid.
Zielsetzung:
Wir bestimmten die mukosale Expression von IL-23p19 und IL-27p28, um zu überprüfen, ob eine Korrelation dieser Zytokine mit der entzündlichen Aktivität bei CED besteht.
Material und Methoden:
Die mRNA-Expression in der Colon-Mukosa von Patienten mit M. Crohn (MC; n=37), Colitis ulcerosa (CU; n=19), nicht-CED-Kontrollen (spezifische Colitis (SC), n=16) und gesunden Kontrollen (n=12) wurde durch real-time PCR gemessen.
Ergebnisse:
IL-23p19 war in entzündeten Arealen bei MC signifikant erhöht (p=0,0377), in einem geringeren Ausmaß bei CU-Patienten, nicht aber bei SC-Patienten. Die Erhöhung der IL-23p19-Konzentrationen korreliert mit der Schwere der endoskopischen Läsionen. IL-27p28 war hingegen nur bei aktivem MC signifikant erhöht, während sich für IL-12p35 und IL-12p40 keine Unterschiede zeigten.
Schlussfolgerung:
IL-23p19- und IL-27p28-Transkripte sind bei M. Crohn deutlich hochreguliert. Die stimulatorischen Effekte dieser Zytokine auf naive T-Zellen könnten, zusätzlich zu einer starken Synergie hinsichtlich der IL-12 stimulierten IFN-α-Produktion zur Perpetuierung des inflammatorischen Prozesses bei CED-Patienten beitragen. Darüberhinaus legt die erhöhte Expression von IL-23- und IL-27-Transkripten eine Th1-dominierte Rolle bei dieser Erkrankung nahe.
In der Pathogenese des M. Crohn wird einer überschießenden Immunantwort, die u.a. durch eine verstärkte Produktion von IL-18 bedingt ist, eine zentrale Rolle beigemessen. In einer kleinen Gruppe von ...Patienten mit MC konnten erhöhte IL-18-Spiegel nachgewiesen werden (J Immunol 1999;163:143). IL-18-Bindungsproteine (IL-18bp) fungieren als natürliche Inhibitoren. Wir untersuchten in einer großen Gruppe von Patienten mit aktivem M. Crohn (n=55), Colitis ulcerosa (n=30), infektiöser Colitis (n=20) und Kontrollen (n=16) die IL-18/IL-18bp-Expression. Die anti-inflammatorische Potenz eines IL-18bp-IgG-Fusionsproteins wurde in ex vivo-Kulturen von mononukleären Zellen der Lamina propria (LPMNC) von MC-Pat. überprüft.
Methodik:
IL-18- und IL-18bp-Transkripte wurden in Biopsien per real-time PCR quantifiziert, die Proteinkonzentration im Westernblot bestimmt. Ein IL-18bp(a)-IgG-Fusionsprotein wurde kloniert, in COS-Zellen exprimiert und aus dem Überstand aufgereinigt. Von MC-Patienten wurden LPMNC isoliert und die IL12/IL-18-induzierte IFN-α-Sekretion durch Zusatz des IL-18bp-IgG gehemmt.
Ergebnisse:
Obwohl die IL-18 Transkripte bei MC-Pat. im Vergleich zu CU-Pat. und Kontrollen erhöht waren, ist der Unterschied nicht signifikant. Die densitometrische Analyse der IL-18/α-Aktin-Ratio zeigte erhöhte IL-18-Proteinkonzentrationen beim MC (101% (Med.; min: 29%; max: 394%) im Vergleich zu CU vs. 58% (8–119%))(p<0,01). Bei genauer Betrachtung ist nur bei 33% der MC-Pat. das IL-18 Protein in entzündlich veränderter Mukosa im Vergleich zu nicht-entzündlich veränderter Mukosa deutlich erhöht. Ebenso ist die Expression der IL-18bp bei aktiven M. Crohn erhöht. Das IL-18bp-IgG hemmt die IFN-α-Sekretion in LPMNC-Kulturen nach 72h um 88,7% (Med. 44–98%). Auffällig ist, dass das FP in LPMNC weniger Pat sehr stark, bei anderen deutlich schwächer wirkt.
Diskussion:
Die IL-18 Expression bei aktivem M. Crohn ist heterogen, nur eine geringe Anzahl der Patienten exprimiert erhöhte IL-18-Konzentrationen. Zukünftige therapeutische Strategien gegen IL-18, z.B. IL-18bp-Fusionsproteine, bei aktivem M. Crohn sollten sich auf diese Untergruppe von Patienten konzentrieren.
Recent studies suggested that the clonally unique Ti epitopes defined by non-cross-reactive monoclonal antibodies might represent the variable regions of the antigen receptor. Here we determine ...whether such anti-Ti antibodies could trigger clonal T cell activation. Anticlonotypic monoclonal antibodies to the 49/43-kdalton heterodimer of a given clone or antibodies to the 20/25-kdalton membrane associated monomorphic T3 molecule selectively induce proliferation and IL-2 secretion when linked to a solid support. In contrast, anti-T4 and anti-T8 antibodies under the same conditions have no effect. In conclusion, these results imply that anticlonotypic antibody functions in a fashion analogous to antigen and further support the notion that the T3-Ti molecular complex represents the antigen receptor on human T lymphocytes.
To define mediator profiles in inflamed and noninflamed areas in inflammatory bowel disease (IBD) we analyzed the expression of 35 messenger-RNAs (mRNAs) encoding cytokines, chemokines, and some ...related molecules in transmural gut tissues (n=138) from patients with ulcerative colitis (UC), Crohn's disease (CD), and inflammatory and normal controls by real-time quantitative reverse transcription polymerase chain reaction. Using sample collectives with a comparable degree of inflammation, most parameters investigated showed similarly increased mRNA expression levels in both active UC and CD. This included proinflammatory cytokines, but also interferon (IFN) gamma and several IFN-gamma inducible chemokines. Only macrophage inflammatory protein (MIP)-2alpha mRNA was expressed at higher levels in inflamed UC vs. CD. IH revealed that MIP-2alpha protein was produced mainly by intestinal epithelial cells. Importantly, in histologically noninflamed/inactive IBD samples mRNAs for several mediators were significantly enhanced, accompanied by elevated levels of migration-inhibition factor related protein (MRP) 14 transcripts. CD14 positive macrophages were found especially in noninflamed/inactive UC, many of which coexpressed the RFD-7 antigen. Our results indicate a substantial overlap in cytokine/chemokine mRNA expression in UC and CD. Elevated mediator expression is evident in noninflamed/inactive areas in both diseases. Local recruitment of MRP-14 positive leukocytes might contribute to this phenomenon. In inactive UC a phenotypically altered population of macrophages expressing CD14 might play an additional role.
Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that ...anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.
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